STIMULUS-RESPONSIVE CORE-SHELL PARTICLES

Abstract

There is provided a core-shell particle having pores extending through its shell and a plurality of polymers that are bonded to the outer surface of the shell, wherein the polymers are comprised of repeating monomer units of formula (1): [Formula should be inserted here] wherein the substituents are as defined herein. There is also provided a method of synthesizing the core-shell particle and use of the core-shell particle as a delivery agent.

##STR00001##

Claims

1. A core-shell particle having pores extending through its shell and a plurality of polymers that are bonded to the outer surface of the shell, wherein each polymer is a copolymer comprising repeating monomer units of formulae (1) and (2): ##STR00009## wherein m and n are at least 1; R.sub.a, R.sub.b, R.sub.c and R.sub.e are independently selected from the group consisting of a hydrogen, optionally substituted aliphatic, optionally substituted hetero-aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl and optionally substituted heterocycloalkyl; R.sub.d is selected from the group consisting of a bond, optionally substituted hetero-aliphatic, optionally substituted aliphatic group, optionally substituted alkoxy, carbonyl, optionally substituted ester, optionally substituted amide, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl and optionally substituted heterocycloalkyl; X is selected from the group consisting of a heteroatom or a positively charged ion; R.sub.f and Y are absent when X is a heteroatom but are both present when X is a positively charged ion such that R.sub.f is selected from the group consisting of optionally substituted aliphatic, optionally substituted hetero-aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl and optionally substituted heterocycloalkyl; and Y is a negatively charged ion when bonded to R.sub.f; and R.sub.h is selected from the group consisting of a hydrogen, optionally substituted aliphatic, optionally substituted hetero-aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl and optionally substituted heterocycloalkyl.

2. The core-shell particle according to claim 1, wherein said optionally substituted aliphatic is selected from the group consisting of optionally substituted C.sub.1-30alkylene, optionally substituted C.sub.2-30alkenylene and optionally substituted C.sub.2-30alkynylene.

3. The core-shell particle according to claim 1, wherein said optionally substituted hetero-aliphatic contains one or more heteroatoms selected from N, O, S, Se or Si, and is selected from the group consisting of optionally substituted C.sub.1-30-heteroalkylene, optionally substituted C.sub.2-30-heteroalkenylene and optionally substituted C.sub.2-30-heteroalkynylene.

4. The core-shell particle according to claim 1, wherein X is selected from the group consisting of N, N.sup.+ and P.sup.+ or when X is N.sup.+ or P.sup.+, R.sub.t and Y are present and Y is selected from the group consisting of sulfonate, carboxylate, nitrite and carbonite.

5. (canceled)

6. The core-shell particle according to claim 4, wherein R.sub.f is selected from the group consisting of C.sub.2-alkylene, C.sub.3-alkylene, C.sub.4-alkylene, C.sub.5-alkylene and C.sub.6-alkylene.

7. The core-shell particle according to claim 1, wherein said R.sub.a, R.sub.b, R.sub.c and R.sub.e are independently a hydrogen or an optionally substituted aliphatic.

8. (canceled)

9. (canceled)

10. The core-shell particle according to claim 1, wherein said R.sub.d is an optionally substituted ester having the formula R.sub.gCO.sub.2R.sub.g or an optionally substituted amide having the formula R.sub.gCONHR.sub.g wherein R.sub.g and R.sub.g is each independently selected from the group consisting of an C.sub.1-30alkylene, C.sub.2-30alkenylene, C.sub.2-30alkynylene, C.sub.1-30heteroalkylene, C.sub.2-30-heteroalkenylene and C.sub.2-30-heteroalkynylene.

11. (canceled)

12. The core-shell particle according to claim 1, wherein said polymers are independently selected from the group consisting of Formula (3), Formula (4), Formula (5), Formula (6), Formula (7), Formula (8), Formula (9) and Formula (10): ##STR00010## ##STR00011## wherein m and n are at least 1; R.sub.a, R.sub.b, R.sub.c and R.sub.e are independently selected from the group consisting of a hydrogen, optionally substituted aliphatic, optionally substituted hetero-aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl and optionally substituted heterocycloalkyl; R.sub.d is selected from the group consisting of a bond, optionally substituted hetero-aliphatic, optionally substituted aliphatic group, optionally substituted alkoxy, carbonyl, optionally substituted ester, optionally substituted amide, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl and optionally substituted heterocycloalkyl; X is selected from the group consisting of a heteroatom or a positively charged ion; R.sub.f and Y are absent when X is a heteroatom but are both present when X is a positively charged ion such that R.sub.f is selected from the group consisting of optionally substituted aliphatic, optionally substituted hetero-aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl and optionally substituted heterocycloalkyl; and Y is a negatively charged ion when bonded to R.sub.f; and R.sub.h is selected from the group consisting of a hydrogen, optionally substituted aliphatic, optionally substituted hetero-aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl and optionally substituted heterocycloalkyl; and p is at least 1.

13. The core-shell particle of claim 12, wherein said polymer of formula (9) is poly(dimethyl(methacryloyloxyethyl)ammonium propanesulfonate)-co-poly(polyethylene glycol methacrylate).

14. The core-shell particle of claim 12, wherein said polymer of formula (10) is poly(dimethylamino ethyl methacrylate)-co-poly(polyethylene glycol methacrylate).

15. The core-shell particle according to claim 1, wherein said polymer has a molecular weight selected from the range of 5 kDa to 500 kDa.

16. The core-shell particle according to claim 1, wherein said polymer is coupled to the shell via a bridging group selected from the group consisting of an aminoalkyl silane, haloalkyl silane, mercapto alkyl silane and an aminoalkoxysilane.

17. The core-shell particle according to claim 1, wherein said particle contains 10% to 90% (w/w) of the polymer of formulae (1) and (2), optionally wherein said article is a microparticle or a nanoparticle with a hydrodynamic diameter of 1 nm to 100 m.

18. (canceled)

19. The core-shell particle according to claim 1, wherein the pores of said particle are microporous or mesoporous.

20. The core-shell particle according to claim 1, wherein said pores on said particle alternate between an open position and a closed position in response to an external stimulus, or wherein said external stimulus is selected from temperature or carbon dioxide concentration.

21. (canceled)

22. The core-shell particle according to claim 20, wherein when said temperature is above a critical temperature, said pores are in said open position and when said temperature is below said critical temperature, said pores are in said closed position, or wherein when said polymer is poly)dimethyl(methaacryloyloxyethyl)ammonium propanesulfonate)-co-poly(polyethylene glycol methacrylate), said critical temperature is 37.5 C.

23. (canceled)

24. (canceled)

25. The core-shell particle according to claim 20, wherein when said carbon dioxide is present, said pores are in said open position and when said carbon dioxide is substantially absent, said pores are in said closed position, or wherein when said polymer is poly(dimethylamino ethyl methacrylate)-co-poly(polyethylene glycol methacrylate), said polymer is responsive to carbon dioxide.

26. (canceled)

27. (canceled)

28. A method of synthesizing a core-shell particle having pores extending through its shell and a plurality of polymers that are bonded to the outer surface of the shell, wherein each polymer is a copolymer comprising repeating monomer units of formulae (1) and (2): ##STR00012## wherein m and n are at least 1; R.sub.a, R.sub.b, R.sub.c and R.sub.g are independently selected from the group consisting of a hydrogen, optionally substituted aliphatic, optionally substituted hetero-aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl and optionally substituted heterocycloalkyl; R.sub.d is selected from the group consisting of a bond, optionally substituted hetero-aliphatic, optionally substituted aliphatic group, optionally substituted alkoxy, carbonyl, optionally substituted ester, optionally substituted amide, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl and optionally substituted heterocycloalkyl; X is selected from the group consisting of a heteroatom or a positively charged ion; R.sub.f and Y are absent when X is a heteroatom but are both present when X is a positively charged ion such that R.sub.f is selected from the group consisting of optionally substituted aliphatic, optionally substituted hetero-aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl and optionally substituted heterocycloalkyl; and Y is a negatively charged ion when bonded to R.sub.f; and R.sub.h is selected from the group consisting of a hydrogen, optionally substituted aliphatic, optionally substituted hetero-aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl and optionally substituted heterocycloalkyl, comprising: i) providing a halide-functionalized particle; and ii) reacting said halide-functionalized particle with a monomer of formulae (1) and (2) to form said core-shell particle.

29. The method of claim 28, wherein said halide-functionalized particle is a bromide-functionalized particle.

30-32. (canceled)

33. A pharmaceutical composition comprising a plurality of core-shell particles having pores extending through its shell and a plurality of polymers that are bonded to the outer surface of the shell, wherein each polymer is a copolymer comprising repeating monomer units of formulae (1) and (2): ##STR00013## wherein m and n are at least 1; R.sub.a, R.sub.b, R.sub.c and R.sub.g are independently selected from the group consisting of a hydrogen, optionally substituted aliphatic, optionally substituted hetero-aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl and optionally substituted heterocycloalkyl; R.sub.d is selected from the group consisting of a bond, optionally substituted hetero-aliphatic, optionally substituted aliphatic group, optionally substituted alkoxy, carbonyl, optionally substituted ester, optionally substituted amide, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl and optionally substituted heterocycloalkyl; X is selected from the group consisting of a heteroatom or a positively charged ion; R.sub.f and Y are absent when X is a heteroatom but are both present when X is a positively charged ion such that R.sub.f is selected from the group consisting of optionally substituted aliphatic, optionally substituted hetero-aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl and optionally substituted heterocycloalkyl; and Y is a negatively charged ion when bonded to R.sub.f; and R.sub.h is selected from the group consisting of a hydrogen, optionally substituted aliphatic, optionally substituted hetero-aliphatic, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl and optionally substituted heterocycloalkyl, wherein the core of said particles contains an agent.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0099] 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.

[0100] FIG. 1(a) is a transmission electron microscopy (TEM) image showing an unmodified hollow silica sphere (HSS) of Example 1. The scale bar in FIG. 1(a) is 0.5 m.

[0101] FIG. 1(b) is a TEM image of HSS-graft-(poly(DMAEMA)-co-poly(PEGMA.sub.1.1k)) particle of Example 1. The scale bar in FIG. 1(b) is 100 nm.

[0102] FIG. 2 is a graph showing the thermal gravimetric analyses of pristine HSS, HSS-Br and HSS-graft-(PDMAEMA-co-PPEGMA.sub.1.1k) of Example 1.

[0103] FIG. 3(a) is a TEM image of pristine hollow silica nanoparticle of Example 2. The scale bar in FIG. 2(a) is 100 nm.

[0104] FIG. 3(b) is a TEM image of HSi-graft-(poly(DMAEMA)-co-poly(PEGMA.sub.1.1k)) particle of Example 2. The scale bar in FIG. 2(b) is 100 nm.

[0105] FIG. 4 is a graph showing the thermal gravimetric analyses of pristine HSi, HSiBr, HSi-graft-(PDMAEMA-co-PPEGMA.sub.1.1k) of Example 2 and that of HSi-graft-(PDMAPS-co-PPEGMA.sub.1.1k) of Example 3.

[0106] FIG. 5(a) is an optical microscope image of oxygen filled HSS-graft-(PDMAEMA-co-PPEGMA.sub.1.1k) of Example 4 in PBS at a concentration of 1 mg/mL. FIG. 5(a) was taken at 100 magnification.

[0107] FIG. 5(b) is an optical microscope image of oxygen filled HSS-graft-(PDMAEMA-co-PPEGMA.sub.1.1k) of Example 4 in PBS at a concentration of 1 mg/mL that has been bubbled with carbon dioxide for 60 minutes. FIG. 5(b) was taken at 100 magnification.

[0108] FIG. 6(a) is a graph showing the measured dissolved oxygen content against time for a degassed PBS solution when an equal volume of O.sub.2 saturated PBS (.square-solid.) or O.sub.2 loaded nanoparticle solution () was added at t=3 minutes as indicated by the blue arrow as demonstrated in Example 4. Solid lines indicate fitted exponential decay function from t=7 minutes.

[0109] FIG. 6(b) is a schematic diagram showing the change in the structure of the nanoparticle in the presence of carbon dioxide.

[0110] FIG. 7 is a graph showing the relative cell viability in L929 cell line after exposure to HSS-graft-polymer (nanoparticles) and positive control polymers, PEI and PDMAEMA (0.03125-1 mg/mL). Relative cell viability was determined using the MTT Assay and expressed as a percentage of untreated cells (control) as demonstrated in Example 5.

[0111] FIG. 8(a) is a graph showing the normalized fluorescence measurements of collected filtrate showing the drug release profiles of Rhodamine B loaded nanoparticles as demonstrated in Example 6.

[0112] FIG. 8(b) is a schematic diagram showing the change in the structure of the nanoparticle in response to a change in temperature.

[0113] FIG. 9 is a graph showing the dynamic light scattering (DLS) results showing the change in the hydrodynamic radius of the particles in PBS and deionized water with respect to temperature according to Example 6.

EXAMPLES

[0114] Non-limiting examples of the invention and a comparative example 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.

[0115] All precursors/chemicals used in the Examples were obtained from Sigma-Aldrich of St. Louis of Missouri of the United States of America.

Example 1Synthesis of HSS-Graft-(Poly(DMAEMA)-Co-Poly(PEGMA.SUB.1.1k.))

Synthesis of Hollow Silica Spheres (HSS)

[0116] 0.5 m sized hollow silica spheres (HSS) were synthesized by firstly dissolving 0.5 g of polyvinyl pyrrolidone (M.sub.n=40,000 g.Math.mol.sup.1) in deionized water (100 mL) and stirring at room temperature for 4 hours. To this, 11 g of styrene (which had been pre-treated with basic alumina to remove inhibitor) and 0.26 g of 2,2-azobis(2-methylpropionamide) dihydrochloride was added and degassed with argon for 30 minutes, then allowed to react for 24 hours at 70 C. The mixture was cooled at the end of the reaction, and 18 mL of the mixture was extracted and mixed with 240 mL of ethanol and 12 mL of aqueous ammonia (25 wt %). Separately, 3.2 mL of tetraethylorthosilicate was mixed with 5 mL of ethanol and added dropwise to the mixture of polystyrene colloid, ethanol and ammonia at 50 C. This mixture was then allowed to react for 24 hours. The solution was centrifuged to collect the suspended particles, washed three times with ethanol, then dried, followed by calcination in a furnace at 550 C. The particles were visualized by transmission electron microscopy (TEM) and as shown in FIG. 1(a), the particle size of the HSS was approximately 500 nm, with a 50 nm silica shell. The particles were also characterized by BET which showed that HSS had a surface area of 230 m.sup.2/g and an average pore size of 45 .

Bromide Functionalization of HSS (HSS-Br) for Atom-Transfer Radical Polymerization

[0117] 0.2 g of the HSS obtained above were dispersed in 10 mL of p-xylene, followed by the addition of 0.3 mL of 3-amino propyl triethoxy silane. The mixture was stirred for 24 hours at 90 C. under argon, after which the mixture was washed several times with diethyl ether, and then filtered. The collected solids were dried in a vacuum oven, then redispersed in 40 mL of anhydrous chloroform and 1.2 mL of triethylamine in a large round bottom flask. This flask was immersed in ice while 0.6 mL of 2-bromoisobutyryl bromide in 4 mL anhydrous chloroform was added dropwise over a period of 1 hour, after which the reaction mixture was removed from ice and allowed to react at ambient temperature for a further 18 hours. At the end of the reaction, the solids were filtered and washed several times with chloroform, then dried under vacuum.

Synthesis of HSS-Graft-(Poly(DMAEMA)-Co-Poly(PEGMA.SUB.1.1k.))

[0118] A monomer solution was first prepared by adding 4 g (25.5 mmol) of dimethyl amino ethyl methacrylate (DMAEMA) and 0.4 g (0.36 mmol) of poly(ethylene glycol) methacrylate (M.sub.n=1,100, PEGMA.sub.1.1k) (both of which had been pre-treated with basic alumina to remove inhibitor) to 5 mL of anhydrous anisole in a sealed flask. This solution was purged with nitrogen gas for 1 hour. The bromide functional HSS (0.2 g) were first dispersed and sonicated in 5 mL of anhydrous anisole in a schlenk flask. To this, 0.02 g of copper bromide was added. Before use, copper bromide was purified by refluxing in glacial acetic acid for 18 hours, washed with ether and dried extensively under vacuum. The flask was then sealed and purged with nitrogen gas for 30 minutes. After 30 minutes, 40 L of N,N,N,N,N-Pentamethyl diethylenetriamine (PMDETA) (which was distilled before use) was added to the schlenk flask via a gas tight syringe, and the solution was purged with nitrogen for a further 30 minutes. At the end of this time period, the monomer solution was transferred to the schlenk fast via a cannula, and the flask was reacted at 90 C. for 9 hours. The overall reaction scheme for this polymerization is shown in Scheme 1 below.

##STR00007##

[0119] The product was then centrifuged and washed with acetone to remove the solvent, copper and unreacted monomer. To further remove the copper catalyst, the solids were redispersed in acetone and mixed with Dowex Marathon MSC (H.sup.+) ion exchange resin. The washed solids were dried under vacuum to yield the nanoparticles. The HSS-graft-(PDMAEMA-co-PPEGMA.sub.1.1k) particles were visualized by TEM and shown in FIG. 1(b). As shown in FIG. 1(b), the diameter of the particle ranges from 585 nm to 615 nm and the shell thickness of the particle ranges from 10 to 75 nm. The pristine HSS, HSS-Br and HSS-graft-(PDMAEMA-co-PPEGMA.sub.1.1k) particles were analyzed for the weight percent of polymer by thermogravimetric analysis (TGA) as shown in FIG. 2. The TGA results show that approximately 37.4% of the polymer grafted hollow silica was made up of the initiator and the polymer.

Example 2Synthesis of HSi-Graft-(Poly(DMAEMA)-Co-Poly(PEGMA.SUB.1.1k.))

Synthesis of Hollow Silica (HSi)

[0120] Hollow silica (HSi) with a diameter of 150 nm was synthesized and functionalized via the procedure described by Lay et al. In a typical process, 3.0 g of polyvinyl pyrrolidone was dissolved in 100 mL of HPLC grade water under stirring for 24 hours at room temperature. Then, 11.0 mL of styrene and 0.26 g of 2,2-azobis(2-methylpropionamide) dihydrochloride were added to the solution under stirring at 100 rpm and 70 C. under argon. After 24 hours, 18 mL of polystyrene colloid solution was mixed with 240 mL of ethanol and an 12 mL of aqueous solution of ammonia (25 wt %). Then, 3.18 mL of tetraethyl orthosilicate in 5 mL of ethanol was added dropwise, and the mixture was stirred at 50 C. for 24 hours. The solid was collected by centrifugation and was calcinated at 550 C. to get hollow silica spheres. The particles were visualized by TEM and as shown in FIG. 3(a), the particle size of the HSi was approximately 150 nm, with a 13 nm silica shell. The particles were also characterized by BET which showed that HSi had a surface area of 401 m.sup.2/g and an average pore size of 55 .

Synthesis of HSi-Graft-(Poly(DMAEMA)-Co-Poly(PEGMA

[0121] [S&F: Please advise on the process used to form the HSi-graft-polymer as your TD only focuses on HSS as the template. If the process to form the HSi-graft-polymer is the same as that to form the HSS-graft-polymer, we can state it as such here. If this is the case, please check that the amounts of the chemicals used, reaction parameters (temperature, time, etc) are the same as those for the HSS process.

[0122] The HSi-graft-(poly(DMAEMA)-co-poly(PEGMA.sub.1.1k)) particles were visualized by TEM and shown in FIG. 3(b). As shown in FIG. 3(b), the particles have an approximate diameter of about 231 nm and an approximate shell thickness of about 18 nm.

[0123] The pristine HSi, HSiBr and HSi-graft-(PDMAEMA-co-PPEGMA.sub.1.1k) particles were analyzed for the weight percent of polymer by TGA as shown in FIG. 4. The TGA results show that the polymer content by weight of the HSi-graft-(PDMAEMA-co-PPEGMA.sub.1.1k) was 37%.

Example 3Synthesis of HSi-Graft-(P(DMAPS)-Co-P(PEGMA.SUB.1.1k.))

[0124] To a 100 mL roundbottom flask, 50 mg of HSi-graft-(P(DMAEMA)-co-P(PEGMA.sub.1.1k)) was dispersed in 50 mL of tetrahydrofuran (THF). To this, 50 mg (36 L) of 1,3-propane sultone was added and the mixture was reacted at 60 C. for 24 hours. The pendant tertiary amine moieties on the polymer then under betainization. The overall reaction scheme for this polymerization if shown in Scheme 2.

##STR00008##

[0125] At the end of the reaction time, the product was centrifuged, and subjected to 3 cycles of redispersion in THF and centrifugation before the collected pellet was redispersed in water. The aqueous solution was freeze dried to yield the zwitterionic HSi-graft-(P(dimethyl(methaacryloyloxyethyl)ammonium propanesulfonate)-co-P(PEGMA.sub.1.1k)). The HSi-graft-(PDMAPS-co-PPEGMA.sub.1.1k) was then subjected to TGA and the result is shown together with the other HSi particles in FIG. 4. The final polymer content on the HSi-graft-(PDMAPS-co-PPEGMA.sub.1.1k) particles was 53% which showed that the increase in the polymer content of the particles could be attributed to the complete betanization of the tertiary amines to the zwitterionic moiety.

Example 4Responsiveness of HSS-Graft-(Poly(DMAEMA)-Co-Poly(PEGMA.SUB.1.1k.)) Particles

[0126] The HSS-graft-(poly(DMAEMA)-co-poly(PEGMA.sub.1.1k)) nanoparticles were placed in a sealed schlenk flask and placed under vacuum for 3 hours to ensure full evacuation of air from the nanoparticles. After the three hours, the schlenk flask was backfilled with oxygen gas at 1 atmosphere and left to equilibrate for 2 hours. Separately, PBS was sparged with nitrogen gas for 1 hour to remove dissolved gases. The degassed PBS was then transferred to the schlenk flask to make up a 10 mg/mL solution of oxygen filled nanoparticles. These particles were further diluted to 1 mg/mL for optical microscopy, the image of which is shown in FIG. 5(a). In FIG. 5(a), multiple bubble-like features can be observed, which are the oxygen filled particles and are present in this manner due to the difference in refractive index of the particle due to the transition of light from liquid to gas and back to liquid. The 1 mg/mL solution was then bubbled with carbon dioxide for 60 minutes, after which a sample was viewed under the optical microscope again, the image of which is shown in FIG. 5(b). In FIG. 5(b), significantly fewer bubble-like features were visible, and the ones visible appeared to be less bright, as highlighted by the arrows in FIG. 5(b). The reduction of these features suggest that the liquid-filled polymer-HSS particles were not visible by optical microscope due to low contrast and only the presence of a gas allowed for their visualization by optical microscopy.

[0127] Dissolved oxygen studies were performed on a Rank Brothers Digital Model 10 dissolved oxygen meter that was calibrated with oxygen saturated PBS solution. Nitrogen sparged solution of PBS was first placed in the oxygen sensor to measure the baseline oxygen content of degassed PBS (which was 19.5%). The solution was allowed to equilibrate for 3 minutes after which an equal volume of the oxygen-filled nanoparticle solution was added to the PBS. The dissolved oxygen content was measured every minute over 30 minutes. As a control, the experiment was repeated with oxygen saturated PBS instead of the nanoparticle solution. The normalized results are shown in FIG. 6a.

[0128] When the O.sub.2 loaded nanoparticle solution was added, the dissolved oxygen levels of the solution continued to increase for 3 minutes. It plateaued for an additional 3 minutes after which the oxygen levels began to decrease slowly over time. Conversely, when the O.sub.2 saturated PBS solution was added, the dissolved oxygen level rose immediately, was stable for 1 minute, and then began a steady decline. While there was no specific dosage of carbon dioxide to the solutions, carbon dioxide would still be present due to the exposure of the sample cell to the atmosphere.

[0129] The oxygen saturation curve for the control experiment suggested that the oxygen levels equilibrated with the atmosphere and thus decline. Furthermore, it is known that the oxygen sensor also consumed dissolved oxygen, thereby contributing to the decline of dissolved O.sub.2. This decay can be regarded as the baseline decay of dissolved oxygen in solution. For the control system, a first order exponential decay was fitted to the decay portion of the curve from t=6 minutes, and is mathematically described by Math. 1. In the case of the O.sub.2 loaded nanoparticles, the maximum O.sub.2 concentration was only achieved 3 minutes after the addition of the solution. This thereby suggested that O.sub.2 was continually being released from the nanoparticles. A first order exponential decay is fitted to the decay curve from t=6 minutes and is mathematically described by Math. 2.

[00001]$\begin{array}{cc}y=34.6.Math.{}^{\left(-\frac{x}{16.2}\right)}+28.6.Math..Math.\left(R^2=0.9978\right)& \left[\mathrm{Math}..Math.1\right]\\ y=34.7.Math.{}^{\left(-\frac{x}{39}\right)}+29.8.Math..Math.\left(R^2=0.9916\right)& \left[\mathrm{Math}..Math.2\right]\end{array}$

[0130] It is noted that the two equations show that the decay of dissolved oxygen over time as slower for the O.sub.2 loaded nanoparticles (Equation 2) as compared to the control experiment with the addition of saturated O.sub.2 PBS (Equation 1). The exponential decay constant was almost 2.5 times higher in the control compared to the decay in the presence of O.sub.2 loaded nanoparticles. This result suggested that O.sub.2 was still continually released from the nanoparticles up to 30 minutes and even beyond.

[0131] The mechanism of the response of the nanoparticles to carbon dioxide is shown in FIG. 6(b). In FIG. 6(b), the nanoparticle 20 is a core-shell particle which has a core 2 surrounded by a silica shell 4 with pores 6 extending from the core 2 through the silica shell 4. The polymer brushes 8 are bonded to the outer surface of the silica shell 4. In the presence of carbon dioxide, the polymer brushes 8 grafted to the surface of the silica shell 4 (namely the PDMAEMA) respond to the carbon dioxide by uncoiling and exposed the silica pores 6. This allows any oxygen 10 present in the core 2 of the nanoparticle 20 to be released from the nanoparticle 20. In the absence of carbon dioxide, the grafted polymers 8 collapse to form a layer that seal the silica pores 6, thus encapsulating the oxygen 10 within the core 2 of the nanoparticle 20.

[0132] The particle size in water was then characterized by dynamic light scattering (DLS). For the HSS-graft-polymer particles, 1 mg/mL solution of the nanoparticles in degassed (nitrogen) deionized water was prepared. Particle size measurements were taken over 10 readings and were found to be 128749 nm. The solution was then bubbled with carbon dioxide for 2 minutes and the particle size was re-measured. The new particle size after carbonation was found to be 154843 nm. The hydrodynamic particle size increased by approximately 260 nm thereby suggesting that the carbonation had an effect on the polymer brush. This showed that PDMAEMA ionized and increased in solubility in the presence of carbon dioxide.

Example 5Cytotoxicity of HSS-Graft-(Poly(DMAEMA)-Co-Poly(PEGMA.SUB.1.1k.)) Particles

[0133] Cytotoxicity of the nanoparticles was assessed on L929 mouse fibroblast cells (obtained from American Type Culture Collection of Rockville of Maryland of the United States of America). Cells were maintained in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/mg penicillin, 100 g/mL streptomycin at standard conditions, 37 C., 5% CO.sub.2 and 95% relative humidity. The cells were seeded in 96-well cell culture plates (110.sup.5 cells/mL) and incubated for 24 hours.

[0134] The cell culture medium was then replaced with medium containing serial dilutions of the nanoparticle or control polymers, Poly(dimethylaminoethyl methacrylate) (PDMAEMA) and polyethylenimine (PEI), (0.03125-1 mg/mL) and incubated for 4 hours. PDMAEMA and PEI are positive control polymers that are commonly used in gene transfection studies and were used here for comparative purposes. The culture medium was then removed and replaced with fresh DMEM and incubated for 42 hours. 10 L of filtered 1-(4,5-dimethyl-thiazol-2-yl)-3,5-diphenylfor-mazan (MTT, obtained from Sigma-Aldrich of St. Louis of Missouri of the United States of America) (5 mg/mL) in PBS (pH 7.4) was added and cells were incubated for an additional 4 hours. Unreacted dye was removed by aspiration and formazan crystals were dissolved with 100 L/well DMSO and the absorbance was measured at 570 nm (SpectraMax Plus384, Molecular Devices). 6 replicate measurements were performed and the relative cell viability is expressed as: [A.sub.570 (polymer+cells)A.sub.570 (blank)]/[A.sub.570 (cells only)A.sub.570 (blank)]100% and shown in FIG. 7.

[0135] As shown in FIG. 7, for the nanoparticle solution, 90-100% of viable cells were detected at the lower concentration range tested (0.03125-0.125 mg/mL) while the relative cell viability ranges between 45-55% at higher concentrations (0.25-1 mg/mL). This was significantly less toxic than PDMAEMA where the cell viability rapidly declined from 55% (at 0.03125 mg/mL) to below 10% and for PEI where even at exposure to the lowest concentration resulted in less than 25% cell viability. The particles were fairly benign at low concentrations thereby showing the vast potential of this material as an oxygen delivery vehicle. Carbon dioxide responsive polymers are an emerging class of materials where its clinical potential has not been fully realized and therefore the toxicity of these materials could be lowered further via polymer modifications.

Example 6Thermoresponsiveness of HSi-Graft-(P(DMAPS)-Co-P(PEGMA.SUB.1.1k.)) Particles

[0136] 10 mg of HSi-(P(DMAPS)-co-P(PEGMA.sub.1.1k)) nanoparticles was weighed into 5 mL of deionized water and sonicated for 10 minutes to fully disperse all the particles. 10 mg of rhodamine B was then introduced into the solution and the mixture was stirred for 18 hours at 50 C. After this period, the solution was cooled to 5 C. The solution was centrifuged and the supernatant was discarded. The remnant solids were washed with cold deionized water several times to remove excess rhodamine B, and the solids were eventually freeze dried to yield a pink powder.

[0137] The lypholized nanoparticles were then dissolved into three batches of water at ambient temperature and stirred for 18 hours. A sample was taken initially and at 18 hours, filtered to remove the particles, and then tested for its fluorescence. After 18 hours, the solution batches were heated up to (i) 38 C.; (ii) 40 C.; and (iii) 42 C.; in order to obtain the drug release profiles at 3 different temperatures. The samples were taken immediately, and at 2 hour intervals 3 times, followed by a final sample at 24 hours after heating. Similarly, the samples were filtered immediately and analyzed for the fluorescence of released Rhodamine B, with an excitation wavelength of 552 nm, and an emission wavelength of 572 nm. The normalized fluorescence measurements of the collected filtrate showing the drug release profiles of Rhodamine B loaded nanoparticles are reported in FIG. 8(a). As seen from FIG. 8(a), the release profile of rhodamine B from the nanoparticles shows that the rhodamine B was released from the particles when the temperature of the particles was elevated beyond a critical temperature (as will be discussed further below). This shows the thermoresponsive properties of the particles.

[0138] The mechanism of the response of the nanoparticles to changes in temperature is shown in FIG. 8(b). The elements in FIG. 8(b) are similar to the elements in FIG. 6(b) and hence, like reference numerals will be used to denote like elements but with an additional prime (). In FIG. 8(b), the nanoparticle 20 is a core-shell particle which has a core 2 surrounded by a silica shell 4 with pores 6 extending from the core 2 through the silica shell 4. The polymer brushes 8 are bonded to the outer surface of the silica shell 4. At a temperature above a critical temperature, the polymer brushes 8 grafted to the surface of the silica shell 4 (namely the PDMAPS) respond to the elevated temperature by uncoiling and exposing the silica pores 6. This allows any agent/load 10 present in the core 2 of the nanoparticle 20 to be released from the nanoparticle. At a temperature below the critical temperature, the grafted polymers 8 collapse to form a layer that seal the silica pores 6, thus encapsulating the agent/load 10 within the core 2 of the nanoparticle 20.

[0139] To determine the critical temperature of the nanoparticle, particularly, the upper critical solubility temperature (UCST), DLS measurements were conducted at a range of temperatures for the zwitterionic polymer grafted HSi in both deionized water and PBS at a concentration of 1 mg/mL since ionic strength can potentially affect the thermoresponsive characteristics. The DLS measurements are shown in FIG. 9. Here, the change in the hydrodynamic radius of the particles in PBS and deionized water with respect to temperature can be observed. As a median point of size change can be seen at 37.5 C. irrespective of solvent, it was determined that the UCST is approximately 37.5 C.

[0140] In traditional stimuli responsive polymeric systems, the solubility is often regarded as when the chains are fully extended and solubilized, and insolubility is regarded as when the chains are collapsed. Therefore, in terms of the hydrodynamic radius of nanoparticles, the particle size would increase with an increase in solubility. Therefore, based on the DLS results shown in FIG. 9, could suggest that there was a decrease in polymer solubility with increasing temperature. However, it is likely that the insolubility of the polymeric chains at a lower temperature caused the aggregation of particles, which resulted in the higher particle size measurement, and as the polymer chains uncoiled, the particles dispersed and thus there was a decrease in the recorded particle size. This is further illustrated by the scheme within FIG. 9 which shows aggregated hollow silica particles with a collapsed polymer layer on the left side of the graph, and dispersed hollow silica with fully extended polymeric brushes on the right side of the graph.

[0141] Hence, it has been shown that the HSi-graft-(P(DMAPS)-co-P(PEGMA.sub.1.1k)) particles are thermoresponsive.

INDUSTRIAL APPLICABILITY

[0142] The core-shell particle may be used as a delivery agent for delivering an agent, a cargo or a load into an environment which triggers the release of the agent, cargo, or load from within the core-shell particle to the external environment.

[0143] The core-shell particle may be used as a delivery agent for a therapeutic agent. The core-shell particle may be injected or ingested by a patient and upon reaching the critical temperature (which can be the patient's normal body temperature or higher in the case of a fever), the core-shell particle can release the therapeutic agent in vivo. The core-shell particle can also be used as a delivery agent in skincare products. The core-shell particle can be formulated as a nanocapsule for sustained deliver of the agent to the skin of a patient during topical administration. The core-shell particle may be used as detergent additives.

[0144] Where the agent is a gas such as oxygen gas, the core-shell particle may be injected into a patient and serves as a nano-sized breathing apparatus by releasing oxygen from the core-shell particle when the particle is exposed to dissolved carbon dioxide. This then results in the oxygenation of blood in the patient and provides medical workers with more time to stabilise the patient. This may be useful at times of cardiac thoracic arrest to give medical workers a longer window to seek medical intervention, thereby reducing mortality rates. The core-shell particle can be used in sports medicine to provide direct oxygen administration; in defense to allow naval divers to function without scuba gear; or in stroke victims to oxygenate the blood vessels in the brain.

[0145] 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.