HYDROGEL

Abstract

A hydrogel comprising a particle physically entrapped within a hydrogel matrix, a method for making the hydrogel, a particle for use in the hydrogel and the use of the hydrogel to sense a chemical species, especially anions in solution. The particle comprises an active material and a plurality of chains of a first polymeric material, each of the chains of the first polymeric material having a first end and a second end. The active material and the first ends of the chains form a core; and the second ends of the chains extend outwardly away from the core to form a shell. The hydrogel matrix comprises chains of a second polymeric material in the form of a three dimensional cross-linked network.

Claims

1. A hydrogel comprising a particle physically entrapped within a hydrogel matrix, the particle comprising an active material and a plurality of chains of a first polymeric material, each of the chains of the first polymeric material having a first end and a second end, wherein the active material and the first ends of the chains form a core; the second ends of the chains extend outwardly away from the core to form a shell; and the hydrogel matrix comprises chains of a second polymeric material in the form of a three dimensional cross-linked network.

2. The hydrogel of claim 1, wherein the active material comprises a luminescent material

3. The hydrogel of claim 1, wherein the active material comprises a metal complex or a non-protein organic fluorophore.

4. The hydrogel of claim 1, wherein the active material comprises an MRI contrast agent, a catalyst or a pharmaceutical.

5. The hydrogel of claim 1, wherein the active material and the first ends of the chain form a core by means of physical interactions only.

6. The hydrogel of claim 1, wherein the three dimensional cross-linked network is a chemically cross-linked network.

7. The hydrogel of claim 1, wherein the particle has a diameter of 50 to 1000 nm when measured in solution by dynamic light scattering.

8. The hydrogel of claim 1, wherein the first polymeric material or the second polymeric material comprises a co-polymer formed from at least one (meth) acrylate monomer.

9. The hydrogel of claim 8, wherein (i) the at least one (meth) acrylate monomer has the general structure H.sub.2C═CR—C(═O)—OR.sup.1 where R is H or CH.sub.3 and R.sup.1 is an organic group having a total of from 1 to 12 carbon atoms; or (ii) the at least one (meth) acrylate monomer is selected from one or more of 2-hydroxyethyl methacrylate, poly(ethylene glycol) methyl ether methacrylate, 2-(diethylamino) ethyl methacrylate, methyl methacrylate, butyl methacrylate, glycidyl methacrylate (2,3-epoxypropyl methacrylate, and/or ethylene glycol dimethacrylate.

10. A method for the preparation of a hydrogel the method comprising providing a particle comprising an active material and a plurality of chains of a first polymeric material, each of the chains of the first polymeric material having a first end and a second end; the active material and the first ends of the chains forming a core and the second ends of the chains extending outwardly away from the core to form a shell; and cross-linking polymer chains of a second polymeric material in the presence of the particle to produce a hydrogel matrix having the particle physically entrapped therein.

11. The method of claim 10, comprising an initial step before cross-linking of the polymer chains of the second polymeric material of preparing the particle from the active material and the chains of the first polymeric material.

12. The method of claim 10, wherein the chains of the first polymeric material are formed by co-polymerising at least two monomers in the presence of the active material.

13. The method of claim 11, wherein preparing the particle comprises purification.

14. The method of claim 10, wherein the cross-linking of the polymer chains of the second polymeric material comprises covalently bonding.

15. A particle comprising a luminescent material and a plurality of chains of a first polymeric material, each chain of the first polymeric material having a first end and second end, wherein the luminescent material and the first ends of the chains form a core and the second ends of the chains extend outwardly away from the core to form a shell.

16. A method for measuring the concentration of a chemical species in a sample, the method comprising contracting the sample with the luminescent hydrogel of claim 2; measuring a change in an optical property of the luminescent material produced by contacting the sample with the luminescent material; and converting said change in optical property to said concentration.

17. The method of claim 16, wherein the sample is an aqueous sample.

18. The method of claim 16, wherein the chemical species is a nucleoside polyphosphate (NPP) anion.

19. The hydrogel of claim 2, wherein the luminescent material comprises a metal complex, wherein the metal is selected from europium, terbium, gadolinium and ytterbium.

20. The hydrogel of claim 2, wherein the luminescent material comprises a the Eu(III) complex.

21. The hydrogel of claim 2, wherein the luminescent material comprises and Eu(III) complex has the structure: ##STR00013## wherein each R is independently selected from hydrogen, C1-C12 alkyl, and NHCOR.sup.1, where R.sup.1 is H or C1-C12 alkyl.

Description

[0103] Embodiments of the invention will now be described with reference to the following figures:

[0104] FIGS. 1A and 1B are schematic diagrams of a particle and a hydrogel in accordance with embodiments of the invention;

[0105] FIG. 2 shows short term fluorescence leaching analysis of a HEMA hydrogel loaded with HEMA armed Ru(bpy).sub.3 particles (left) and analysis of the water phase (right);

[0106] FIG. 3 shows long-term fluorescence leaching analysis of a HEMA hydrogel loaded with PEGMA particles (left) and the percentage fluorescence of the gel (right);

[0107] FIG. 4 short-term fluorescence leaching analysis of a HEMA hydrogel entrapping free Ru(bpy).sub.3 (not in particles) (left) and analysis of the water (right);

[0108] FIG. 5 shows fluorescence spectroscopy leaching analysis of fluorescent HEMA-shelled particles entrapped within p(HEMA-co-EGDMA) hydrogels of different crosslinking densities (as determined by monomer feed): 10 vol % (A) and 50 vol % (B) (λex=460 nm, measurements recorded every 2 nm, at integration time of 500 ms);

[0109] FIG. 6 shows fluorescence spectroscopy analysis for the long-term leaching of p(HEMA-co-EGDMA) hydrogels, crosslinked with 10 vol % EGDMA, entrapping non-particle Ru(bpy)3 (λex=440 nm, measurements recorded every 2 nm, at integration time of 500 ms); and

[0110] FIG. 7 shows fluorescence spectroscopy sensing analysis for a p(HEMA-co-EGDMA) hydrogel entrapping Sodium Green™ encapsulated in HEMA-shelled particles, equilibrated in solutions of sodium chloride of differing concentrations, (λ.sub.ex=488 nm, measurements recorded every 0.5 nm, at integration time of 500 ms).

EXAMPLES

Results and Discussion

[0111] Polymeric particles (polymeric “stars”) were made encapsulating fluorophores. The polymeric particles were prepared by cross-linking an “outer” methacrylate monomer with an “inner” methacrylate monomer using a cross-linker.

[0112] Hydrogels were made with 10, 20 and 30 vol % crosslinking density, loaded with PEGMA and HEMA armed particles, with cores of both DEAEMA (pH responsive) and MMA (non-responsive).

[0113] Leaching was prevented through the use of the “double entrapment” method, i.e. through the entrapment (encapsulation) of a fluorophore in particles and subsequent entrapment in the hydrogel. This is evidenced by FIG. 2, which shows a peak at 0.1=620 nm in the hydrogel and a lack of a peak at =620 nm in the solution analysis (the absorption maximum for the ruthenium complex), and a minimal decrease in the intensity of the gel fluorescence (with the slight decrease attributed to photobleaching).

[0114] Long-term stability analysis indicated little/no leaching over the course of 4 days (FIG. 3).

[0115] In contrast a control (using hydrogels with non-particle trapped fluorophore) indicated significant leaching after 20 minutes, highlighting the improved stability of the complex in the hydrogel imparted by the encapsulation (FIG. 4). 0 minutes=uppermost line and 100 minutes=lowermost line.

[0116] Evaluation of different methods indicated the “secondary entrapment method” using a HEMA gel was the best method to prevent fluorophore leaching:

TABLE-US-00003 Decrease in fluorescence after Method of entrapment 90 minutes (%) Comp. Ex. 1 Trapping in a DMAm gel* 94 Comp. Ex. 2 Trapping in a charged DMAEMA gel* 64 Ex. 1 Trapping in polymeric particles in a 40 DMAm gel* Comp. Ex.3 Trapping in a HEMA gel# 46 Ex.3 Trapping in polymeric particles in a 15 (±8)  HEMA gel# *fluorescein used as sensor, #Ru(bpy).sub.3 used as sensor,  8% (±8%) over 72 hours. Errors generated though the measurement error calculated by 3 repeat measurements of a single gel within a cuvette.

Fluorescein Dimethacrylate

[0117] The polymeric particles were synthesised based on a modified literature procedure (Mabire, A. B., Brouard, Q., Pitto-Barry, A., Williams, R. J., Willcock, H., Kirby, N., Chapman, E., O'Reilly, R. K., Polym. Chem. 2016, 7, 5943). In order to attribute any leaching to the escape of polymeric particles from the hydrogel, fluorescein dimethacrylate was used to covalently attach the fluorophore into the polymeric particle. The inventors would prefer to avoid chemical bonding between the active material and the polymer chains, i.e. to ensure that the active material and the polymer chains remain separate entities, but this example provides a useful demonstrator model. It was hypothesised that any reduction in fluorescence intensity would therefore indicate leaching of the particles from the gel. The resultant HEMA-shelled or PEGMA-shelled polymeric particles were entrapped within a HEMA-based hydrogel, synthesised at both 10 vol % and 50 vol % crosslinking EGMDA.

[0118] Following polymerisation, hydrogels were dialysed in a beaker of distilled water for 24 hours prior to analysis. This step of pre-dialysis prior to analysis allows for removal of non-trapped particles and unreacted monomer, and was hypothesised to remove any effect of swelling on the fluorescence intensity recorded. Leaching analysis of the 10 vol % crosslinking EGDMA gels (A) and the 50 vol % crosslinking EGDMA gels (B) loaded with HEMA particles (500 μl of particle solution), using fluorescence spectroscopy, indicated significant retention of fluorophore over the 90 minutes, evident in a strong fluorescence intensity after 90 minutes (FIGS. 5, A and B respectively, 0 minutes=uppermost line and 90 minutes=lowermost line). The presence of two peaks in the fluorescence spectrum of fluorescein has been demonstrated in the literature to be as a result of S1 to S0 transitions, with polymerisation shown to broaden the peaks.

Thermally Initiated Polymerisation

[0119] To ensure that entrapment of the particles within the hydrogel matrix using a thermally initiated method of polymerisation did not have a detrimental effect on the fluorescence of the probe, the fluorescence behaviour of the particles directly after synthesis, and after heating at 90° C. for 16 hours was evaluate. Results indicated that thermally initiated polymerisation did not impact on the fluorescence, with a slight increase in fluorescence intensity attributed to the slightly higher concentration of the heated sample owing to slight water evaporation during the heating.

Tris(Bipyridine) Ruthenium (II) Complex (Ru(Bpy).SUB.3.)

[0120] The fluorophore for development and optimisation of the demonstrator model was changed to tris(2,2′-bipyridyl)dichlororuthenium (II) complex, (Ru(bpy).sub.3). This fluorophore is significantly closer in structure, charge and hydrophilicity to the europium complexes described above. Whilst being metal centred, Ru(bpy).sub.3 is highly water soluble. Therefore, in preventing leaching of the Ru(bpy).sub.3 complex, it would be expected that the europium complex would not leach owing to its more amphipathic nature.

[0121] Owing to the significant effect of pH on the fluorescence intensity of the HEMA hydrogels when encapsulating fluorescent particles, Ru(bpy).sub.3-containing particles were synthesised with the permanently hydrophobic monomer methyl methacrylate (MMA) in place of the pH responsive DEAEMA. To ensure that the system was an accurate model, non-functionalised Ru(bpy).sub.3 was encapsulated within the polymeric particles without the use of chemical bonds.

[0122] Ru(bpy).sub.3 particles were incorporated into HEMA gels at different particle loadings and crosslinking densities achieved through varying the vol % of EGMDA and the volume of particle solution added to the hydrogel formulation prior to polymerisation, using the previously described method for the synthesis of p(HEMA-co-EGDMA) hydrogels.

TABLE-US-00004 Particle Loading (μL of particle Crosslinking Density solution, either HEMA or Gel (vol % EGDMA) PEGMA shelled) A 10 50 B 10 100 C 10 250 D 25 50 E 25 100 F 25 250 G 50 50 H 50 100 I 50 250

[0123] Similarly to the previous leaching analysis, following polymerisation the gels were dialysed against deionised water for 24 hours prior to analysis, to ensure removal of unreacted monomer and non-trapped fluorescent particles. Analysis of the water phase of the leaching analysis for all of the gels, regardless of the particle loading or crosslinking density, indicated no fluorophore present in the solution over the course of analysis, with no peak at the emission maxima for Ru(bpy).sub.3 at λ.sub.em=620 nm.

[0124] Moreover, no peak was present corresponding to the polymeric particles entrapping Ru(bpy).sub.3 at λ.sub.em=608 nm, with the blue shift in emission between the free Ru(bpy).sub.3 and the entrapped Ru(bpy).sub.3 consistent with the literature. Moreover, visual analysis of the gels within the UV light box following the 90-minute leaching evaluation further confirmed with continued presence of red emission of the RU(II) complex from the fluorophore loaded particles within the hydrogel.

[0125] Using gels synthesised with 250 μl of particle solution and 10 vol % crosslinking EGMDA, the leaching analysis of HEMA-shelled and PEGMA-shelled Ru(bpy).sub.3 loaded particles entrapped within the HEMA gel were evaluated, for both short- and long-term stability. Analysis of the liquid phases for both studies indicated no loss of fluorophore from within the gel, with no peaks at the emission maximum for Ru(bpy).sub.3 (λ.sub.em=620 nm) observed.

[0126] Moreover, analysis of the gels indicated significant retention of the Ru(bpy).sub.3 fluorophore over the course of two hours, with an overall decrease in fluorescence intensity of 40% and 24% for the HEMA-shelled and PEGMA-shelled particles, respectively. Additionally, the new cuvette method of analysis was demonstrated to produce more consistent results, without anomalous measurements of significantly higher, or lower/fluorescence intensities than the bulk of the fluorescence spectroscopy measurements during the study.

[0127] Repeating the leaching analysis over a longer period of time, through recording of the fluorescence intensity every 24 hours for a period of 3 days, allowed for evaluation of leaching over a time frame potentially applicable to longer-term continuous monitoring applications, for example waste water contamination monitoring. The long-term analysis indicated lower levels of leaching over the course of 72 hours, with a decrease in fluorescence intensity of 3% and 9% for the HEMA-shelled and PEGMA-shelled particles, respectively.

[0128] This lower degree of leaching over a longer period of analysis is attributed to less frequent water changes creating a lower concentration gradient therefore reducing leaching. Additionally, owing to more frequent measurements, the short-term leaching experiments may have a greater decrease in fluorescence intensity of the hydrogel over the course of the study owing to photobleaching. Regardless, these results strongly indicate minimal leaching of the fluorescent particles from the hydrogel over practically useful timescales.

[0129] To confirm that the secondary encapsulation method was indeed preventing leaching of the fluorophore from the gel, a control experiment was carried out using a HEMA-based hydrogel incorporating free (not encapsulated within particles) Ru(bpy).sub.3).

[0130] Leaching analysis on the resultant fluorescent gel clearly indicated significant loss of the fluorophore from the hydrogel over the period of 72 hours, with a large peak in the fluorescence spectrum attributable to the Ru(bpy).sub.3 at λ.sub.em=610 nm clearly visible in the water phase after 24 and 72 hours (FIG. 6, 24 hours=upper line and 72 hours=lower line). Moreover, fluorescence spectroscopy analysis of the hydrogel indicates a decrease in fluorescence intensity of approximately 70% over the course of 72 hours, significantly greater than the 3% for the Ru(bpy).sub.3 when utilising the method of secondary encapsulation.

Sodium Green™

[0131] To allow for production of a demonstrator model with minimal leaching and the ability to sense ions, HEMA-shelled polymeric particles were synthesised entrapping the sodium ion-sensing fluorophore Sodium Green™. Particles were incorporated into HEMA-based hydrogels crosslinked with 10 vol % EGDMA, using the same synthetic procedure as previously described, and by the addition of 750 μl of particle solution to the hydrogel formulation.

[0132] To confirm a reduction in the leaching when using the method of secondary encapsulation of the fluorophore, the leaching behaviour of HEMA-based hydrogels loaded with Sodium Green™ encapsulated within the polymeric particles was also investigated. Repeating the same analysis procedure, as previously described, analysis indicated complete retention of the fluorophore over the course of 90 minutes, with no decrease in fluorescence intensity for the gel observed. Moreover, the fluorescence spectroscopy analysis of the water phase indicated the absence of Sodium Green™ in the solution, with no peak at its maximum emission wavelength in solution (λ.sub.max=530 nm), and the absence of the Sodium Green™ loaded polymeric particles which have an emission maximum of λ.sub.max=516 nm, with the blue shift in emission wavelength consistent with the literature. The absence of these peaks in the fluorescence spectrum of the water phases further confirm fluorescent particle retention in the hydrogel.

[0133] In order to again confirm that leaching is reduced owing to the secondary encapsulation of the Sodium Green™ within the polymeric particles, a control gel was synthesised using the previously described procedure, in which non-particle encapsulated, free Sodium Green™ was directly immobilised within the hydrogel.

[0134] Evaluation of the leaching behaviour of the Sodium Green™ incorporating hydrogels (through either secondary encapsulation or direct entrapment within the hydrogel) was carried out as previously. Analysis of the HEMA-based hydrogels entrapping non-particle Sodium Green™ indicated significant leaching of the fluorophore from within the gel after only 20 minutes, and a decrease in fluorescence intensity of 60% over the course of the 90 minutes, and with the loss of the fluorophore from within the gel confirmed by its presence within the water phase of the leaching analysis set-up.

[0135] Having successfully demonstrated that the method of secondary encapsulation prevents leaching of the sodium-responsive fluorophore, attention was directed to confirming the sensing ability of the encapsulated fluorophore. Initial studies were carried out using the solution of Sodium Green™ HEMA-shelled polymeric particles. Fluorescence spectroscopy analysis confirmed a slight increase in fluorescence intensity as the concentration of sodium ions added increased (FIG. 7, 5 mM NaCl=uppermost line and 0=lowermost line), confirming that encapsulation of the fluorophore has not impacted on the ability of the luminescent sensor to function (a commonly reported problem in other sensing materials).

CONCLUSION

[0136] There is provided a system in which there is relatively little leaching of the ion-responsive fluorophore from within a hydrogel matrix over a clinically relevant timescale. Moreover, the range of fluorophores (metal and non-metal centred) and the different monomers used in the production of the hydrogels and particles indicate the potential scope of producing a universally applicable system for fluorophore entrapment for sensing applications. Moreover, we have been able to successfully entrap an ion-sensing fluorophore within a polymeric particle, and confirm that entrapment does not prevent the ion-sensing of the fluorophore.

[0137] Whilst this project has mainly focused on the development of a demonstrator model capable of sodium sensing, incorporation of other responsive sensors could produce systems with relevance to clinical diagnostics, medical devices, biosensors and other applications.

Methodology

Materials

[0138] The following reagents were used as received: Fluorescein sodium salt (BioReagent, Sigma-Aldrich), ethylene glycol dimethacrylate (EGDMA, 98%, Sigma-Aldrich), poly(ethylene glycol) methylether methacrylate (PEGMA, M.sub.n-300 g/mol), 2-(diethylamino)ethyl methacrylate (DEAEMA, 99%, Sigma-Aldrich), potassium persulfate (KPS, >99%, Sigma-Aldrich), Sodium Green™ (Fisher Scientific), sodium chloride (NaCl, >98%, Sigma-Aldrich), methyl methacrylate (MMA, 99%, Sigma-Aldrich), 2-hydroxyethyl methacrylate (HEMA, 97%, Sigma-Aldrich) and Tris(2,2′-bipyridyl)dichlororuthenium complex (Ru(bpy).sub.3, 99.95%, Sigma-Aldrich). Inhibitor for all monomers was removed by passing through a plug of basic alumina. DMSO was used as received from Sigma-Aldrich. Silicone Isolators were received from Grace Bio-Labs and the plastic layers removed.

TABLE-US-00005 Abbreviation Name CAS HEMA 2-hydroxyethyl methacrylate 868-77-9 PEGMA (Mn − Poly(ethylene glycol) methyl ether 26915- 300 Da) methacrylate 72-0 DEAEMA 2-(diethylamino) ethyl methacrylate 105-16-8 MMA Methyl methacrylate 80-60-6 EGDMA Ethylene glycol dimethacrylate 97-90-5 Ru(bpy).sub.3 Tris(2,2′-bipyridyl)dichlororuthenium 50525- (II) hexahydrate 27-4 Fluorescein Fluorescein sodium salt 518-47-8 (sodium salt) Sodium Green Sodium Green, cell impermeant — Eu-complex [Eu.L.sup.1].sup.+(Chem. Commun., 2015, 51, — 10879) DMAm N,N-dimethylacrylamide 2680-03-7 HPMA 2-hydroxypropyl methacrylamide 21442-01-3 DMAEMA 2-(dimethylamino)ethyl methacrylate 2867-47-2 BuMA Butyl methacrylate 97-88-1 GlyMA Glycidyl methacrylate 106-91-2 Glycerol Glycerol monomethacrylate 5919-74-7 methacrylate

Instrumentation

[0139] Fluorescence spectroscopy measurements were carried out using a Fluoromax fluorometer. For fluorescein sodium salt: λex=460 nm, λem=490-620 nm, for Sodium Green™: λex=488 nm, λem=500-625 nm, for fluorescein dimethacrylate: λex=460 nm, λem=490-620 nm: and for Ru(bpy).sub.3: λex=440 nm, λem=500-800 nm. For leaching studies, measurements were recorded at 2 nm intervals, with an integration time of 500 ms. For all other analyses, measurements were recorded at 0.5 nm intervals, with an integration time of 500 ms. Silicone isolators were used to produce hydrogels of uniform sizes, with the isolator placed between 2 slides, with a 2 mm gap between the top of the isolator and the top covering slide to allow for injection of the reaction solution (425 μl). Following curing, the top slide was removed, the gel removed from the slide and dialysed against deionised water to remove unreacted monomer and un-trapped fluorophore.

Synthetic Methods

Synthesis of Polymeric Particles:

[0140] PEGMA Armed Particles with Covalently Attached Fluorescein (Large Particles, pH Responsive, Non-Ion-Sensing)

[0141] PEGMA (M.sub.n-300 g/mol, 42 μL) was first dissolved in 50 mL deionised water; EGDMA (26 μL), fluorescein dimethacrylate (14 mg) and DEAEMA (2.75 mL) were added to the stirring solution. Whilst stirring (490 RPM), the mixture was heated to 65° C. and purged with nitrogen gas for 30 minutes. Separately, the initiator potassium persulfate (0.025 g) was dissolved in water (1 mL) and degassed with nitrogen, prior to addition to the reaction mixture. The solution was stirred at 65° C. for 16 hours under a nitrogen atmosphere. The resultant particles were purified by exhaustive dialysis (MWCO 3.5 kDa) against deionised water.

HEMA Armed Particles with Covalently Attached Fluorescein (Large Particles, pH Responsive, Non-Ion-Sensing)

[0142] HEMA (100 μL) was first dissolved in 50 mL deionised water; EGDMA (26 μL), fluorescein dimethacrylate (14 mg) and DEAEMA (2.75 mL) were added to the stirring solution. Whilst stirring (490 RPM), the mixture was heated to 65° C. and purged with nitrogen gas for 30 minutes. Separately, the initiator potassium persulfate (0.025 g) was dissolved in water (1 mL) and degassed with nitrogen, prior to addition to the reaction mixture. The solution was stirred at 65° C. for 16 hours under a nitrogen atmosphere. The resultant particles were purified by exhaustive dialysis (MWCO 3.5 kDa) against deionised water.

PEGMA Armed Particles Loaded with Ru(Bpy).sub.3 (Large Particles, Non-Responsive, Non-Ion-Sensing)

[0143] PEGMA (M.sub.n-300 g/mol, 42 μL) was first dissolved in 50 mL deionised water; EGDMA (52 μL), MMA (1.5 mL), and Ru(bpy).sub.3 (2 mg) were added to the stirring solution. Whilst stirring (490 RPM), the mixture was heated to 65° C. and purged with nitrogen gas for 30 minutes. Separately, the initiator potassium persulfate (0.025 g) was dissolved in water (1 mL) and degassed with nitrogen, prior to addition to the reaction mixture. The solution was stirred at 65° C. for 16 hours under a nitrogen atmosphere. The resultant particles were purified by exhaustive dialysis (MWCO 3.5 kDa) against deionised water.

HEMA Armed Particles Loaded with Ru(Bpy).sub.3 (Large Particles, Non-Responsive, Non-Ion-Sensing)

[0144] HEMA (100 μL) was first dissolved in 50 mL deionised water; EGDMA (52 μL), MMA (1.5 mL), and Ru(bpy).sub.3 (2 mg) were added to the stirring solution. Whilst stirring (490 RPM), the mixture was heated to 65° C. and purged with nitrogen gas for 30 minutes. Separately, the initiator potassium persulfate (0.025 g) was dissolved in water (1 mL) and degassed with nitrogen, prior to addition to the reaction mixture. The solution was stirred at 65° C. for 16 hours under a nitrogen atmosphere. The resultant particles were purified by exhaustive dialysis (MWCO 3.5 kDa) against deionised water.

HEMA Armed Particles Loaded with Sodium Green™ (Large Particles, Non-Responsive, Ion-Sensing)

[0145] HEMA (100 μL) was first dissolved in 50 mL deionised water; EGDMA (52 μL), MMA (1.5 mL), and Sodium Green™ (500 μl of 60 μM solution made up in DMSO) were added to the stirring solution. Whilst stirring (490 RPM), the mixture was heated to 65° C. and purged with nitrogen gas for 30 minutes. Separately, the initiator potassium persulfate (0.025 g) was dissolved in water (1 mL) and degassed with nitrogen, prior to addition to the reaction mixture. The solution was stirred at 65° C. for 16 hours under a nitrogen atmosphere. The resultant particles were purified by exhaustive dialysis (MWCO 3.5 kDa) against deionised water.

Hydrogel Synthesis:

[0146] Please note, in the following methods, hydrogels are immediately removed from the silicone mould when still hot. If the gels are allowed to cool within the silicon mould and sandwiched between the two glass slides it is very difficult to remove the gels from the slides without cracking the gel. If immediate removal is not possible and the gel has cooled and is now hard/brittle, submerge the entire silicone mould and glass slide set up in a petri dish filled with distilled water. Check periodically to ensure that the gel is hydrating. Once hydrated remove the gel from within the silicone moulds and glass slides, and continue with the re-dialysis step.

Typical Procedure for the Synthesis of p(DMAm-Co-MBAc) Hydrogels Loaded with Fluorescein Sodium Salt (Comparative Example 1)

[0147] DMAm (0.6 g, 1 eq.), MBAc (2 wt %) and Irgacure 184 (0.1 wt %) were dissolved in a solution of fluorescein sodium salt (100 μM in deionised water, 5 mL). Following purging with nitrogen, 100 μL of reaction solution was placed into the silicone mould and the mixture cured for 90 minutes.

Typical Procedure for the Synthesis of p(DMAm-Co-EGDMA) Hydrogels Loaded with Fluorescein Sodium Salt (Comparative Example 2)

[0148] DMAm (0.6 g, 1 eq.), EGDMA (2 wt %) and Irgacure 184 (0.1 wt %) were dissolved in a solution of fluorescein sodium salt (100 μM in deionised water, 5 mL). Following purging with nitrogen, 100 μL of reaction solution was placed into the silicone mould and the mixture cured for 90 minutes.

Typical Procedure for the Synthesis of p(DEAEMA-Co-EGDMA-Co-PEGMA) Polymeric Stars Loaded with Fluorescein Sodium Salt.

[0149] PEGMA (42 μL, 1 eq.) was first dissolved in 50 mL deionised water, stirred for 2 minutes, and EGDMA (2 wt %) and DEAEMA (10 wt %) were added to the solution. The mixture, whilst stirred, was degassed by purging with nitrogen for 30 minutes, and further heated at 65° C. whilst purging was continued. The initiator (KPS, 0.1 wt %) was dissolved separately in water (1 mL) and the solution purged with nitrogen before being added to the reaction mixture. The reaction was stirred at 65° C. for 16 hours and cooled to room temperature. The particles were purified by exhaustive dialysis (MWCO=3.5 kDa) against deionised water.

Typical Procedure for the Synthesis of a HEMA Hydrogel Loaded with Fluorescent PEGMA Fluorescein Particles (10 Vol % Crosslinking EGDMA)

[0150] AIBN (0.002 g) was dissolved in a solution of HEMA (0.9 mL) and EGDMA (0.1 mL). Fluorescein dimethacrylate-PEGMA shelled particles (250 μl of particle solution) were added and the solution purged with nitrogen for 20 minutes. The solution (425 μl) was placed into the silicone mould and the mixture cured in the oven at 85° C. for 3 hours. Whilst still warm, the slides were removed from the oven, the gel removed from the silicone mould and dialysed in water for 24 hours.

Typical Procedure for the Synthesis of a HEMA Hydrogel Loaded with Fluorescent HEMA Fluorescein Particles (10 Vol % Crosslinking EGDMA)

[0151] AIBN (0.002 g) was dissolved in a solution of HEMA (0.9 mL) and EGDMA (0.1 mL). Fluorescein dimethacrylate-HEMA shelled particles (250 μl of particle solution) were added and the solution purged with nitrogen for 20 minutes. The solution (425 IA) was placed into the silicone mould and the mixture cured in the oven at 85° C. for 3 hours. Whilst still warm, the slides were removed from the oven, the gel removed from the silicone mould and dialysed in water for 24 hours.

Typical Procedure for the Synthesis of a HEMA Hydrogel Loaded with Fluorescent PEGMA-Ru(Bpy).sub.3 Particles (10 Vol % Crosslinking EGDMA)

[0152] AIBN (0.002 g) was dissolved in a solution of HEMA (0.9 mL) and EGDMA (0.1 mL). PEGMA shelled Ru(bpy).sub.3 entrapping polymeric particles (250 μl of particle solution) were added and the solution purged with nitrogen for 20 minutes. The solution (425 μl) was placed into the silicone mould and the mixture cured in the oven at 85° C. for 3 hours. Whilst still warm, the slides were removed from the oven, the gel removed from the silicone mould and dialysed in water for 24 hours.

Typical Procedure for the Synthesis of a HEMA Hydrogel Loaded with Fluorescent HEMA-Ru(Bpy).sub.3 Particles (10 Vol % Crosslinking EGDMA)

[0153] AIBN (0.002 g) was dissolved in a solution of HEMA (0.9 mL) and EGDMA (0.1 mL). HEMA shelled Ru(bpy).sub.3 entrapping polymeric particles (250 μl of particle solution) were added and the solution purged with nitrogen for 20 minutes. The solution (425 μl) was placed into the silicone mould and the mixture cured in the oven at 85° C. for 3 hours. Whilst still warm, the slides were removed from the oven, the gel removed from the silicone mould and dialysed in water for 24 hours.

Typical Procedure for the Synthesis of a HEMA Hydrogel Loaded with Fluorescent Ru(Bpy).sub.3 (10 Vol % Crosslinking EGDMA)

[0154] AIBN (0.002 g) was dissolved in a solution of HEMA (0.9 mL) and EGDMA (0.1 mL). Ru(bpy).sub.3 (500 μl of 60 μM solution made up in water) was added and the solution purged with nitrogen for 20 minutes. The solution (425 μl) was placed into the silicone mould and the mixture cured in the oven at 85° C. for 3 hours. Whilst still warm, the slides were removed from the oven, the gel removed from the silicone mould and dialysed in water for 24 hours.

Typical Procedure for the Synthesis of a HEMA Hydrogel Loaded with Fluorescent HEMA-Sodium Green™ Particles (10 Vol % Crosslinking EGDMA)

[0155] AIBN (0.002 g) was dissolved in a solution of HEMA (0.9 mL) and EGDMA (0.1 mL). HEMA shelled Sodium Green™ entrapping polymeric particles (250 μl of particle solution) were added and the solution purged with nitrogen for 20 minutes. The solution (425 μl) was placed into the silicone mould and the mixture cured in the oven at 85° C. for 3 hours. Whilst still warm, the slides were removed from the oven, the gel removed from the silicone mould and dialysed in water for 24 hours.

Typical Procedure for the Synthesis of a HEMA Hydrogel Loaded with Fluorescent Sodium Green™ (10 Vol % Crosslinking EGDMA)

[0156] AIBN (0.002 g) was dissolved in a solution of HEMA (0.9 mL) and EGDMA (0.1 mL). Sodium Green™ (500 μl of 60 μM solution made up in DMSO) was added and the solution purged with nitrogen for 20 minutes. The solution (425 μl) was placed into the silicone mould and the mixture cured in the oven at 85° C. for 3 hours. Whilst still warm, the slides were removed from the oven, the gel removed from the silicone mould and dialysed in water for 24 hours.