A MICELLAR PARTICLE

Abstract

There is provided a micellar particle having at least a core-shell configuration, wherein a fluorescent molecule is encapsulated within said core and wherein said shell is formed from an inorganic compound.

Claims

1. A micellar particle having at least a core-shell configuration, wherein a fluorescent molecule is encapsulated within said core, said core being formed from a hydrophobic component of an amphiphilic polymer and wherein said shell is formed from an inorganic compound.

2. The micellar particle as claimed in claim 1, further comprising a corona that extends from said shell.

3. The micellar particle as claimed in claim 2, wherein a hydrophilic component of said amphiphilic polymer forms the corona of said micellar particle.

4. The micellar particle as claimed in claim 3, wherein said amphiphilic polymer is a block copolymer or a block terpolymer.

5. The micellar particle as claimed in claim 3, wherein said amphiphilic polymer comprises a polyethylene-oxide monomer.

6. The micellar particle as claimed in claim 5, wherein said polyethylene-oxide functionalized polymer is selected from the group consisting of polyhedral oligosilsesquioxanes-PEO (POSS-PEO), PEO-polyhydroxybutyrate-PEO (PEO-PHB-PEO), polylactic-acid-PEO (PLA-PEO), PEO-PLA-PEO, PEO-polypropyleneoxide-PEO (PEO-PPO-PEO), polydimethylsiloxane-graft-PEO (PDMS-graft-PEO) and polystyrene-PEO (PS-PEO).

7. The micellar particle as claimed in claim 1, wherein said inorganic compound is a ceramic selected from the group consisting of hydroxyapatite, zirconia, silica, titanium oxide and alumina.

8. The micellar particle as claimed in claim 1, wherein said fluorescent molecule is an organic molecule.

9. The micellar particle as claimed in claim 8, wherein said fluorescent molecule is selected from the group consisting of Coumarin545T, DCJTB, acridine orange, proflavine, N-(30sulfopropyl)acridinium, phenylalanine, tryptophan, tyrosine, anthracene, 9-cyanoanthracene, 9,10-Diphenylanthracene, naphthalene, 1-anilino-8-naphthalene sulfonate, 6-propionyl-2-(dimethylaminonaphthalene), perylene, phenanthrene, pyrene, puranine, p-quaterphenyl, rubrene, p-terphenyl, [60] fullerene, [70] fullerene, auramine O, malachite green, crystal violet, 1,3,5,7,8-pentamethylpyrromethene-difluoroborate, disodium-1,3,5,7,8-pentamethylpyrromethene-2,6-disulfonate-difluoroborate, 1,3,5,7,8-pentamethyl-2,6-diethyl pyrromethene-difluoroborate, 8-Acetoxymethyl-2,6-diethyl-1,3,5,7-tetramethyl pyrromethene fluoroborate, 1,2,3,5,6,7-hexamethyl-8-cyanopyrromethene-difluoroborate, 2,3,5,6-1H,4H-Tetrahydro-8-methylquinolizino-[9,9a,1-gh]-coumarin, 7-amino-4-methylcoumarin, 7-amino-4-trifluoromethylcoumarin, 7-dimethylamino-4-trifluoromethylcoumarin, 7-diethylamino-4-trifluoromethylcoumarin, 2,3,5,6-1H,4H-Tetrahydro-8-trifluormethylquinolizino-[9,9a,1-gh]coumarin, 7-diethylaminocoumarin, 7-ethylamino-4-trifluormethylcoumarin, cryptocyanine, Cy3, Cy5, Cy7, 1,10diethyl-2,2-dicarbocyanine iodide, HITCI, indocyanine green, IR 140, cresyl violet, nile blue, nile red, oxazeine 1, oxazine 170, oxazine 750, 2,5-Bis-(4-biphenylyl)-1,3,4-oxadiazole, 2,5-Bis-(4-biphenylyl)-oxazole, 2-(1-Naphthyl)-5-phenyloxazole, 2-(4-Biphenylyl)-6-phenylbenzoxazole, 1,4-Di[2-(5-phenyloxazolyl)]benzene, 2,5-Diphenyloxazole, thionine, methylene blue, eosin Y, erythrosine B, fluorescein disodium salt uranin, dichlorofluorescein disodium salt, tetrachlorotetraiodofluorescein, pyronine Y, pyronine B, rhodamine B, Rhodamine 6G, rhodamine 101, rhodamine 110, sulforhodamine, sulforhodamine 101, tetramethylrhodamine, quinine sulfate, DAPI, N-methylcarbazole, 4-dicyanomethylene-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran, DCM-OH and N-acetyl-L-tryptophanamide.

10. The micellar particle as claimed in claim 2, wherein said corona of said micellar particle is functionalized with a linker group.

11. The micellar particle as claimed in claim 10, wherein said linker group is selected from a carboxylic acid group or an amine group.

12. The micellar particle as claimed in claim 1, wherein said micellar particle has a particle size in the nano-sized range.

13. The micellar particle as claimed in claim 12, wherein the particle size of said micellar particle is less than 10 nm.

14. The micellar particle as claimed in claim 1, wherein said shell has a thickness that is in the range of 5% to 95% of the diameter of said micellar particle.

15. The micellar particle as claimed in claim 1, further comprising a therapeutic agent encapsulated within said core.

16. A process for forming a micellar particle having at least a core-shell configuration comprising: a. mixing a reactant solution comprising an amphiphilic polymer or monomers thereof, a fluorescent molecule and shell precursors in an organic solvent; and b. mixing said reactant solution from operation (a) with an aqueous liquid to form said micellar particle in which said fluorescent molecule is encapsulated within said core, said core being formed from a hydrophobic component of said amphiphilic polymer and wherein said shell is formed from said shell precursors.

17. The process as claimed in claim 16, wherein said organic solvent is selected from the group consisting of tetrahydrofuran, dioxane, chloroform and dichloromethane.

18. The process as claimed in claim 16, wherein said shell precursor is selected from the group consisting of a hydroxyapatite precursor, a zirconia precursor, a silica precursor, a titanium oxide precursor and an alumina precursor.

19. The process as claimed in claim 16, wherein said reactant solution further comprises a swelling agent.

20. Use of a micellar particle having at least a core-shell configuration, wherein a fluorescent molecule is encapsulated within said core, said core being formed from a hydrophobic component of an amphiphilic polymer and wherein said shell is formed from an inorganic compound, as a bioimaging agent or a biodetection agent.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0065] The accompanying drawings illustrate a disclosed embodiment and serves to explain the principles of the disclosed, embodiment. It is to be understood, however, that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the invention.

[0066] FIG. 1(a) is a transmission electron microscopy (TEM) image having a scale bar of 100 nm showing Coumarin545T@SiO.sub.2 nanocapsules while FIG. 1(b) is a TEM image having a scale bar of 100 nm showing DCJTB@SiO.sub.2 nanocapsules.

[0067] FIG. 2 is a graph showing the UV-Vis and photoluminescent spectra of Coumarin545T@SiO.sub.2.

[0068] FIG. 3 is a graph showing the UV-Vis and photoluminescent spectra of DCJTB@SiO.sub.2.

[0069] FIG. 4 is a graph comparing the time-resolved photoluminescence decay of Coumarin545T dissolved in THF with that of Coumarin545T encapsulated inside the core of the nanocapsules.

[0070] FIG. 5 is a graph comparing the time-resolved photoluminescence decay of DCJTB dissolved in THF with that of DCJTB encapsulated inside the core of the nanocapsules.

[0071] FIG. 6(a) is a graph showing the hydrodynamic sizes of Coumarin545T@SiO.sub.2 in the stability study while FIG. 6(b) shows, the hydrodynamic sizes of Coumarin545T@F127.

[0072] FIG. 7 is a graph showing the hydrodynamic diameters of Coumarin545T@SiO.sub.2 and DCJTB@SiO.sub.2 dispersed in 1PBS containing 10% FBS as a function of incubation time at 37 C.

[0073] FIG. 8 is a bar graph showing the viabilities of Hela cells cultured in the presence of Coumarin545T@SiO.sub.2 or DCTJB@SiO.sub.2.

[0074] FIG. 9a to FIG. 9d are confocal images obtained at different channels of Hela cells after being incubated with Coumarin545T@SiO.sub.2. FIG. 9a was obtained under bright field; FIG. 9b was obtained under FITC channel; FIG. 9c was obtained under DAPI channel and FIG. 9d was obtained under FITC and DAPI channels (showing a merged image).

[0075] FIG. 10a to FIG. 10d are confocal images obtained at different channels of Hela cells after being incubated with DCJTB@SiO.sub.2. FIG. 10a was obtained under bright field; FIG. 10b was obtained under Texas Red channel; FIG. 10c was obtained under DAPI channel and FIG. 10d was obtained under FITC and DAPI channels (showing a merged image).

EXAMPLES

[0076] Non-limiting examples of the invention will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

Example 1

Preparation of Green Light Emitting Coumarin@SiO.SUB.2 .Nanocapsule

[0077] 75 mg of Pluronic F127 (PEO.sub.106PPO.sub.65PEO.sub.100) was first dissolved in 800 L dioxane to form a clear solution. 50 L (1 mg/mL in dioxane) of Coumarin 545T and 65 L of tetramethoxysilane (TMOS) were then added. All chemicals were obtained from Sigma-Aldrich of Missouri of St. Louis of the United States of America, except for Coumarin 545T and DCJTB (used in Example 2) which was obtained from Tokyo Chemical. Industry of Japan. Coumarin 545T is an organic dye molecule having the chemical structure below.

##STR00002##

[0078] The above mixture solution was injected into 10 mL of deionized water immersed in a water-bath under ultra sonication for 15 minutes. The solution was further sonicated for another 5 minutes, followed by stirring at room temperature for 4 days to evaporate off dioxane to ensure a complete hydrolysis of the TMOS at the interface between the core and corona of the F127 micelles. Fluorescent silica nanoparticles or nanocapsules consisting of a poly(propylene oxide) core, a silica shell and an exterior free poly(ethylene oxide) layer are formed. Due to the hydrophobicity of the Coumarin 545T molecules, the Coumarin 545T molecules are encapsulated inside the hollow core of the nanocapsules as discrete particles. Due to the presence of the exterior free poly(ethylene oxide) layer, the nanoparticles have excellent colloidal stability in an aqueous environment and are photostable.

Example 2

Preparation of Red Light Emitting Nanocapsule DCJTB@SiO.SUB.2.Nanocapsule

[0079] 75 mg of Pluronics F127 was first dissolved into 600 L tetrahydrofuran (THF) to form a clear solution. 50 L (500 g/mL in THF) of DCJTB, 20 L of trimethylbenzene and 65 L of TMOS were then added. Trimethylbenzene functions as a swelling agent to enlarge the hollow core of the nanocapsules to form the nanocapsule morphology of the micellar particle (DCJTB@SiO.sub.2). DCJTB is an organic dye molecule having the chemical structure below.

##STR00003##

[0080] The above mixture solution was injected into 10 mL of deionized water immersed in a water-bath under ultra sonication for 15 minutes. The solution was further sonicated for another 5 minutes, followed by stirring at room temperature for 4 days to evaporate off THF to ensure a complete hydrolysis of TMOS at the interface between the core and corona of the F127 micelles. Similar to Example 1, fluorescent silica nanoparticles or nanocapsules consisting of a poly(propylene oxide) core, a silica shell and an exterior free poly(ethylene oxide) layer are formed. Due to the hydrophobicity of the Coumarin 545T molecules, the DCJBT molecules are encapsulated inside the hollow core of the nanocapsules as discrete particles.

Preparation of Dicarboxylic Acid Modified F127

[0081] 12.6 g of F127 was first dried in vacuum at 100 C. for 24 hours in order to remove adsorbed water. It was then dissolved in 60 mL of anhydrous dimethylacetamide (DMAC) while being stirred and heated to 70 C. Upon complete dissolution, 0.3 g of succinic anhydride was added into the solution and stirred rapidly at 70 C. under nitrogen atmosphere. The reaction mixture was then heated rapidly to 90 C. and stirred for 24 hours at this temperature. After cooling to room temperature, the final dark brown reaction mixture was precipitated against excess cold diethyl ether in a dropwise manner. The precipitates were dissolved in deionized water and dialyzed for 48 hours using dialysis tube with molecular weight cut-off (MWCO) of 1000 g/mol. The final solution was then freeze-dried to obtain dicarboxylic acid modified F127 block copolymer. The dicarboxylic acid modified F127 block copolymer may be used in place of the F127 of any of Example 1 or 2 above. As an Example, to introduce 20% carboxylic acid to the nanocapsule surface, 20% of F127 was replaced by dicarboxylic acid modified F127.

Example 3

Characterization of Silica Nanocapsules

[0082] The morphologies, optical properties, stabilities, antifouling properties, cytotoxicities and fluorescence images of the above silica nanocapsules formed from Examples 1 and 2 were characterized in this Example 3.

Morphology of Nanocapsules

[0083] The morphologies of the Coumarin545T@SiO.sub.2 and DCJTB@SiO.sub.2 samples were determined using transmission electron microscopy (TEM) at a magnification of 36,000. FIG. 1(a) is a TEM image of Coumarin545T@SiO.sub.2 nanocapsules while FIG. 1(b) is a TEM image of DCJTB@SiO.sub.2 nanocapsules. As can be seen from FIG. 1(a) and FIG. 1(b), all of the nanocapsules have a core-shell structure with uniform size. The average core diameter of the samples was about 9 nm while the average outer diameter of the samples was about 15 nm.

Optical Property Study

[0084] Quantum yield was measured with respect to the fluorescence standard solution, namely fluorescein (Ethanol; QY=0.79), and rhodamine B (Ethanol; QY=0.49). Fluorescein was used as a reference for Coumarin545T@SiO.sub.2, whereas rhodamine B was used for DCJTB@SiO.sub.2. The procedures involved diluting the fluorescent silica nanocapsules and the fluorescence standard solution with their respective solvent to have the same absorbance at excitation wavelength (460 nm for fluorescein; 490 nm for rhodamine B). Quantum yields for fluorescent silica nanocapsules were measured by dividing, the integrated emission area of their fluorescent spectrum against that of fluorecein or rhodamine B in Ethanol. As shown in Table 1, both of the nanoparticles exhibit high quantum yield of 0.99 for Coumarin545T@SiO.sub.2 and 0.56 for DCJTB@SiO.sub.2, suggesting that the fluorescent nanocapsules demonstrate the required high fluorescence brightness for fluorescence imaging applications. In addition, the lifetime of Coumarin545T@SiO.sub.2 was determined to be 3.38 ns, while the lifetime of coumarin545T in THF was determined to be 2.86 ns. Similarly, the lifetime of DCJTB@SiO.sub.2 was determined to be 3.40 ns, while the lifetime of DCJTB in THF was determined to be 2.50 ns.

TABLE-US-00001 TABLE 1 Optical properties of dye in THF and encapsulated in silica nanocapsules Coumarin 545T DCJTB nanocap- nanocap- THF sules THF sules .sub.em (nm) 504 520 605 610 Fluorescence Lifetime (, ns) 2.86 3.38 2.50 3.40 Quantum Yield (QY) 0.99 0.99 0.99 0.56 fwhm (nm) 35 70 73 90

[0085] The photoluminescence lifetimes of the Coumarin545T@SiO.sub.2 and DCJTB@SiO.sub.2 samples were measured using a Time Correlated Single Photon Counting (TCSPC) module (PicoQuant PicoHarp 300 of PicoQuant of Berlin, Germany). The Coumarin545T@SiO.sub.2 and DCJTB@SiO.sub.2 samples were excited by using 405 nm line of a picosecond pulsed laser diode (PicoQuant PDL 800-B). The photoluminescence was dispersed through a monochromator (Acton SpectroPro 2300i) and detected with a micro channel plate photomultiplier tube (MCP-PMT) detector (Hamamatsu R3809U-50).

[0086] The UV-Vis and photoluminescent spectra of Coumarin545T@SiO.sub.2 are shown in FIG. 2 while those of DCJTB@SiO.sub.2 are shown in FIG. 3.

[0087] FIG. 4 is a graph showing the time-resolved photoluminescence decay of Coumarin545T dissolved in THF and that when encapsulated inside the core of the nanocapsules. FIG. 5 is a graph showing the time-resolved photoluminescence decay of DCJTB dissolved in THF and that when encapsulated inside the core of the nanocapsules. It was observed that all photoluminescence decay curves could be well fitted with a single exponential decay function and the obtained lifetimes of fluorescent nanocapsules are significantly larger than that of their corresponding dye solutions. Based on these figures, when compared to the photoluminescence behavior of the dye solutions (Coumarin545T or DCJTB) in their respective solvents, there is a significant red-shift in the emission peak for both of the silica nanocapsules, which should be ascribed to the - stacking of the dye molecules by encapsulation. The emission spectra of the nanocapsules are also broader than the dye solutions in their respective solvents. The silica coating could lead to improvement in the photostability of the dye molecules by limiting their contact with the outside environment, especially soluble oxygen molecules.

Stability Study

[0088] The aqueous suspensions of the Coumarin545T@SiO.sub.2 nanocapsules and Coumarin545T@F127 were diluted for 5 to 100 times against deionized water, and then stirred at room temperature for 24 hours before measurements of the hydrodynamic sizes by dynamic light scattering. The Coumarin545T@F127 sample was prepared in a similar way to Coumarin545T@SiO.sub.2 nanocapsules but without the use of TMOS silica precursor. From FIG. 6(b), it can be observed that the hydrodynamic size of Coumarin545T@F127 significantly increased with a much more broader size distribution when diluted from 5 to 100 times, indicating the disintegration of the polymeric micelles. On the contrary, FIG. 6(a) showed that the hydrodynamic size of Coumarin545T@SiO.sub.2 remained consistent with dilution from 5 to 100 times, indicating that the silica shell could successfully inhibit the dissociation of the polymeric micelles during dilution. Hence, unlike polymeric micelles which spontaneously dissociate when they are diluted below the critical micelle concentration (CMC), the silica shell of the fluorescent nanocapsules effectively prevented the micelles from dissociation even though the polymer concentration fell below the CMC.

Antifouling Property Study

[0089] The antifouling behaviors of Coumarin545T@SiO.sub.2 and DCJTB@SiO.sub.2 nanocapsules were evaluated by monitoring their hydrodynamic size changes upon incubating the nanocapsules in 1PBS with 10 vol % FBS at 37 C. The final concentration of the nanocapsules in the solution was 1 mg/mL. Dynamic Light Scattering (DLS) was used to monitor the hydrodynamic size changes during the incubation period.

[0090] As shown in FIG. 7, there was no significant variation in the hydrodynamic diameters of the Coumarin545T@SiO.sub.2 and DCJTB@SiO.sub.2 nanocapsules during the incubation period of 22 hour, indicating that the nanocapsules remained stable without aggregation in the presence of the serum proteins, demonstrating their antifouling behavior due to the presence of free hydrophilic poly(ethylene oxide) chains on the exterior surface that prevents nonspecific adsorption of biomolecules.

[0091] On the other hand, nanoparticles without suitable surface modification can easily aggregate into large particles and absorb protein on the surface, which unfortunately speeds up reticuloendothelial clearance and shortens blood circulation lifetime, limiting their use in biological studies. In addition, the particle size of the nanoparticles would increase significantly due to the nonspecific binding of proteins on silica surface.

Cytotoxicity Study

[0092] The cytotoxicity of the fluorescent nanocapsules was studied by determining the viability of cervical cancer cells (Hela) using MTS assay. The Hela cells obtained from American Type Culture Collection (ATCC) were grown in culture medium at 37 C. in 5% CO.sub.2. The culture medium contains completed DMEM with glucose (4500 mg/L), sodium pyruvate and L-glutamin, 10% FBS, 100 units Penicillin and 100 mg/ml of Streptomycin. The Hela cells were seeded into a 96 well plate at a density of 15,000 cells/well. The cells were then cultured at 37 C. in 5% CO.sub.2 in the presence of growth medium containing fluorescent nanocapsules from 0 to 6 mg/mL. After incubation for 24 hours, the growth medium was discarded and replaced by MTS/phenazine methosulfate solution followed by incubation for another 1 hour. The optical absorbance of each well was then measured at 490 nm on a SpectraMax M2 Multi-Mode Microplate Reader (Molecular Devices). The results were normalized with respect to the result obtained without the addition of fluorescent nanocapsules. The data were averaged from three experiments for each fluorescent nanocapsules concentration.

[0093] As shown in FIG. 8, the cell viability of Hela cells were close to 100% at the concentration of 6 mg/mL after incubation for 24 hours, suggesting that the nanocapsules are non-cytotoxic and safe for biomedical applications.

Fluorescence Imaging Study

[0094] Hela cells were cultured in a 96-well plate with culture medium at 37 C. in 5% CO.sub.2. The culture medium contains completed DMEM with 4500 mg/L of glucose, sodium pyruvate and L-glutamin, 10% FBS, 100 units of Penicillin and 100 mg/mL of Streptomycin. The Hela cells were seeded into chamber slides at a density of 15,000 cells/well. After 24 hours of incubation, the culture medium was replaced by fresh DMEM containing fluorescent nanocapsules at 2.4 mg/mL. After 1 hour of incubation in the presence of fluorescent nanocapsules, each well was washed with PBS for three times to remove supernatant nanoparticles. The cells were then mounted using culture medium with DAPI. The cells then were imaged by using an inverted microscope (Nikon, Eclipse Ti). The corresponding fluorescent image was acquired at FITC channel (ex 488 nm, em 525 nm) for coumarin545T@SiO.sub.2 and Texas Red Channel (ex 595 nm, em 615 nm) for DCJTB@SiO.sub.2.

[0095] As can be seen in FIG. 9 and FIG. 10, the fluorescent images acquired at FITC channel (ex 488 nm, em 525 nm) for Coumarin545T@SiO.sub.2 (FIG. 9) and Texas Red Channel (ex 595 nm, em 615 nm) for DCJTB@SiO.sub.2 (FIG. 10) clearly demonstrated that both of the green and red nanoparticles can efficiently internalize in the cytoplasm region of the Hela cells, which showed great potential to be used as fluorescence cellular imaging probes.

Comparative Example

[0096] A stock solution of poly(2-methoxy-5-(2-ethyl-hexyloxy)-1,4-phenylene vinylene) (MEH-PPV), poly(2-(2-phenyl-4,5-di(3-methyl-butoxy)-phenyl-1,4-phenylenevinylene)) (BP-PPV) and poly(9,9-dihexylfluorenyl-2,7-diyl) (C6PF) in THF were prepared by dissolving the respective polymers in THF to a concentration of 1.0 mg/ml. 75 l of this stock solution was mixed with 75 mg of Pluronic F127 (PEO.sub.106PPO.sub.70PEO.sub.106) in 825 l of THF to form a clear solution. 65 l of TMOS was then added. The mixture solution was injected into a 10 g of deionized water immersed in water-bath under ultra sonication in three minutes. The solution was further sonicated for ten minutes, followed by stirring at room temperature for four days to evaporate off THF and ensure a complete hydrolysis of TMOS at the interface between the core and corona of the micelles.

[0097] The particles formed in this comparative example exhibited very low quantum yield of less than 10%, which significantly limits the use of these particles as effective emitters especially in experiments requiring high brightness of the fluorophores such as long-term cell tracing, animal study and clinical surgery. Hence, the applications of such fluorophores are severely limited due to the low quantum yields.

Applications

[0098] The disclosed micellar particles may be used in biomedical applications such as bioimaging or biodetection.

[0099] The disclosed micellar particles may have low toxicity, good colloidal stability and may be capable of emitting distinctive fluorescence light upon excitation with high quantum yield and good photostability. Advantageously, the use of fluorescent molecules as disclosed herein may result in nanocapsules having a high quantum yield not seen in the prior art, which then increases the range of applications that can be employed with the disclosed nanocapsules.

[0100] The disclosed micellar particles may have a hydrophilic group on the corona portion of the particles to impart the desired colloidal stability, low cytotoxicity and enhanced blood circulation property. In addition, the disclosed micellar particles may be in the nano-size and may demonstrate good antifouling property, which could prevent the nanocapsules from non-specific binding to circulating proteins in a host and hence avoid clearance by the reticuloendothelial system of a host.

[0101] Fluorescent molecules that are encapsulated in the core of the micellar particles are protected from environmental degradation, leading to the micellar particles having good photostability.

[0102] The amphiphilic polymer making up the micellar polymer may be modified with a chemical group that can react with or bind to a ligand, which in turns can bind to a target receptor or substrate in vivo, allowing for bioimaging or biodetection.

[0103] The disclosed micellar particles may be made in a facile one-pot method whereby fluorescent silica nanocapsules encapsulating fluorescent molecules are formed.

[0104] It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.