CALCIUM PEROXIDES NANOPARTICLES AS ADJUVANT THERAPY

Abstract

The invention provides CaO.sub.2 nanoparticles having a pH-responsive coating for use in a method of adjuvant therapy of hypoxic tumour cells or tissues. The nanoparticles find particular use in enhancing cancer therapies that depend on oxygen to exert their effect, such as photodynamic therapy (PDT), sonodynamic therapy (SDT), and radiotherapy. The invention also provides pharmaceutical compositions containing the coated CaO.sub.2 nanoparticles, together with at least one photosensitiser, sonosensitiser, or radiosensitiser and, optionally, at least one pharmaceutical carrier or excipient.

Claims

1. CaO.sub.2 nanoparticles having a pH-responsive coating for use in a method of adjuvant therapy of hypoxic tumour cells or tissues.

2. CaO.sub.2 nanoparticles as claimed in claim 1 for use as an adjuvant therapy to at least one of the following cancer therapies: photodynamic therapy, sonodynamic therapy and radiotherapy.

3. CaO.sub.2 nanoparticles for use as claimed in claim 1 or claim 2 having a diameter of up to about 1 m, preferably from 5 to 900 nm, more preferably from 10 to 500 nm, e.g. in the range from 10 to 300 nm.

4. CaO.sub.2 nanoparticles for use as claimed in any one of the preceding claims which contain at least 10 wt. %, preferably at least 25 wt. %, e.g. at least 40 wt. % of active CaO.sub.2 (based on the total weight of the particles).

5. CaO.sub.2 nanoparticles for use as claimed in any one claims 1 to 3 which consist essentially of (e.g. consist of) calcium peroxide.

6. CaO.sub.2 nanoparticles for use as claimed in any one of the preceding claims having a pH-responsive coating which is stable at physiological pH but which degrades at a pH which is less than physiological pH, e.g. at a pH of less than about 7.4.

7. CaO.sub.2 nanoparticles for use as claimed in claim 6, wherein said pH-responsive coating degrades in the range of pH 6.0 to 7.4, preferably 6.2 to 7.4.

8. CaO.sub.2 nanoparticles for use as claimed in any one of the preceding claims, wherein said pH-responsive coating comprises one or more basic units (e.g. amine groups) which ionise at a pH of less than about 7.4.

9. CaO.sub.2 nanoparticles for use as claimed in any one of the preceding claims, wherein said pH-responsive coating comprises a copolymer derived from esters of acrylic and methacrylic acid and which has one or more basic functional groups (e.g. amine groups).

10. CaO.sub.2 nanoparticles for use as claimed in claim 9, wherein said pH-responsive coating is a Eudragit E-type polymer or a derivative thereof.

11. CaO.sub.2 nanoparticles for use as claimed in any one of claims 1 to 9, wherein said pH-responsive coating is obtained by polymerisation of methyl methacrylate, ethyl acrylate and 2-(dimethylamino)ethyl methacrylate.

12. CaO.sub.2 nanoparticles for use as claimed in claim 11, wherein said pH-responsive coating comprises a terpolymer having the following structure: ##STR00002## wherein x, y and z are integers representing the molar ratio of the monomeric units in the polymer material.

13. CaO.sub.2 nanoparticles for use as claimed in any one of the preceding claims, wherein said pH-responsive coating is linked to one or more of the following active agents: a photosensitiser, a sonosensitiser, a radiosensitiser, or an enzyme capable of catalysing the degradation of hydrogen peroxide to oxygen, e.g. catalase.

14. CaO.sub.2 nanoparticles for use as claimed in any one of the preceding claims, wherein said nanoparticles are embedded in a polymeric matrix comprising a physiologically acceptable, biodegradable polymer, e.g. poly(D,L-lactide-co-glycolide) (PLGA).

15. CaO.sub.2 nanoparticles for use as claimed in claim 14, wherein said polymeric matrix further comprises one or more of the following active agents: a photosensitiser, a sonosensitiser, a radiosensitiser, or an enzyme capable of catalysing the degradation of hydrogen peroxide to oxygen, e.g. catalase.

16. A pharmaceutical composition comprising CaO.sub.2 nanoparticles having a pH-responsive coating as defined in any one of claims 1 to 15, together with at least one photosensitiser, sonosensitiser, or radiosensitiser and, optionally, at least one pharmaceutical carrier or excipient.

17. A product comprising (i) CaO.sub.2 nanoparticles having a pH-responsive coating as defined in any one of claims 1 to 15, and (ii) a photosensitiser, a sonosensitiser, or a radiosensitiser for simultaneous or sequential use in a method of photodynamic therapy, sonodynamic therapy, or radiotherapy.

18. A kit comprising the following components: (i) CaO.sub.2 nanoparticles having a pH-responsive coating as defined in any one of claims 1 to 15, (ii) a photosensitiser, a sonosensitiser, or a radiosensitiser; and optionally (iii) instructions for the use of said components in a method of photodynamic therapy, sonodynamic therapy, or radiotherapy.

19. A method of treatment of hypoxic tumour cells or tissues in a patient, said method comprising the following steps: (i) administering to said cells or tissues of said patient an effective amount of CaO.sub.2 nanoparticles having a pH-responsive coating as defined in any one of claims 1 to 15; (ii) if required, administering to said cells or tissues of said patient at least one of the following active agents: a photosensitizer, a sonosensitizer, or a radiosensitizer; and (iii) subjecting said cells or tissues to one or more of the following: light, ultrasound and ionising radiation whereby to treat said tumour cells or tissues.

20. A method as claimed in claim 19, wherein said steps are carried out in the following order: (ii), (i), (iii).

21. CaO.sub.2 nanoparticles having a pH-responsive coating, wherein said coating comprises (e.g. consists essentially of) a polymer obtainable (or obtained) by polymerisation of the following monomers: methyl methacrylate, ethyl acrylate and 2-(dimethylamino) ethyl methacrylate.

22. CaO.sub.2 nanoparticles having a pH-responsive coating, wherein said coating is linked to at least one of the following active agents: a photosensitiser, a sonosensitiser, a radiosensitiser, or an enzyme capable of catalysing the degradation of H.sub.2O.sub.2 to oxygen, e.g. catalase.

23. A polymeric (nano)particle having embedded therein CaO.sub.2 nanoparticles which optionally are provided with a pH-responsive coating, wherein said polymeric (nano)particle further comprises and/or is linked to one or more of the following active agents: a photosensitiser, a sonosensitiser, a radiosensitiser, or an enzyme capable of catalysing the degradation of H.sub.2O.sub.2 to oxygen, e.g. catalase.

Description

[0113] The invention will now be described further with reference to the following non-limiting Examples and the accompanying figures in which:

[0114] FIG. 1 shows a representative SEM image (a) and DLS plot (b) of CaO.sub.2 NPs prepared in Example 1;

[0115] FIG. 2 shows stacked .sup.1H NMR spectra of (i) methyl methacrylate; (ii) ethyl acrylate; (iii) 2-(dimethylamino) ethyl methacrylate; and (iv) pH-responsive polymer 1 prepared in Example 3;

[0116] FIG. 3 shows a representative SEM image (a) and DLS plot (b) of polymer coated CaO.sub.2 NPs prepared in Example 3;

[0117] FIG. 4 is a plot of increase in dissolved oxygen for a solution of PBS against time over a period of 20 minutes in the absence (diamonds) or presence (circles) of uncoated CaO.sub.2 NPs;

[0118] FIG. 5 is a plot of % dissolved oxygen for solutions of de-oxygenated PBS solution at pH 6.0 and pH 7.4 in the absence and presence of polymer coated CaO.sub.2 NPs;

[0119] FIG. 6 is a plot of % increase in SOSG fluorescence at 510 nm for solutions containing (i) de-oxygenated PBS (ii) de-oxygenated PBS and CaO.sub.2 NPs (35.6 M) (iii) de-oxygenated PBS and 5 M Rose Bengal (RB) and (iv) de-oxygenated PBS, 5 M Rose Bengal and CaO.sub.2 NPs (35.6 M);

[0120] FIG. 7 is a plot of cell viability for BxPC-3 cells, cultured under hypoxic conditions with (i) no treatment (CTRL dark bar) or after treatment with (ii) light only (CTRL light bar) (iii) RB only in an EtOH vehicle (RB+EtOH black bar) (iv) RB in an EtOH vehicle+light (RB+EtOH light bar) (iv) RB+CaO.sub.2 NP in an EtOH vehicle (RB+NP dark bar) (vi) RB+CaO.sub.2 NP+ light in an EtOH vehicle (RB+NP dark bar);

[0121] FIG. 8 shows: (a) Plot of average tumour pO.sub.2 in mice bearing ectopic MiaPaca-2 pancreatic tumours recorded for 20 min before and 40 min following an IV injection of polymer coated CaO.sub.2 NPs in a PBS (pH 7.40.1) vehicle (unbroken line) or vehicle only (dashed line) (injection occurred at t=20 mins); and (b) Plot showing the mean tumour pO.sub.2 for various time intervals before and following IV administration of polymer coated CaO.sub.2 NPs, obtained from integration of the plot shown in (a). n=3, *p0.05;

[0122] FIG. 9 shows: (a) Plot of % change in tumour volume against time for SCID mice bearing human xenograft Mia-PaCa2 pancreatic tumours treated with (i) no treatment (squares) (ii) PDT only (circles) (iii) polymer coated CaO.sub.2 NPs only (diamonds) and (iv) polymer coated CaO.sub.2 NPs and PDT (triangles); and (b) Plot of average body weight for each group of mice over the time course of the experiment. ***p0.01.

EXAMPLES

Reagents and Equipment:

[0123] Calcium chloride, PEG 200, 1M ammonia solution, 35% hydrogen peroxide, sodium hydroxide, phosphate buffered saline (PBS), luminol, methanol, ethanol, hexane, chloroform, Rose Bengal, singlet oxygen sensor green (SOSG), anhydrous tetrahydrofuran (THF), 2-(dimethylamino)ethyl methacrylate, methyl methacrylate, ethyl acrylate and 1,1-Azobis(cyclohexanecarbonitrile) (ABCN) were purchased from commercial sources at the highest possible grade. BxPC-3 and MiaPaCa cells were obtained from the American Type Culture Collection (ATCC) and matrigel from BD Biosciences, Erembodegem, Belgium. SCID mice (C.B-17/IcrHanHsd-PrkdcSCID) were bred in house. Scanning electron microscopy (SEM) analysis was conducted using an FEI Quanta scanning electron microscope while dynamic light scattering (DLS) measurements were performed using a Malvern Zetasizer 3000HSA (Malvern, Worcs., UK). Dissolved oxygen measurements were recorded using a Thermo Scientific DO Probe Orion 083005MD (Fisher Scientific, Ottawa, ON, Canada) while nanoparticle solutions were mixed using a Silverson homogenizer (Silverson Machines Ltd, Chesham, U.K.). Fluorescence measurements were undertaken using a Cary Eclipse spectrophotometer while 96 well plates were analysed using a Fluostar Omega plate reader. Tumour pO.sub.2 measurements were performed using an Oxylite oxygen electrode sensor (Oxford Optronics, Oxford, UK). NMR spectra were obtained on Varian 500 MHz instrument at 25.01 C. and processed using Bruker software. Mass spectra were obtained using a Finnegan LCQ-MS instrument. Error in measurements was expressed as % standard error of the mean while statistical analysis was undertaken using 2-tailed Students t-test.

Example 1Preparation of Uncoated CaO.SUB.2 .NPs

[0124] CaO.sub.2 nanoparticles (CaO.sub.2 NPs) were prepared following a hydrolysis-precipitation procedure similar to that developed by Khodaveiside et al. (Journal of hazardous materials, 192(3): 1437-1440, 2011). This utilised CaCl.sub.2 as a calcium precursor and polyethylene glycol 200 (PEG200) as a surface modifier.

[0125] Ammonia solution (15.0 mL, 1M) and PEG 200 (120.0 mL, 0.6744 mol) was added to a stirred solution of calcium chloride (3.0 g, 0.027 mol) in distilled water (30 mL). A solution of 35% H.sub.2O.sub.2 (15 mL, 0.17 mol) was then added to the mixture at a rate of 3 drops per minute and the colourless solution stirred for a further 2 hours at room temperature. A NaOH solution (0.1 M) was then added until a pH value of 11.5 was achieved when the solution changed to a white coloured suspension. The precipitate was separated by centrifugation and the resulting pellet washed three times with NaOH (25 mL, 0.1 M). The powder was then washed with distilled water until the filtrate pH reached 8.4 and the powder dried in vacuo at 80 C. for 2 hours. The resulting particles were suspended in ethanol and sonicated for 5 minutes. The suspension was passed through a Millex Filter Unit (0.45 m) to isolate larger particles and the filtrate concentrated to dryness affording the CaO.sub.2 nanoparticles as a white powder. The size and size distribution of the nanoparticles was determined by SEM and DLS.

[0126] The resulting particles were analysed by SEM and found to be spherical in shape with an average diameter of 116.07.6 nm (FIG. 1a). Closer inspection of the particle morphology revealed the appearance of several smaller particles coalesced together to form the larger sized NPs. This was confirmed when an ethanol solution containing the NPs was analysed using DLS where the particle diameter was found to be much smaller at 21.0 nm11 nm (FIG. 1b).

Example 2Determination of CaO.SUB.2 .Content in the Uncoated CaO.SUB.2 .NPs

[0127] The amount of active CaO.sub.2 contained within the NP powder produced in Example 1 was determined by measuring the luminescence generated when a fixed amount of the NP powder was dissolved in an aqueous luminol solution. The hydrogen peroxide generated from reaction of CaO.sub.2 with the aqueous medium subsequently reacts quantitatively with luminol to produce a chemiluminescent signal that is proportional to the amount of hydrogen peroxide present.

[0128] The active CaO.sub.2 content of the NPs was determined by reaction with luminol in PBS. A chemiluminescence intensity/concentration calibration curve for the reaction of H.sub.2O.sub.2 with luminol was performed according to the procedure adopted by Komagoe et al. (Analytical sciences, 22(2): 255-258, 2006). CaO.sub.2 NPs in ethanol solution (50 L, 35.6 M) were added to a luminol solution (50 L, 10 mg/mL) and the chemiluminescence intensity determined using a plate reader. The CaO.sub.2 content was determined by indirectly measuring the number of moles of H.sub.2O.sub.2 produced (by reference to the calibration graph) from the fixed mass of CaO.sub.2 powder and assuming all the available CaO.sub.2 was converted to H.sub.2O.sub.2. Using this approach, the amount of active CaO.sub.2 present in the NP powder was determined as 44.92.3% with the remaining mass due to excipients such as PEG.

Example 3Coating of CaO.SUB.2 .NPs with Polymer

[0129] Polymer 1 was prepared by the free radical co-polymerisation of 2-(dimethylamino)ethyl methacrylate, methyl methacyrlate and ethyl methacrylate in a 2:1:1 ratio. Polymer 1 contains a tertiary amine side chain making it possess low aqueous solubility as the free base but which becomes soluble once ionised. The successful preparation of polymer 1 was confirmed by .sup.1H NMR spectroscopy with the stacked spectra of each monomer and polymer 1 shown in FIG. 2. The olefinic protons present in the spectra of the monomers between 5.5 and 6.5 ppm were not present in polymer 1 indicating they had been successfully polymerised to form the backbone of polymer 1. In addition, the peaks were much broader in the spectrum of polymer 1 than in the monomers, which is characteristic of protons in or near the backbone of polymers due to an ineffective averaging of their chemical shift anisotropies.

[0130] Polymer 1 was then used to coat the CaO.sub.2 NPs produced in Example 1 using a modified oil-in-water emulsion technique. A SEM image of the resulting polymer coated CaO.sub.2 NPs is shown in FIG. 3a and again reveals spherical particles with an average diameter of 24817 nm which was similar to the hydrodynamic diameter (27871 nm) determined by DLS (FIG. 3b).

Preparation of Polymer 1:

[0131] 2-(dimethylamino)ethyl methacrylate (157.2 mg, 1 mmol), methyl methacrylate (100.1 mg, 2 mmol) and ethyl acrylate (100.1 mg, 1 mmol) and a catalytic amount of the free radical initiator (ABCN) were dissolved in anhydrous THF (5 mL) and placed in a Carious reaction vessel. The contents were then subjected to three freeze-pump-thaw cycles, sealed under vacuum and placed in a Carious oven at 80 C. for 72 hours. The contents were removed and hexane (20 mL) added to the facilitate precipitation followed by centrifugation for 5 min at 6000 rpm. The supernatant was removed, the pellet containing 1 re-dissolved in anhydrous THF and precipitated again with hexane a centrifuged at 6000 rpm. This purification procedure was repeated twice further before the pellet was dried in vacuo at 80 C. and characterised by .sup.1H NMR spectroscopy.

Coating of CaO.sub.2 NPs with Polymer 1 to Form 1-CaO.sub.2 NPs:

[0132] CaO.sub.2 NPs were coated with polymer 1 using a modified single emulsion method (Choi et al., International journal of pharmaceutics, 311(1): 223-228, 2006). CaO.sub.2 NPs (10 mg) were dispersed in hexane (10 mL) and sonicated for 5 min. The NP suspension was then added dropwise at a rate of 2 mL/min to a solution of polymer 1 (100 mg, 0.36 mol) in ethanol (40 mL) using a Silverson homogeniser at 9000 RPM for 5 mins to ensure efficient mixing. After a further mixing period for 6 hours, the emulsion was freeze dried and reconstituted in sterile water when required for use. The 1-CaO.sub.2 NPs were characterised using SEM and DLS.

Example 4Dissolved Oxygen Experiments Using Uncoated and Coated CaO.SUB.2 .NPs

[0133] For the dissolved oxygen experiments involving the uncoated CaO.sub.2 NPs, an ethanol solution containing the NPs (10.0 mg, 24 mmol) was added to 10 mL of de-oxygenated PBS solvent. The dissolved oxygen was then measured and recorded every 1 min using a dissolved oxygen meter. For dissolved oxygen experiments involving the 1-CaO.sub.2 NPs, separate solutions of de-oxygenated water were pH adjusted to pH 7.4 or 6.2. The 1-CaO.sub.2 NPs (2 mg) were then added to each solution and the dissolved oxygen was measured using a dissolved oxygen meter 3 min following addition. Results were compared against identical solutions in the absence of 1-CaO.sub.2 NPs. Both sets of experiments were repeated in triplicate.

[0134] To determine the ability of the uncoated CaO.sub.2 NPs to generate molecular oxygen upon contact with water and improve oxygen levels in the resulting solution, a simulated hypoxic environment was generated by de-oxygenating a solution of PBS (pH=7.40.1). A fixed amount of the CaO.sub.2 NPs was added to the solution and the amount of dissolved oxygen present in the solution determined as a function of time. The results are shown in FIG. 4 and reveal a rapid increase in dissolved oxygen level (37.25%) 10 minutes after NP addition with no further increase observed over the next 16 min suggesting all the CaO.sub.2 NPs were used up. In contrast, a degassed PBS solution that was exposed to the open atmosphere increased by only 4.10% over the same time period. These results demonstrate that the CaO.sub.2 NPs rapidly decompose when they come into contact with aqueous medium generating a significant enhancement in the oxygen levels of the resulting solution.

[0135] The ability of the 1-CaO.sub.2 NPs to generate oxygen as a function of solution pH was determined by monitoring the increase in dissolved oxygen. De-oxygenated aqueous solutions containing 1-CaO.sub.2 NPs (1 mg/mL) were pH adjusted to either pH 7.4 or pH 6.0 and the change in dissolved oxygen measured at each pH 5 minutes later. The results are shown in FIG. 5 and reveal a 45% increase in dissolved oxygen at pH 6.0 compared to only 7% increase at pH 7.4. This increase in dissolved oxygen results from dissolution of the polymer coating at lower pH that exposes the CaO.sub.2 core to the aqueous environment.

[0136] The pH of an aqueous suspension containing 1-CaO.sub.2 NPs (2 mg in 10 mL) was lowered from pH 7.4 to pH 6.2 in approximate 0.1 pH increments. The visual appearance of the resulting suspensions/solutions was photographed at pH 7.4, 6.9 and 6.2. As the pH was adjusted from pH 7.4 to pH 6.2, the visual appearance changed from a milky suspension to a more transparent solution. These results suggest that the 1-CaO.sub.2 NPs should remain stable in the systemic circulation at pH=7.4 but become activated to release O.sub.2 when in the more acidic tumour microenvironment.

Example 5Determination of Singlet Oxygen Generation

[0137] The ability of the uncoated CaO.sub.2 NPs to enhance PDT mediated singlet oxygen generation was determined using the singlet oxygen probe sensor green (SOSG). SOSG is inherently non-fluorescent but reacts with singlet oxygen to generate a fluorescent product with the fluorescence intensity being proportional to the amount of singlet oxygen generated (Faulkner et al., Free Radical Biology and Medicine, 15(4): 447-451, 1993).

[0138] A de-oxygenated PBS solution (2:98; EtOH:H.sub.2O) containing SOSG (2.5 M) and the sensitiser Rose Bengal (5 M) was prepared and an ethanol solution containing CaO.sub.2 NPs (35.6 M) added. Immediately, the solution was then irradiated with white light for 5 min at which point the fluorescence intensity at 530 nm was measured using a fluorescence spectrometer. Control experiments in the absence of the CaO.sub.2 NPs (i.e. RB, SOSG and light) and CaO.sub.2NPs only (i.e. CaO.sub.2 NPs, SOSG, and light) were also conducted for comparative purposes. The results are shown in FIG. 6 and reveal a significant increase (324.8%, p<0.001) in the amount of SOSG fluorescence observed for the solution containing CaO.sub.2 NPs, RB and treated with light compared to the control experiments, indicating the ability of the NPs to provide oxygen during the photodynamic event and enhance ROS generation in this simulated hypoxic environment.

Example 6In Vitro PDT Experiments Using Uncoated CaO.SUB.2 .NPs

[0139] Having determined the ability of the CaO.sub.2 NPs to improve the light induced ROS generation of Rose Bengal in a simulated hypoxic environment, the next step was to determine if this improved ROS generation would also result in increased Rose Bengal mediated PDT killing of human BxPC-3 pancreatic cancer cells.

[0140] BxPc3 cells were seeded in a 96 well plate at a density of 510.sup.4 cells per well. The cells were cultured in an anaerobic cabinet (O.sub.2/CO.sub.2/N.sub.2, 0.1:5:94.9 v/v/v) for 3 hours to generate a hypoxic environment and then spiked with RB (1 M) and incubated for a further 3 hours under anaerobic conditions. This concentration of RB is much lower than normally used in PDT cell-based studies (5 M) and was chosen to enable a moderate PDT effect in the absence of CaO.sub.2 NPs so that any beneficial effect provided by the NPs could be determined (Chan et al., Tissue engineering, 13(1): 73-85, 2007). The cells were then incubated with an ethanol: PBS (50:50) solution of the NPs (50 M) for 5 min before being exposed to light from a 532 nm emitting LED for 30 sec. The NP solution was then removed, the cells washed with fresh PBS and incubated in fresh media under normoxic conditions for a further 21 hours before cell viability was determined using a MTT assay. The use of a 50% ethanolic NP solution in these experiments was not ideal but care was taken to ensure that contact time with the cells was kept to a minimum. We also conducted vehicle only, RB only, light only and NP only controls for comparative purposes. The results are shown in FIG. 7 and reveal a significant (p<0.01) reduction in viability for those cells treated with PDT in the presence of CaO.sub.2 NPs (33.3%) compared to PDT treatment alone (10.8%). In addition, there was no observable toxicity exhibited by the nanoparticles themselves at the concentration used in this experiement. These results suggest that treatment of hypoxic BxPC3 with CaO.sub.2 NPs prior to PDT treatment can enhance oxygen levels improving the PDT mediated efficacy.

Example 7Determination of the Effect of CaO.SUB.2 .NPs on Tumour pO.SUB.2

[0141] To determine the ability of the 1-CaO.sub.2 NPs to enhance tumour oxygenation in an in vivo model, ectopic human xenograft MiaPaca-2 tumours were established in SCID mice. The Mia-PaCa-2 model is known to form hypoxic tumours and has previously been used in efficacy experiments involving hypoxia activated prodrugs. Unlike the BxPC-3 cell line, MiaPaca-2 cells also express the KRAS mutation making it a more representative model of the disease in vivo.

[0142] Mia-Paca-2 cells were maintained in RPMI-1640 medium supplemented with 10% foetal calf serum. Cells were cultured at 37 C. under 5% CO.sub.2 in air. The cells (510.sup.6) were re-suspended in 100 L of Matrigel (BD Biosciences, Erembodegem, Belgium) and implanted subcutaneously into the rear dorsum of male SCID mice. Animals were treated humanely and in accordance with licensed procedures under the UK Animals (Scientific Procedures) Act, 1986. Tumour formation occurred approximately 5 weeks after implantation and tumour measurements were taken every other day using callipers. Once the tumours had reached an average volume of 250 mm.sup.3 calculated from the geometric mean diameter using the equation tumour volume=(W*H*L/2), animals were randomly distributed into two groups (n=3): (i) 1-CaO.sub.2 NPs and (ii) vehicle only. Following induction of anaesthesia via intraperitoneal injection of Hyponym/Hypnovel (150 l i.p) of a mixture of 2:1:1; PBS: Hypnorm (0.315 mg/ml fentanyl citrate and fluanisone 10 mg/ml, VetaPharma Ltd, U.K.): Hypnovel (10 mg/ml midazolom, Roche, UK) the oxygen partial pressure (pO.sub.2) of tumours was recorded using an Oxylite oxygen electrode sensor. A fibre optic probe was inserted into a 21-gauge needle before insertion into the centre of the tumour tissue. The needle was withdrawn and the probe readings allowed to stabilise for 5 minutes. The oxygen level in the tumours was recorded every second for 20 min. 100 L aliquots of 1-CaO.sub.2 NPs in a PBS vehicle (2 mg/mL) or PBS alone were administered to the respective groups by tail-vein injection with pO.sub.2 recorded every second for a further 40 minutes. This time period was chosen to avoid the need for re-administering anaesthesia.

[0143] The results are shown in FIG. 8 and reveal no significant change in the pO.sub.2 reading in the 20 min period before injection for either group with a mean pO.sub.2 reading of 2.0 mm Hg. However, approximately 10 min after injection, tumour pO.sub.2 levels in the 1-CaO.sub.2 NP group increased dramatically reaching a peak of 16 mmHg before levelling off at 6 mm Hg 30 min after injection. In contrast, mice treated with vehicle alone showed no noticeable change in tumour pO.sub.2 over the time course of the experiment.

Example 8Effect of Polymer Coated CaO.SUB.2 .NPs on PDT Efficacy In Vivo

[0144] All animals employed in this study were treated humanely and in accordance with licenced procedures under the UK Animals (Scientific Procedures) Act 1986. Mia-PaCa 2 xenograft tumours were established as described above. Once the tumours had reached an average volume of 25417 mm.sup.3 the mice were randomly separated into 4 groups (n=5). Group 1 involved untreated animals, group 2 the PDT only group, Group 3 the 1-CaO.sub.2 NPs only group and group 4 the PDT+1-CaO.sub.2 NPs group. For group 2, mice received an intratumoural injection (100 L) of Rose Bengal (0.1 mg/mL) in a PBS solvent and the tumour was then exposed to a LED emitting white light for 3 min (0.05 J/cm.sup.2). Group 3 received a tail vein injection (100 L) of 1-CaO.sub.2 NPs in a PBS vehicle (2 mg/mL) while group 4 also received a tail vein injection (100 L) of 1-CaO.sub.2 NPs in a PBS vehicle (2 mg/mL) in addition to an intratumoural injection (100 L) of Rose Bengal (0.1 mg/mL) and light exposure as described for group 2. The tumour volume was measured daily over the course of 6 days using callipers.

[0145] The results for tumour volume are shown in FIG. 9a. The results demonstrate that there was no significant difference in tumour volume for mice treated with PDT alone or with 1-CaO.sub.2 NPs alone 5 days after treatment, relative to the untreated control animals. In contrast, a significant reduction (p<0.0005) of 70.5% was observed for animals treated with the 1-CaO.sub.2 NPs and PDT over the same time period. In addition, there was no significant change in body weight in animals treated with the 1-CaO.sub.2 NPs alone or in combination with PDT suggesting the treatment was well tolerated (FIG. 9b).

[0146] These results highlight the benefit of 1-CaO.sub.2 NPs in improving the PDT-mediated treatment of hypoxic tumours such as pancreatic adenocarcinoma. The same approach would be expected to enhance other therapies that depend on ROS generation to provide a therapeutic effect, such as SDT and radiotherapy.