SONODYNAMIC THERAPY

Abstract

The invention provides a method of preparing a microbubble covalently attached to at least one therapeutic agent which comprises: (i) providing a lipid (e.g. a phospholipid) capable of forming a microbubble; covalently linking at least one therapeutic agent to said lipid to produce a functionalised lipid; and preparing a microbubble from said functionalised lipid. Microbubble-therapeutic agent complexes which comprise a microbubble shell formed from a plurality of lipids (e.g. phospholipids) in which at least a proportion of the lipids are covalently linked to at least one therapeutic agent are also provided. Examples of therapeutic agents which may be attached to the microbubble include chemotherapeutic agents and sonosensitising agents. The complexes find use in methods of sonodynamic therapy and, in particular, in methods of combined sonodynamic therapy and chemotherapy.

Claims

1. A method of preparing a microbubble covalently attached to at least one therapeutic agent, said method comprising the following steps: providing a lipid capable of forming a microbubble; covalently linking at least one therapeutic agent to said lipid whereby to produce a functionalised lipid; and preparing a microbubble from said functionalised lipid.

2. A method as claimed in claim 1, wherein said lipid is selected from the group consisting of phospholipids, fatty acids, triglycerides, diglycerides, monoglycerides, sterols and sterol derivatives, sphingolipids, and combinations thereof.

3. A method as claimed in claim 1, wherein said lipid is a phospholipid, preferably a phospholipid selected from any of the following: phosphatidylcholines, e.g. dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, and 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC); phosphatidic acids; phosphatidylethanolamines, e.g. dioleoylphosphatidylethanolamine, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE); phosphatidylserines; phosphatidylglycerols, e.g. diphosphatidylglycerols such as cardiolipin; and phosphatidylinositols.

4. A method as claimed in claim 3, wherein said phospholipid is a compound of formula (I), or a pharmaceutically acceptable salt thereof: ##STR00013## wherein: R.sup.1 and R.sup.2, which may be the same or different, are either saturated, or mono- or polyunsaturated, C.sub.10-30 acyl groups, for example —CO—C.sub.10-25 alkyl or —CO—C.sub.10-25 alkenyl groups; and R.sup.3 is ##STR00014##

5. A method as claimed in claim 3, wherein said phospholipid is selected from 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and combinations thereof.

6. A method as claimed in any one of the preceding claims, wherein the microbubble is prepared from a mixture of said functionalised lipid and one or more other lipids which are linked to one or more biocompatible polymers or polysaccharides, e.g. polyethylene glycol (PEG).

7. A method as claimed in any one of the preceding claims, wherein said functionalised lipid is produced by enzyme-catalysed phosphatidylation of a hydroxyl-containing therapeutic agent.

8. A method as claimed in claim 7, wherein said functionalised lipid is a compound of formula (II), or a pharmaceutically acceptable salt thereof: ##STR00015## wherein R.sup.1 and R.sup.2, which may be the same or different, are as defined in claim 4; and Y is the residue of a therapeutic agent.

9. A method as claimed in any one of claims 1 to 6, wherein the step of covalently linking at least one therapeutic agent to said lipid whereby to produce said functionalised lipid comprises the following steps: chemical modification of said lipid to form a modified lipid; and subsequent covalent linkage of said modified lipid to said therapeutic agent.

10. A method as claimed in claim 9, wherein said lipid is a phospholipid and the step of chemical modification of said lipid comprises reacting the lipid with succinic anhydride, or a derivative thereof.

11. A method as claimed in claim 10, wherein said functionalised lipid is a compound of formula (III), or a pharmaceutically acceptable salt thereof: ##STR00016## wherein R.sup.1 and R.sup.2, which may be the same or different, are as defined in claim 4; and Y is the residue of a therapeutic agent.

12. A method as claimed in any one of the preceding claims, wherein the step of preparing a microbubble from said functionalised lipid comprises high speed mixing or sonication of an aqueous solution comprising said functionalised lipid and one or more stabilising agents.

13. A method as claimed in any one of the preceding claims, wherein said therapeutic agent is selected from a chemotherapeutic agent, a sonosensitising agent, and a combination of such agents.

14. A method as claimed in claim 13, wherein said chemotherapeutic agent is selected from the following: antifolates (e.g. methotrexate); 5-fluoropyrimidines (e.g. 5-fluorouracil or 5-fluorouridine); cytidine analogues (e.g. gemcitabine); purine antimetabolites (e.g. mercaptopurine); alkylating agents (e.g. cyclophosphamide); non-classical alkylating agents (e.g. dacarbazine); platinum analogues (e.g. cisplatin); antitumour antibiotics (e.g. actinomycin D, bleomycin, mitomycin C); bioreductive drugs (e.g. mitomycin C, Banoxantrone (AQ4N)); anthracyclines (e.g. doxorubicin, mitoxantrone); topoisomerase I inhibitors (e.g. irinotecan); topoisomease II inhibitors (e.g. etoposide); antimicrotubule agents such as vinca alkaloids (e.g. vincristine), taxols (e.g. paclitaxel), and epothilones (e.g. ixabepiline); antioestrogens (e.g. tamoxifen); antiandrogens (e.g. biclutamide, cyproterone acetate); aromatase inhibitors (e.g. anastrazole, formestan); antiangiogenic or hypoxia targeting drugs (either naturally occuring, e.g. endostatin, or synthetic, e.g. gefitinib, lenalidomide); antivascular agents (e.g. cambretastatin); tyrosine kinase inhibitors (e.g. gefitinib, erlotinib, vandetanim, sunitinib); oncogene or signalling pathway targeting agents (e.g. tipfarnib, lonafarnib, naltrindole, rampamycin); agents targeting stress proteins (e.g. geldanamycin and analogues thereof); autophagy targeting agents (e.g. chloroquine); proteasome targeting agents (e.g. bortezomib); telomerase inhibitors (targeted oligonucleotides or nucleotides); histone deacetylase inhibitors (e.g. trichostatin A, valproic acid); DNA methyl transferase inhibitors (e.g. decitabine); alkyl sulfonates (e.g. busulfan, improsulfan and piposulfan); aziridines (e.g. benzodopa, carboquone, meturedopa, and uredopa); ethylenimines and methylamelamines (e.g. altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine); nitrogen mustards (e.g. chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard); nitrosureas (e.g. carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine); purine analogues (e.g. fludarabine, 6-mercaptopurine, thiamiprine, thioguanine); pyrimidine analogues (e.g. ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine); androgens (e.g. calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone); anti-adrenals (e.g. aminoglutethimide, mitotane, trilostane); immune checkpoint inhibitors (e.g. the PD-1/PDL-1 interaction inhibitors BMS-1001 and BMS-1166); immune response modifiers (e.g. imiquimod and resiquimod); and pharmaceutically acceptable salts, derivatives or analogues of any of these compounds.

15. A method as claimed in claim 14, wherein the chemotherapeutic agent is an anti-metabolite, e.g. 5-fluorouracil, 5-fluorouridine, or gemcitabine.

16. A method as claimed in any one of claims 13 to 15, wherein said sonosensitising agent is selected from the group consisting of phenothiazine dyes (e.g. methylene blue, toluidine blue), Rose Bengal, porphyrins (e.g. Photofrin®), chlorins, benzochlorins, phthalocyanines, napthalocyanines, porphycenes, cyanines (e.g. Merocyanine 540 and indocyanine green), azodipyromethines (e.g. BODIPY and halogenated derivatives thereof), acridine dyes, purpurins, pheophorbides, verdins, psoralens, hematoporphyrins, protoporphyrins and curcumins.

17. A method as claimed in any one of the preceding claims which further comprises the step of loading a hydrophobic chemotherapeutic agent into the hydrophobic tail region of the lipid, preferably wherein said hydrophobic chemotherapeutic agent is an anti-microtubule agent, e.g. a taxol such as paclitaxel (PTX).

18. A method as claimed in any one of the preceding claims, wherein the microbubble comprises a shell which retains a gas selected from perfluorobutane, perfluoropropane, and oxygen.

19. A method as claimed in any one of the preceding claims, wherein the microbubble has a diameter in the range of from 0.05 to 100 μm.

20. A microbubble-therapeutic agent complex obtained or obtainable by a method as claimed in any one of the preceding claims.

21. A microbubble-therapeutic agent complex which comprises a microbubble shell formed from a plurality of lipids (e.g. from a plurality of phospholipids), wherein at least a proportion of said lipids are covalently linked to at least one therapeutic agent.

22. A pharmaceutical composition comprising a microbubble-therapeutic agent complex as claimed in claim 20 or claim 21, together with at least one pharmaceutical carrier or excipient.

23. A microbubble-therapeutic agent complex or a pharmaceutical composition as claimed in any one of claims 20 to 22 for use as a medicament.

24. A microbubble-therapeutic agent complex or a pharmaceutical composition as claimed in any one of claims 20 to 22 for use in a method of sonodynamic therapy, preferably for use in a method of combined sonodynamic therapy and chemotherapy.

25. A microbubble-therapeutic agent complex or a pharmaceutical composition for use as claimed in claim 24 in the treatment of cancer, metastasis or micrometastasis derived from said cancer, or in the treatment of circulating tumour cells (CTCs), preferably in the treatment of a deep-sited tumour such as pancreatic cancer.

Description

[0127] The invention will now be described further with reference to the following non-limiting Examples and the accompanying figures in which:

[0128] FIG. 1 is a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) spectrum of “Lipid-Gem” (m/z 500-2000).

[0129] FIG. 2 shows stacked .sup.1H NMR spectra of 1,2-dibehenoyl-sn-glycero-3-phosphocholine standard (top), gemcitabine standard (middle) and “Lipid-Gem” (bottom) recorded in a CDCl.sub.3/MeOD mixed solvent system.

[0130] FIG. 3 shows (a) a representative optical micrograph of “Lipid-Gem” microbubbles (1:25 dilution, ×40 magnification); and (b) representative size distribution analysis of images of “Lipid-Gem” microbubbles.

[0131] FIG. 4 shows (a) a representative optical micrograph of “Lipid-Gem+DSPE-PEG (2000)+PTX” microbubbles (1:25 dilution, ×40 magnification); and (b) representative size distribution analysis of images of “Lipid-Gem+DSPE-PEG (2000)+PTX” microbubbles.

[0132] FIG. 5 shows representative images of human pancreas ductal adenocarcinoma cell line (Panc-1) spheroids treated with: (a) no treatment +(left) and −(right) ultrasound; (b) “Lipid-Gem” microbubbles ([Lipid-Gem]=10 μM) +(left) and −(right) ultrasound; and (c) “Lipid-Gem+PTX” microbubbles ([Lipid-Gem]=10 μM, [PTX]=6.2 μM)+(left) and −(right) ultrasound.

[0133] FIG. 6 is a plot of cell viability of the Panc-1 spheroids shown in FIG. 5 treated with: (a) no treatment; (b) “Lipid-Gem” microbubbles ([Lipid-Gem]=10 μM); and (c) “Lipid-Gem+PTX” microbubbles ([Lipid-Gem]=10 μM, [PTX]=6.2 μM) in the absence (grey bar) and presence (black bar) of ultrasound.

[0134] FIG. 7 is a plot of tumour growth in mice bearing BxPC-3 tumours that were either: (i) untreated (upward triangles); or treated with (ii) 100 μL I.V injection of “Lipid-Gem” microbubble suspension plus ultrasound (circles); (iii) 100 μL I.V injection of “Lipid-Gem+PTX” microbubble suspension plus ultrasound (squares); (iv) 100 μL I.P injection of Gem HCl (120 mg/kg) (filled diamonds); or (v) 100 μL of IP injection of Gem HCl (120 mg/kg)+Free PTX (15 mg/kg) (hollow diamonds).

[0135] FIG. 8 is a plot of % change in body weight for animals that were either: (i) untreated (triangles); or treated with (ii) 100 μL I.V injection of “Lipid-Gem” microbubble suspension plus ultrasound (circles); (iii) 100 μL I.V injection of “Lipid-Gem+PTX” microbubble suspension plus ultrasound (squares); (iv) 100 μL I.P injection of Gem HCl (120 mg/kg) (filled diamonds); or (v) 100 μL of I.P injection of Gem HCl (120 mg/kg)+Free PTX (15 mg/kg) (hollow diamonds).

[0136] FIG. 9 shows stacked .sup.1H NMR spectra of 1,2-dibehenoyl-sn-glycero-3-phosphocholine standard (top), 5-flourouridine standard (middle) and “Lipid-5FUR” (bottom).

[0137] FIG. 10 is a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) spectra of “Lipid-5FUR” (m/z 199-2000) in negative mode.

[0138] FIG. 11 is a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) spectra of “Lipid-Gem amide (18:0)” (m/z 199-2000) in negative mode. The base peak at 1090.33 corresponds to the [M−H].sup.− ion.

[0139] FIG. 12 shows the .sup.1H NMR spectra of “Lipid-Gem amide (18:0)”.

[0140] FIG. 13 is a plot of cell viability of PANC-1 cells treated with (a) gemcitabine hydrochloride (0-40 μM) and (b) DSPE-Gem (0-40 μM).

[0141] FIG. 14 shows chemical structures of (a) DSPC; (b) DSPE-RB; (c) DSPE-Gem; (d) DSPE-PEG (2000); (e) cholesterol. Images of (f) pre-microbubble lipid suspension and (g) freshly prepared microbubble suspension. 3D schematic representation of (h) Gem-RB-MB.

[0142] FIG. 15 is an optical microscope image of Gem-RB-MBs.

[0143] FIG. 16 is a size distribution analysis of Gem-RB-MBs.

EXAMPLES

Example 1—Preparation of Gemcitabine-functionalised microbubbles

[0144] 1.1 Synthesis of “Lipid-Gem”

[0145] A Gemcitabine-functionalised lipid (“Lipid-Gem”) was produced by reaction of gemcitabine hydrochloride and 1,2-dibehenoyl-sn-glycero-3-phosphocholine in a biphasic emulsion of chloroform and aqueous buffer (200 mM sodium acetate, 200 mM calcium chloride). The reaction was catalysed through the addition of phospholipase D to the aqueous buffer prior to mixing.

##STR00007##

[0146] A chloroform solution (20 mL) of 1,2-dibehenoyl-sn-glycero-3-phosphocholine (500 mg, 480 μmol) was added to a stirred solution of gemcitabine hydrochloride (400 mg, 1.5 mmol) and phospholipase D from Streptomyces sp (3 mg, 900 units) in sodium acetate buffer (200 mM, pH 4.5, 5 mL) containing calcium chloride (200 mM). The mixture was stirred vigorously at 45° C. for 6 hours after which a solution containing chloroform (10 mL) and methanol (15 mL) was added. The organic layer was separated and the aqueous layer washed twice with a chloroform/methanol mixture (2:1). The organic extracts were combined, dried using anhydrous sodium sulphate, filtered and concentrated in vacuo to yield a waxy, off-white solid. The crude product was purified using preparative thin layer chromatography (chloroform:methanol:(7N) ammonium hydroxide, 65:25:4, Rf −0.41) to give “Lipid-Gem” (25% yield).

[0147] Formation of “Lipid-Gem” was confirmed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) spectroscopy and .sup.1H NMR. The MALDI-TOF spectrum of “Lipid-Gem” (m/z 500-2000) is shown in FIG. 1. The base peak at 1084.7758 corresponds to the [M+Na].sup.+ ion. The peak at 1062.7800 corresponds to the [M+H].sup.+ ion. The peak at 1106.7753 corresponds to the [M+2Na—H].sup.+ ion. FIG. 2 shows the stacked .sup.1H NMR spectra of 1,2-dibehenoyl-sn-glycero-3-phosphocholine standard (top), gemcitabine standard (middle) and “Lipid-Gem” (bottom) recorded in a CDCl.sub.3/MeOD mixed solvent system. The resonances corresponding to the choline moiety present on the phospholipid polar head group (3.2 ppm) and the neighbouring methylene protons (3.6 ppm) shown in the top spectrum are no longer present in the spectrum corresponding to “Lipid-Gem” indicating a successful cleavage of the choline group. In addition, characteristic protons from gemcitabine such as the two aromatic ring protons which appear at 5.9 and 7.8 ppm respectively and the 3′ hydroxyl group which appears at 5.2 ppm are clearly visible in the spectrum corresponding to “Lipid-Gem”.

[0148] 1.2 Preparation of Perfluorobutane (PFB) Loaded “Lipid-Gem” Microbubbles

[0149] “Lipid-Gem” (5 mg, 4.71 μmol) was dissolved in a mixture of chloroform and methanol (2:1, 100 L) and then placed in a vacuum oven at 40° C. for 1 hour to allow the solvent to evaporate. The dried lipid film was rehydrated in a mixture of PBS, glycerol and propylene glycol (8:1:1, 2 mL) and stirred at 90° C. for 1 hour. The liposomal suspension was then sonicated using a probe sonicator at power setting 25% for 1 minute. The suspension was then further sonicated at power setting 90% under an atmosphere of perfluorobutane gas for 30 seconds to form a milky-white microbubble suspension. This suspension was transferred to a centrifuge tube and centrifuged (5 min, 100 rcf). The infranatant was discarded and the microbubbles were then re-suspended in a mixture of PBS, glycerol and propylene glycol (8:1:1) and used immediately.

[0150] 1.3 Preparation of PFB Loaded “Lipid-Gem+Paclitaxel (PTX)” Microbubbles

[0151] “Lipid-Gem” (5 mg, 4.71 μmol), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (1.43 mg, 0.51 mol) and paclitaxel (2.5 mg, 2.93 μmol) were dissolved in a mixture of chloroform and methanol (2:1, 100 μL) and then placed in a vacuum oven at 40° C. for 1 hour to allow the solvent to evaporate. The dried lipid film was rehydrated in a mixture of PBS, glycerol and propylene glycol (8:1:1) and stirred at 90° C. for 1 hour. The liposomal suspension was then sonicated suing a probe sonicator at power setting 25% for 1 minute. The suspension was then further sonicated at power setting 90% under an atmosphere of perfluorobutane gas for 30 seconds to form a milky white microbubble suspension. This suspension was transferred to a centrifuge tube and centrifuged (5 min, 100 ref). The infranatant was discarded and the microbubbles were then re-suspended in a mixture of PBS, glycerol and propylene glycol (8:1:1) and used immediately.

Example 2—Determination of Mean Diameter and Concentration of Microbubbles Produced in Example 1

[0152] A sample of freshly prepared microbubble suspension (10 μL) was diluted in PBS (990 μL) and a sample of this suspension (10 μL) was loaded into a haemocytometer chamber. Using an optical microscope (×40 objective), 20 images of the microbubbles were taken. The microbubble size distribution and concentration was then determined through image analysis using ImageJ software. The brightfield image was converted to 8-bit greyscale before an automated threshold strategy was applied. Particle diameter was then calculated relative to the scale bar present in the brightfield image.

[0153] FIG. 3 shows (a) a representative optical micrograph of the “Lipid-Gem” microbubbles (1:25 dilution, ×40 magnification); and (b) a representative size distribution analysis of images of the “Lipid-Gem” microbubbles. The mean concentration of the microbubbles was 1.8×10.sup.9+1.5×10.sup.8 microbubbles/mL. The mean diameter of the microbubbles was determined to be 2.4±1.9 μm.

[0154] FIG. 4 shows (a) a representative optical micrograph of the “Lipid-Gem+DSPE-PEG (2000)+PTX” microbubbles (1:25 dilution, ×40 magnification); and (b) representative size distribution analysis of images of “Lipid-Gem+DSPE-PEG (2000)+PTX” microbubbles. The mean concentration of the microbubbles was 1.7×10.sup.9+1.1×10.sup.8 microbubbles/mL. The mean diameter of the microbubbles was determined to be 2.3±1.7 μm.

Example 3—In Vitro Cytotoxicity of “Lipid-Gem” Microbubbles and “Lipid-Gem+PTX” Microbubbles in Panc-1 Spheroids

[0155] The human primary pancreatic carcinoma cell line PANC-1 was maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 1 g/L glucose and supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% fetal bovine serum (FBS). Cells were incubated at 37° C. in a humidified atmosphere with 5% CO.sub.2. Spheroids were prepared by seeding 2000 cells (200 μL) into a pre-coated 96-well plate (60 μL 1.5% agarose per well). Cells were incubated for 5 days to allow for spheroid formation. The media in each well was then replaced with either fresh media, “Lipid-Gem” microbubbles ([Lipid-Gem]=10 μM) or “Lipid-Gem+PTX” microbubbles ([Lipid-Gem]=10 μM, [PTX]=6.2 μM). Selected wells were then treated individually with ultrasound (Sonidel SP100 sonoporator, 30 s, frequency—1 MHz, ultrasound power density—3.0 W/cm.sup.2, duty cycle—40%). Two days after initial treatment spheroids were washed four times with PBS. Spheroids were then placed into Eppendorf tubes and re-suspended in 90 μL of media. To this suspension was added 10 μL of MTT (5 mg/mL in PBS) and the cells incubated for 3 hours. Each tube was then centrifuged and the supernatant was discarded. The pellet was then dissolved in DMSO (100 μL) and the contents of each tube was transferred to a 96 well plate. The absorbance of the formazan dye metabolite was measured at 570 nm using a plate reader.

[0156] Results are shown in FIG. 5 by way of representative images of the Panc-1 spheroids. There was no apparent effect on spheroid morphology when the spheroids were treated with ultrasound in the absence of microbubbles. Spheroids treated with the “Lipid-Gem” microbubbles with no ultrasound stimulus also did not show any degradation in morphology. However, upon application of the ultrasound stimulus, the tightly packed spheroid structure began to show signs of degradation with small areas of cell debris becoming apparent. This effect was further amplified when PTX was added to the formulation. Significant structural damage is apparent in spheroids treated with the “Lipid-Gem+PTX” microbubbles in the absence of ultrasound stimulus. The most evidential morphological degradation of spheroids occurred upon ultrasound treatment of spheroids treated with the “Lipid-Gem+PTX” microbubbles.

[0157] FIG. 6 shows the cell viability of the Panc-1 spheroids in FIG. 5. No statistically significant cell death was observed in spheroids treated with either “Lipid-Gem” microbubbles or “Lipid-Gem+PTX” microbubbles in the absence of ultrasound. However, a statistically significant decrease in cell viability of 38% was observed for spheroids treated with “Lipid-Gem” microbubbles plus ultrasound. A further 31% decrease in cell viability was observed when spheroids were treated with “Lipid-Gem+PTX” microbubbles plus ultrasound.

Example 4—In Vivo Cytotoxicity of “Lipid-Gem” Microbubbles and “Lipid-Gem+PTX” Microbubbles in a BxPC-3 Xenograft Model

[0158] All animals in these studies were treated humanely and in accordance with licensed protocols under the Animals (Scientific Procedures) Act 1986 (UK). BxPc-3 cells were maintained in RPMI 1640 media supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% fetal bovine serum (FBS) in a humidified 5% CO.sub.2 atmosphere at 37° C. Cells (1×10.sup.6) were re-suspended in Matrigel® and implanted subcutaneously into the rear dorsum of SCID (C.B-17/IcrHan@Hsd-Prkdcscid) mice. Tumours reached treatable size within 3 weeks. Tumour measurements were taken daily using callipers. Once the tumours had reached an average volume of 150 mm.sup.3 animals were randomly assigned into treatment groups. Animals were anaesthetised using isoflurane with medical grade oxygen as the carrier gas. Animals were treated with a 100 μL I.V. injection of either “Lipid-Gem” microbubble ([Lipid-Gem]=70.1 μg/10.sup.8 MB) or “Lipid-Gem+PTX” microbubbles ([Lipid-Gem]=75.1 μg/10.sup.8 MB, [PTX]=21.3 μg/10.sup.8 MB). As control groups, animals were treated with a 100 μL I.P injection of either (i) gemcitabine hydrochloride (120 mg/kg) or (ii) gemcitabine hydrochloride (120 mg/kg) and PTX (15 mg/kg). Ultrasound was applied directly to the tumour immediately following injection at an ultrasound frequency of 1 MHz, an ultrasound power density of 3.5 W/cm.sup.2 and a duty cycle of 30% for 3.5 min. Tumour volume was measured using callipers and calculated using the formula: V=L×W×H/2.

[0159] Results are shown in FIGS. 7 and 8. The tumour growth plot in FIG. 7 reveals that the scaled clinical dose of Gem HCl significantly controls tumour growth progression. “Lipid-Gem” microbubbles without PTX showed significant improvement in tumour growth control compared to Gem HCl. The greatly enhanced tumour inhibitory effect delivered by treatment with the “Lipid-Gem” microbubbles clearly demonstrates one of the advantages of the invention since the amount of Gem in this formulation is some 120-fold lower than that administered to the group treated with the clinical dose of Gem (i.e. Gem-HCl). There was a further small but non-significant decrease in tumour growth progression for mice treated with “Lipid-Gem+PTX” microbubbles compared to animals treated with non-PTX containing microbubbles. The plot in FIG. 8 of % change in body weight against time during treatment shows that no significant reduction in body weight was observed in any animal from any of the treatment groups. In contrast, administration of an equivalent “control” for the combination of free Gem HCl and PTX (i.e. a scaled clinical dose) was found to result in a significant drop in body weight of the animals. This confirms the microbubble delivery of Gem and PTX in accordance with the invention is improved compared to a conventional treatment and well tolerated.

Example 5—Preparation of 5-Flourouridine-Functionalised Lipid

[0160] A 5-fluorouridine-functionalised lipid (“Lipid-5FUR”) was produced by reaction of 5-fluorouridine and 1,2-dibehenoyl-sn-glycero-3-phosphocholine in a biphasic emulsion of chloroform and aqueous buffer (200 mM sodium acetate, 200 mM calcium chloride). The reaction was catalysed through the addition of phospholipase D to the aqueous buffer prior to mixing.

##STR00008##

[0161] A chloroform solution (20 mL) of 1,2-dibehenoyl-sn-glycero-3-phosphocholine (500 mg, 480 μmol) was added to a stirred solution of 5-flourouridine (395 mg, 1.5 mmol) and phospholipase D from Streptomyces sp (3 mg, 900 units) in sodium acetate buffer (200 mM, pH 4.5, 5 mL) containing calcium chloride (200 mM). The mixture was stirred vigorously at 45° C. for 6 hours after which a solution containing chloroform (10 mL) and methanol (15 mL) was added. The organic layer was separated, and the aqueous layer washed twice with a chloroform/methanol mixture (2:1). The organic extracts were combined, dried using anhydrous sodium sulphate, filtered and concentrated in vacuo to yield a white solid. The crude product was purified using preparative thin layer chromatography (chloroform:methanol:(7N) ammonium hydroxide, 65:25:4) to give “Lipid-5FUR”.

[0162] FIG. 9 shows the stacked .sup.1H NMR spectra of 1,2-dibehenoyl-sn-glycero-3-phosphocholine standard (top), 5-flourouridine standard (middle) and Lipid-5FUR (bottom). The resonances corresponding to the choline moiety present on the phospholipid polar head group (3.2 ppm) and the neighbouring methylene protons (3.6 ppm) shown in the top spectra are no longer present in the spectra corresponding to “Lipid-5FUR” indicating a successful cleavage of the choline group. In addition, characteristic protons from the uracil moiety of 5-flourouridine such as the imide proton which appears at 8.0 ppm and the aromatic ring proton which appears at 5.9 ppm are clearly visible in the spectra corresponding to “Lipid-5FUR”. FIG. 10 is a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) spectra of “Lipid-5FUR” (m/z 199-2000) in negative mode. The base peak at 1069.48 corresponds to the [M−H].sup.− ion. The peak at 603.30 peak is from the matrix.

Example 6—Preparation of Gemcitabine-Functionalised Lipid

[0163] ##STR00009##

[0164] 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (3) was used as the commercially available starting material. The primary amine of 3 was reacted with succinic anhydride in basic conditions to form an amide bond and terminal carboxylic acid group. In parallel, the 3- and 5-hydroxyl functionalities of 1 were protected with tert-butyl dimethyl silyl ethers by reacting 1 with TBDSM-Cl and imidazole in basic conditions. This was followed by an amidation reaction between the primary amine group of 2 and the pendant carboxylic acid of 4 to form 5. The tert-butyl dimethyl silyl ethers of the gemcitabine moiety of 5 were then deprotected using TBAF to yield “Lipid-Gem amide (18:0)”.

[0165] 6.1 Synthesis of TBDMS-Gem (2)

[0166] To a stirred solution of gemcitabine hydrochloride (1, 1 g, 3.4 mmol), imidazole (2.32 g, 34 mmol) and TBDMS-Cl (5.3 g, 20 mmol) in anhydrous dimethylformamide (50 mL) was added dropwise triethylamine (1.12 mL, 6.8 mmol). The solution was stirred at room temperature under a nitrogen atmosphere for 24 hours before which the solvent was removed in vacuo. The residue was taken up in aqueous sodium hydrogen carbonate (10% w/v) solution and extracted with ethyl acetate (5×100 mL). The organic extracts were combined and washed with aqueous sodium hydrogen carbonate (10% w/v) solution (1×50 mL) and brine (2×50 mL) before the solvent was removed in vacuo. The crude solid was purified using column chromatography (dichloromethane:methanol 9:1) to give 2 as a white crystalline solid (1.5 g, 91%).

[0167] 6.2 Synthesis of N-succinyl DSPE (4)

[0168] To a stirred solution of DSPE (3, 0.9 g, 1.2 mmol) and triethylamine (669 μL, 4.8 mmol) in chloroform (40 mL) was added succinic anhydride (0.15 g, 1.4 mmol). The solution was stirred under a nitrogen atmosphere for 24 hours after which the solvent was removed in vacuo. The crude residue was precipitated in ice cold acetone, filtered and washed thoroughly with ice cold acetone to give 4 (0.8 g 78%).

[0169] 6.3 Synthesis of TBDMS-Gem-N-DSPE (5)

[0170] To a stirred solution of 4 (0.7 g, 0.8 mmol) in anhydrous dichloromethane (40 mL) was added EDC HCl (0.29 g, 1.5 mmol) and HOBt (0.2 g, 1.5 mmol). This solution was stirred at room temperature under a nitrogen atmosphere for 30 minutes after which time 2 (0.43 g, 0.88 mmol) was added. The solution was stirred for a further 24 hours after which time the solvent was removed in vacuo. The residue was precipitated in ice cold acetone, filtered and washed thoroughly with ice cold acetone to yield 5 (0.6 g). This was used in the next step without further purification.

[0171] 6.4 Synthesis of DSPE-Gem (6)

[0172] To a stirred suspension of 5 (0.6 g, 0.45 mmol) in anhydrous tetrahydrofuran (20 mL) in an ice bath was added dropwise TBAF (1M in THF, 1.13 mL, 1.13 mmol). The suspension slowly dissolved over the course of 15 minutes and the ice bath was removed. The solution was stirred for a further 24 hours before the solvent was removed in vacuo. The residue was precipitated in ice cold acetone, filtered and washed thoroughly in ice cold acetone. The crude product was purified using preparative thin layer chromatography (chloroform:methanol:ammonium hydroxide (7N) 65:25:4) to give 6 (60% yield for last two steps).

[0173] FIG. 11 is a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) spectra of “Lipid-Gem amide (18:0)” (m/z 199-2000) in negative mode. The base peak at 1090.33 corresponds to the [M−H].sup.− ion. FIG. 12 shows the .sup.1H NMR spectra of “Lipid-Gem amide (18:0)”. The characteristic aromatic protons from the cytosine moiety of gemcitabine appear as two doublets at 6.10 and 8.15 ppm. The two sets of protons from the ethyl linker appear at 2.4 and 2.65 ppm.

Example 7—Preparation of Gemcitabine-Functionalised Lipid

[0174] ##STR00010##

[0175] An alternative gemcitabine-functionalised lipid may be prepared according to Scheme 4 above. This scheme utilises 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (10) as the commercially available starting material. The primary amine of 10 is reacted with succinic anhydride in basic conditions to form an amide bond and terminal carboxylic acid group. In parallel, the 3-hydroxyl and primary amine functionalities of 7 are protected with tert-butyl carbonate esters by reacting 7 successively with DBDC. This is followed by an esterification reaction between the 5-hydroxyl group of 9 and the pendant carboxylic acid of 11 to form 12. The tert-butyl carbonate esters of the gemcitabine moiety of 12 are then deprotected using TFA to yield “Lipid-Gem ester (18:0)” (13).

Example 8—Preparation of Gemcitabine-Functionalised Lipid

[0176] ##STR00011##

8.1 Synthesis of (2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl tert-butyl carbonate (2)

[0177] Di-tert-butyl dicarbonate (DBDC) (0.36 g, 1.65 mmol) was added to the solution of gemcitabine hydrochloride (1) (0.50 g, 1.67 mmol) and Na.sub.2CO.sub.3 (2.40 g, 8.49 mmol) in a mixture of dioxane and water (5:1, v/v, 10 ml) and the reaction was stirred at room temperature for 50 hours. Subsequent TLC analysis (DCM:acetone:ethanol, 5:4:1, v/v) showed almost complete consumption of gemcitabine. The reaction mixture was then diluted with water (100 mL) and extracted with ethyl acetate (3×30 mL). The combined organic extracts were washed with brine (20 mL), dried over sodium sulfate, filtered and concentrated to dryness under reduced pressure. The residue was washed with diethyl ether to afford compound 2 as a white solid (0.57 g, 95%). .sup.1H NMR (DMSO-d.sub.6): 7.65 (d, J=7.5 Hz, 1H, —CH), 7.39 (brs, 2H, —NH.sub.2), 6.15 (s, 1H, —CH), 5.91 (d, J=7. Hz, 1H, —CH), 5.19 (brs, 1H, —CH), 5.09 (brs, 1H, —OH), 4.09 (d, J=5.5 Hz, 1H, —CH), 3.89-3.79 (m, 2H, —CH.sub.2), 1.42 (s, 9H, —CH.sub.3×3). ESI-MS: calculated for C.sub.14H.sub.19F.sub.2N.sub.3O.sub.6=363.3; found=386.1 [M+Na].sup.+.

8.2 Synthesis of tert-butyl(1-((2R,4R,5R)-4-((tert-butoxycarbonyl)oxy)-3,3-difluoro-5-(hydroxymethyl) tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (3)

[0178] DBDC (3.00 g, 13.74 mmol) was added to a stirred solution of 2 (0.50 g, 1.38 mmol) in dioxane (25 mL) and the resulting mixture was stirred at 40° C. for 70 hours. Subsequent TLC analysis (DCM:Acetone:EtOH, 5:4:1, v/v) showed complete consumption of compound 2. The solvent was removed under reduced pressure and the residue was suspended in water (10 mL) and extracted with DCM (4×20 mL). The combined organic extracts were washed with brine (10 mL), dried over sodium sulfate, filtered and concentrated to dryness under reduced pressure. The crude compound was purified by column chromatography (DCM:Acetone, 5%-20%, v/v) to afford compound 3 as a white powder (0.41 g, 65%). .sup.1H NMR (CDCl.sub.3:MeOH, 2:1 v/v) δ: 8.12 (d, J=7.5 Hz, 1H, —CH), 7.35 (d, J=7.5 Hz, 1H, —CH), 6.39-6.29 (m, 1H, —CH), 5.19-5.11 (m, 1H, —CH), 4.18 (d, J=5.5 Hz, 1H, —CH), 3.99-3.79 (m, 2H, —CH.sub.2), 1.45 (s, 18H, CH.sub.3×6). .sup.13C NMR (CDCl.sub.3:MeOH, 2:1 v/v): 164.0 (C), 154.3 (CO), 152.3 (CO), 151.6 (CO), 145.1 (CH), 121.8 (C), 95.5 (CH), 84.2 (C), 81.7 (C), 79.3 (CH), 59.5 (CH.sub.2), 28.1 (CH.sub.3), 27.5 (CH.sub.3). ESI-MS: Calculated for C.sub.19H.sub.27F.sub.2N.sub.3O.sub.8=463.4; found=486.3 [M+Na].sup.+.

8.3 Synthesis of 4-(((2R,3R,5R)-5-(4-((tert-butoxycarbonyl)amino)-2-oxopyrimidin-1(2H)-yl)-3-((tert-butoxycarbonyl)oxy)-4,4-difluorotetrahydrofuran-2-yl)methoxy)-4-oxobutanoic acid (4)

[0179] To a stirred solution of 3 (1.63 g, 3.5 mmol) in anhydrous DCM (15 mL) was added TEA (1.42 g, 14.0 mmol) and this solution was stirred at room temperature for 10 minutes followed by the addition of succinic anhydride (0.70 g, 7.00 mmol). This solution was stirred at room temperature for 24 hours or until no starting material was visible by TLC (DCM:acetone:acetic acid, 6:2:0.05 v/v). Following completion of the reaction the solution was washed with distilled water (3×20 mL) and brine (3×20 mL) and dried with anhydrous sodium sulphate before removal of the solvent under reduced pressure. The resultant crude product was purified using column chromatography (DCM:acetone:acetic Acid, 6:2:0.05 v/v) to afford compound 4 as an off-white crystalline solid (1.65 g, 82%). .sup.1H NMR (DMSO-d6) δ: 12.25 (brs, 1H, —COOH), 10.51 (brs, 1H, —NH), 8.01 (d, J=7.5 Hz, 1H, —CH), 7.05 (d, J=7.5 Hz, 1H, —CH), 6.13 (m, 1H, —CH), 5.13 (brs, 1H, —CH), 4.55-4.15 (m, 3H, —CH, —CH.sub.2), 2.61-2.33 (m, 4H, —CH.sub.2×2), 1.55 (s, 18H, —CH.sub.3×6). .sup.13C NMR (CDCl.sub.3:MeOH, 2:1 v/v): 164.0 (CO), 154.3 (CO), 152.3 (C), 151.6 (CO), 145.1 (CH), 121.8 (C), 95.5 (CH), 84.2 (C), 81.7 (CH), 79.3 (CH), 59.5 (CH.sub.2), 39.6 (CH.sub.2), 28.1 (CH.sub.3), 27.5 (CH.sub.3). ESI-MS: Calculated for C.sub.23H.sub.31F.sub.2N.sub.3O.sub.11=563.5; found=562.0 [M−H].sup.−.

8.4 Synthesis of 3-(((2-(4-(((2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)-4-oxobutanamido)ethoxy)(hydroxy)phosphoryl)oxy) propane-1,2-diyl distearate (6)

[0180] To a stirred solution of DSPE (0.10 g, 0.13 mmol), 4 (0.09 g, 0.15 mmol) and HBTU (0.06 g, 0.15 mmol), in chloroform (15 mL), was added DIPEA (0.02 g, 0.15 mmol) and the solution was stirred under reflux at 45° C. for 24 hours until no starting material was visible by TLC (CHCl.sub.3:CH.sub.3OH, 8:2 v/v). After completion of the reaction the solvent was removed under reduced pressure to yield the resultant crude compound 5 (0.175 g, 0.13 mmol). To a solution of 5 in anhydrous DCM (10 mL) was added TFA (5 mL) and the resulting solution was stirred at 0° C. for 1 hour followed by further stirring at room temperature for 24 hours until no starting material was visible by TLC (CHCl.sub.3:CH.sub.3OH, 8:2 v/v). Following completion of the reaction, the solvent was removed under reduced pressure and the residue was dissolved in CHCl.sub.3:CH.sub.3OH (2:1 v/v) and washed with an aqueous sodium bicarbonate solution (10% w/v). The organic layer was separated and concentrated under reduced pressure. The resultant crude product was purified using column chromatography (DCM:MeOH 2:1 v/v) to afford compound 6 as a white powder (0.09 g, 61%). .sup.1H NMR (CDCl.sub.3:MeOH, 2:1) 6:7.53 (d, J=7.5 Hz, 1H, —CH), 6.28-6.25 (m, 1H, —CH), 6.01 (d, J=7.5 Hz, 1H, —CH), 5.24-5.22 (m, 1H, —CH), 4.45-4.39 (m, 4H, —CH×2, —CH.sub.2), 4.19-3.89 (m, 6H, —CH.sub.2×3), 3.40 (t, 2H, —CH.sub.2), 2.73-2.67 (m, 2H, —CH.sub.2), 2.59-2.56 (m, 2H, —CH.sub.2), 2.33-2.29 (m, 4H, —CH.sub.2×2), 1.60-1.59 (m, 4H, —CH.sub.2×2), 1.28-1.26 (m, 56H, —CH.sub.2×28), 0.87 (t, 6H, —CH.sub.3×2). .sup.13C NMR (CDCl.sub.3:MeOH, 2:1v/v): 173.9 (CO), 173.5 (CO), 173.4 (CO), 172.2 (CO), 165.9 (C), 156.3 (CO), 140.7 (CH), 124.0 (C), 121.8 (CH), 95.7 (CH), 84.0 (CH), 78.3 (CH), 70.3 (CH), 69.9 (CH.sub.2), 70.3 (CH.sub.2), 63.8 (CH.sub.2), 63.3 (CH.sub.2), 62.5 (CH.sub.2), 62.4 (CH.sub.2), 61.7 (CH.sub.2), 40.3 (CH.sub.2), 34.1 (CH.sub.2), 33.8 (CH.sub.2), 31.8 (CH.sub.2), 31.6 (CH.sub.2), 30.2 (CH.sub.2), 29.5 (CH.sub.2), 29.4 (CH.sub.2), 29.2 (CH.sub.2), 29.0 (CH.sub.2), 28.8 (CH.sub.2), 24.7 (CH.sub.2), 24.6 (CH.sub.2), 22.5 (CH.sub.2), 22.3 (CH.sub.2), 13.7 (CH.sub.3), 13.6 (CH.sub.3). MALDI-TOF-MS: Calculated for C.sub.54H.sub.95F.sub.2N.sub.4O.sub.14P=1093.34; found=1091.45 [M−H].sup.−.

Example 9—Preparation of Rose Bengal-Functionalised Lipid

[0181] ##STR00012##

9.1 Synthesis of 8-((2,3,4,5-tetrachloro-6-(6-hydroxy-2,4,5,7-tetraiodo-3-oxo-3H-xanthen-9-yl)benzoyl)oxy)octanoic acid (7)

[0182] To a stirred solution of Rose Bengal disodium salt (10.00 g, 9.80 mmol) in anhydrous DMF (100 mL) was added 8-bromooctanoic acid (2.20 g, 9.80 mmol) and the solution was stirred at 80° C. for 8 hours or until no starting material was visible by TLC (CHCl.sub.3:CH.sub.3OH 8:2 v/v). Following completion of the reaction, the solvent was removed under reduced pressure and the residue was stirred in diethyl ether (200 mL) for 24 hours after which time the suspension was filtered and the solids were stirred in distilled water (200 mL) for 24 hours. This suspension was then filtered and dried to afford the carboxylic acid derivative 8 as a deep purple powder (8.70 g, 80%). .sup.1H NMR (DMSO-d.sub.6) δ: 7.49 (s, 2H, aromatic-CH×2), 3.89 (brs, 2H, O—CH.sub.2—), 2.09 (brs, 2H, —CH.sub.2—COOH), 1.39 (brs, 2H, —CH.sub.2—), 1.21-1.12 (m, 6H, —CH.sub.2—×3), 0.89 (brs, 2H, —CH.sub.2—). ESI-MS: Calculated for C.sub.28H.sub.18Cl.sub.4I.sub.4O.sub.7=1115.86; found=1114.47 [M−H].sup.−.

9.2 Synthesis of 3-((hydroxy(2-(8-((2,3,4,5-tetrachloro-6-(6-hydroxy-2,4,5,7-tetraiodo-3-oxo-3H-xanthen-9-yl)benzoyl)oxy)octanamido)ethoxy) phosphoryl)oxy)propane-1,2-diyl distearate (8)

[0183] To a stirred solution of DSPE (0.50 g, 0.60 mmol), 8 (0.90 g, 0.80 mmol) and HBTU (0.30 g, 0.8 mmol) in CHCl.sub.3:CH.sub.3OH:H.sub.2O (65:35:8 v/v) was added DIPEA (0.43 g, 3.3 mmol) and the solution was stirred at room temperature for 24 hours or until no starting material was visible by TLC (CHCl.sub.3:CH.sub.3OH 8:2 v/v). After completion of the reaction the solvent was removed under reduced pressure and the resultant crude product was purified using column chromatography (CHCl.sub.3:CH.sub.3OH 8:2 v/v) to afford 9 as a deep purple powder (0.63 g, 43%). .sup.1H NMR (CDCl.sub.3:CD.sub.3OD, 2:1 v/v) δ: 7.74 (s, 2H, aromatic-CH×2), 5.25-5.23 (m, 1H, —CH), 4.19-4.15 (m, 2H, —OCH.sub.2—), 4.01-3.95 (m, 6H, —CH.sub.2×3), 3.53-3.51 (m, 2H, CH.sub.2), 3.36-3.35 (m, 2H, —CH.sub.2), 2.35-2.23 (m, 6H, —CH.sub.2×3), 1.61-1.50 (m, 6H, —CH.sub.2×3), 1.36-1.08 (m, 62H, —CH.sub.2×31), 0.90-0.87 (m, 6H, —CH.sub.3×2). .sup.13C NMR (CDCl.sub.3:CD.sub.3OD, 2:1): 175.8 (CO), 173.9 (CO), 173.5 (CO), 173.2 (C), 163.3 (CO), 157.8 (CH), 150.8 (C), 141.5 (C), 136.8 (C), 135.6 (C), 132.1 (C), 130.3 (C), 129.5 (C), 112.8 (C), 101.6 (C), 96.2 (C), 80.1 (C), 79.3 (C), 77.0 (CH.sub.2), 75.9 (CH.sub.2), 72.0 (CH.sub.2), 49.4 (CH.sub.2), 49.0 (CH.sub.2), 40.0 (CH.sub.2), 36.0 (CH.sub.2), 34.1 (CH.sub.2), 33.9 (CH.sub.2), 31.7 (CH.sub.2), 31.3 (CH.sub.2), 29.5 (CH.sub.2), 29.2 (CH.sub.2), 29.0 (CH.sub.2), 28.6 (CH.sub.2), 28.0 (CH.sub.2), 25.0 (CH.sub.2), 24.7 (CH.sub.2), 22.5 (CH.sub.2), 13.7 (CH.sub.3). MALDI-TOF-MS: Calculated for C.sub.69H.sub.98Cl.sub.4I.sub.4NO.sub.14P=1845.93; found=1844.31 [M−H].sup.−.

Example 10—Determination of the Efficacy of DSPE-Gem in PANC-1 Cells

[0184] PANC-1 cells were maintained in high glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and incubated at 5% CO2 at 37° C. Cells were seeded (3×10.sup.3) in a 96 well plate, the following day cells were treated with several concentrations of gemcitabine hydrochloride or DSPE-Gem prepared according to Example 8 (0, 5, 10, 20 and 40 μM). Cell viability was determined 48 hours later using an MTT assay. Results are shown in FIG. 13.

Example 11—Preparation and Characterisation of Rose Bengal and Gemcitabine Loaded Microbubbles (“RB-Gem-MB”)

[0185] Preparation of RB-Gem-MB:

[0186] To a 5 mL round bottom flask was added DBPC (1.02 mg, 1.13 μmol), DSPE-Gem prepared according to Example 8 (1.03 mg, 0.94 μmol), DSPE-RB prepared according to Example 9 (1.74 mg, 0.94 μmol), DSPE-PEG-2000 (1.06 mg, 0.38 μmol) and cholesterol (0.15 mg, 0.38 μmol) dissolved in a mixture of chloroform and methanol (2:1 v/v) to achieve a total lipid concentration of 5 mg/mL and a molar ratio of 3:2.5:2.5:10:10. The solvent was then removed using a rotary evaporator (45° C., speed setting 10) to yield a thin lipid film. Further drying of the film was carried out by placing the flask in a vacuum oven (20° C.) for 2 hours. The dried film was then hydrated by adding 1 mL of a mixture of PBS, glycerol and propylene glycol (8:1:1 v/v) (PGP) and agitating the suspension on a rotary evaporator (85° C., 45 min) in the dark. The resultant turbid lipid suspension was then sonicated using a probe sonicator (amplitude 25%, 30 seconds) to yield a transparent suspension of lipid vesicles. Once cooled to room temperature the suspension (1 mL) was added to a 3 mL crimp vial and the headspace of the vial was replaced with PFB gas and sealed. The vials were then mechanically agitated using a Vialmix device (45 seconds) to produce a purple suspension of RB-Gem-MBs. A 3D representation of the resulting “RB-Gem-MB” is shown in FIG. 14.

[0187] Characterisation of RB-Gem-MB:

[0188] An appropriate volume of freshly prepared MB-RB-Gem was diluted in cold PGP. A 10 μL sample was then loaded into the viewing chamber of a haemocytometer and viewed under a microscope (×40 objective). Microscope images (n=20) were collected and saved as high resolution TIFF files. These images were then analysed using a bespoke MATLAB algorithm to give the distribution of MB diameters and mean MB concentration. Results are shown in FIGS. 15 and 16.