Technical field The present invention relates generally to methods and materials for use in magnetic resonance imaging (MRI), and in particular for use in saturation transfer mediated imaging, for example of tumours and the like.

Background art

The current standard method for imaging tumours in vivo is [ ¹⁸ F]FDG-PET. The method relies on the administration of [ ¹⁸ F]FDG to patients which is relatively expensive and delivers a major radiation dose, which is undesirable for repeated scanning. Saturation transfer mediated imaging methods represent an alternative to [ ¹⁸ F]FDG-PET.

Several methods exist to detect saturation transfer-related changes in magnetization. One method is chemical exchange saturation transfer (CEST), in which the exchangeable pool is saturated using an off-resonance pulse. A variation of the off resonance CEST technique is the use of an off-resonance spin-lock technique, which in practice does not change the outcome. For rapidly exchanging protons such as hydroxyl groups present in glucose and the like, an on-resonance spin-lock technique can be used, providing an image which is Ti p-weighed. By choosing the amplitude of the spin-lock pulse, one can adjust the sensitivity of the technique to a specific exchange rate regime. Finally, T2- weighted imaging has also been shown to be usable to detect changes due to an increase in fast exchanging protons such as hydroxyl protons and the like.

GlucoCEST is an imaging technique that enables visualisation of natural, non-radioactive glucose ¹ .

GlucoCEST utilizes two properties of hydroxyl protons: first, when exposed to a magnetic field, the magnetic moments of hydroxyl protons precess at a different frequency as those of bulk tissue water and can therefore be selectively labeled using radiofrequency pulses; second, hydroxyl and water protons undergo exchange, thereby allowing magnetic labeling to be transferred from glucose to water and for glucose to be detected from the change in water signal in the MRI images.

The CEST technique thus provides an amplification of detection by using the very large water signal rather than relying on the much smaller signal from glucose. GlucoCEST can therefore provide higher resolution imaging than the existing standard method,

[ ¹⁸ F]FDG-PET, whilst avoiding the expense and radiation dose to the patient associated with the latter technique.

US20160095945A1 relates to the use of non-labeled sugars and detection by MRI for assessing tissue perfusion and metabolism. Summarv of the invention

The present inventors have noted that glucose, or related analogues, when used as CEST or related saturation transfer mediated imaging methods will interact with the body glucose sensing system.

This problem is acknowledged in the art. For example Chan et al. (2012) Magnetic resonance in medicine 68.6 (2012): 1764-1773 discusses the possible use of simple D- glucose as an infusable biodegradable MRI agent for cancer detection. With respect to disadvantages, the authors note that glucose tests may cause hypoglycaemia and glucose may not be suitable for some populations, for instance patients with diabetes. The suggested solution is that glucose monitoring may be used, which in practice leads to additional lines being used in patients while in the scanner, and is therefore undesirable. Nevertheless since each patient will have a different metabolic response to the introduced sugars, this will impact on the reproducibility of the GlucoCEST MRI method.

That will be the case even in healthy volunteers. However the problem is exacerbated in patient populations having abnormal glucose responses.

In addition, as cancer is typically more prevalent in ageing populations, a significant number of patients will not be able to undergo a GlucoCEST MRI exam due to insulin resistance or diabetes Thus it can be seen that imaging methods which avoid the drawbacks of [ ¹⁸ F]FDG-PET, while mitigating problems with glycaemic responses, would provide a contribution to the art.

The present inventors have developed a novel series of imaging agents, for use in saturation transfer mediated imaging methods, based on liposome-encapsulated sugar compounds.

For brevity the term "CEST" is used herein in relation to these methods and compositions. Nevertheless it will be understood that, unless context demands otherwise, any disclosure in relation to CEST applies mutatis mutandis to the other saturation transfer mediated imaging methods (T1 rho or T2-weighted imaging). Furthermore, the encapsulation of any of these sugar compounds in the liposome may be referred herein for brevity a "LipoGluco-CEST" but likewise will be understood to refer mutatis mutandis to the other sugar compounds described herein.

The methods and materials of the invention can not only improve circulation time of the imaging agents, but also aid in protecting the patient from the effects of administration of the sugar, and avoid triggering an acute insulin response. This allows for high resolution GlucoCEST imaging of tumours, metabolic imaging and "dose painting" for radiotherapy available to all patients, and mitigates the variability in the imaging caused by the glycaemic response. The inventors have further investigated the effects of modifying the physical and chemical properties of the liposomes employed in the methods, for example using kinds of lipid constituents, and have demonstrated, unexpectedly, that lipids comprising n-EG or PEG head groups could be formulated into "shielded" liposomes which nevertheless gave excellent CEST signals. The use of these or other modified lipids additionally provides the ability to achieve enhanced cell-targeting, for example based on targeting peptides.

Thus the agents of the invention have particular utility in measurements of glucose or other sugar uptake in tumours, either through the enhanced permeability and retention (EPR) effect or via tumour cell targeting on the liposomal surface.

The invention also provides for the use of sugar compound (e.g. glucose analogue) - loaded liposomes to provide simultaneous tumour imaging and chemotherapy by targeting areas of greater tumour metabolism. In the examples below, the CEST signal arising from 'free' 2-deoxy-D-glucose (2-DG; a well-characterized glycolytic inhibitor that has been shown to inhibit tumour growth in vivo) was compared to liposome- encapsulated 2-DG and to natural D-glucose, respectively. The results showed it was possible to achieve an increase in signal for 2-DG loaded liposomes when compared to both free 2-DG and glucose. This increase in signal adds to the versatility of the LipoCEST techniques, since it means that GlucoCEST may be performed at lower field strengths, and hence may be more clinically accessible. Although the use of nanoparticles or liposomes has previously been discussed in relation to diaCEST agents, these disclosures were not specifically directed towards GlucoCEST and did not concern mitigation of the glycaemic response (see e.g. US20160206760A1 ; Chan et al (2014) Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology 6.1 : 1 1 1 -124; WO 2014/124006; Chan et al (2014) J Control Release: 180:51 -9; WO 2013/158719; US20150133768A1 ; WO2016036735A1 ). Several of these disclosures are focussed on the use of liposomal doxorubicin, with barbituric acid as a diaCEST agent.

Detailed disclosure of the invention

In one aspect the invention provides an agent for use in a method of saturation transfer mediated imaging, such as chemical exchange saturation transfer (CEST) imaging, in a subject, which agent comprises a liposome encapsulating a sugar compound, as defined herein wherein: the liposome is equal to or between 10 to 500 nm in diameter, and the concentration of sugar compound encapsulated in the liposome equal to or between 5 and 100 mM, and the sugar compound is encapsulated in an aqueous solution of pH between 5 and 8.

The method for imaging may comprise administering the agent to the subject and imaging the agent using magnetic resonance imaging as described above. The agent may be administered in an effective amount. The imaging may be performed using convention magnetic resonance imaging and any scanners have appropriate magnetic field strengths e.g. a 1 .5T MRI scanner, a 3.0T MRI scanner, or higher etc.

As explained below, the agent may be used theranostically.

Modulation of glycaemic response

In the present invention the liposome encapsulation can be used to modulate glycaemic response in the subject i.e. the agent may be used for this purpose.

"Modulate" in this context means reducing the effect of the glycaemic response in the subject on the concentration of the sugar compound. For example an acute injection of glucose may lead to transient hyperglycaemia, followed by hypoglycaemia due to the triggered acute insulin response. Modulation here has the effect of reducing variability between different imaging events in a given subject, leading to an enhanced replicability, or between different subjects in a cohort or group, leading to an enhanced reproducibility.

Thus in some embodiments the subject may be one known to have abnormal glucose response. In some embodiments the subject may be one known to have a normal glucose response. The liposome encapsulated agent may be used to reduce variability between subjects

Liposomal encapsulation of the sugar compound (e.g. 2-DG) will therefore mean that elderly patients (who are at higher risk of suffering from type 2 diabetes as well as cancer) and patients with Type 1 diabetes will be able to benefit from effective and more reproducible glucoCEST imaging. Other variability in the glycaemic response arising from e.g. presence or not of comorbidities affecting the general metabolism, muscle mass, general health, age, and pharmaceutical use (e.g. of steroids) will also be mitigated.

Thus in example embodiments of the invention, the subject may be over 60 years old, may have Type 1 or Type 2 diabetes, and\or may be one who is being administered (i.e. prescribed a course of) steroids. In embodiments of the invention, the agent may be used in methods in which the subject or subject group is actively selected in relation to their glycaemic response - for example may be a subject or subject group which is known, diagnosed, or believed to have one or more of the aforementioned characteristics. Sugar compounds

The present invention has particular utility for sugar compounds which are capable of generating a glycaemic response in the subject via triggering a glucose sensing response (see e g. Thorens, B. "Glucose sensing and the pathogenesis of obesity and type 2 diabetes." International journal of obesity 32 (2008): S62-S71 ; Thorens, B. "Brain glucose sensing and neural regulation of insulin and glucagon secretion." Diabetes, Obesity and Metabolism 13.s1 (201 1 ): 82-88.).

As explained above, the sugar compound will be one suitable for diaCEST i.e. having labile protons which are exchangeable with bulk water.

The sugar compound may be one which is subject to enhanced uptake by tumours, such as glucose which is typically taken up by a glucose transporter (see e.g. Vander Heiden, M. G., Cantley, L. C, & Thompson, C. B. (2009). Understanding the Warburg effect: the metabolic requirements of cell proliferation, science, 324(5930), 1029-1033.).

In the Examples below, the inventors have investigated the CEST signals from a variety of sugar compounds, including both monosaccharides and disaccharides,

unmodified and modified (analogue) sugars, and sugars which are known to be potentially beneficial in anti-cancer treatment regimes, or which are of purely diagnostic value.

Unmodified sugars In embodiments of the invention the sugar compound may be a suitable unmodified sugar such as a hexose (e.g. allose, altrose, glucose, mannose, gulose, idose, galactose, talose) or pentose (aldopentoses and ketopentoses). A further example is fructose.

In embodiments of the invention the sugar compound may be a suitable

monosaccharides as discussed above, or a disaccharides (e.g. sucrose, maltose).

In one embodiment the sugar compound is D-glucose.

Such sugars may be used in the present invention to provide suitable CEST signals for imaging e.g. of tumours.

Modified sugars

In embodiments of the invention the sugar compound may be a suitable modified sugar. For example the sugar compound may be a sugar analogue e.g. a glucose analogue. Such analogues may have particular utility in therapeutic or theranostic applications.

Therefore in one embodiment the sugar compound is a D-glucose analogue. In one embodiment the glucose analogue is L-glucose, or an acetate derivative thereof (e.g. L-glucose pentaacetate).

In one embodiment the glucose analogue is 2-deoxy-D-glucose (2-DG; see Figure 2). 2-DG has been shown to be an effective radiosensitizer in vitro, ³ and has also been proposed as a very potent, and longer-lasting, GlucoCEST agent, ^{4 5} . However, it cannot currently be used in humans due to its toxicity, ⁶ and 2-DG has also been shown to trigger the insulin response in vivo ⁷ .

As explained herein, these properties of 2-DG means it can be advantageously employed in the present invention within liposomes for simultaneous imaging for radiotherapy dosing of tumours, targeting of radiation therapy and radiosensitisation.

Example glucose analogues are shown below and include, but not limited to L-glucose, 6- DG, 3-OMG, and FDG:

Glucose 2-deoxyglucose (2-DG) 6-deoxyglucose (6-DG) 3-O-methylglucose (3-OMG) FDG

Conjugated sugars or sugar analogues

As noted above, sugar compounds for use in the invention may be those which have been identified as having potential use in anti-cancer therapies. These include not just sugar analogues, but also conjugates (see e.g. S. R. Punganru et al., Bioorg Med Chem Lett 26, 2829 (2016); C. Granchi et al., MedChemCommun., 7, 1716 (2016); Y.-L Jiang et al., Chem. Biol. Drug. Des., 86, 1017 (2015). Thus the invention also embraces use of e.g. glucose when conjugated to a further therapeutic moiety.

Non-limiting examples are as follows:

Examples may also include conjugates of disaccharides, for example:

Brartemicin is a trehalose-derived metabolite which may have utility as an inhibitor of cancer cell invasion.

Those skilled in the art will readily appreciate, in the light of the present disclosure, that other modified and unmodified sugars, or conjugates thereof, will have utility in the diagnostic or theranostic methods described herein. Lipids for use in liposomes

Suitable lipids for the preparation of liposomes, and methods for preparing liposomes, are described hereinafter. Many lipids are related to ethanolamine, phosphoethanolamine, choline, phosphocholine, and glycerol, shown below.

Two common classes of phospholipids are the phosphatidylethanolamines and the phosphatidylcholines, shown below, wherein R ¹ and R ² denote hydrocarbon chains, for example, derived from fatty acids R ¹ -C(=0)OH and R ² -C(=0)OH, respectively.

Common "saturated" fatty acids are shown in the following table.

Common Name Chemical Formula Code Corresponding Alkyl Cx capric acid CH ₃ (CH ₂ ) ₈ COOH 10:0 CH ₃ (CH ₂ ) ₈ - Cg undecylic acid CH ₃ (CH ₂ ) ₉ COOH 1 1 :0 CH ₃ (CH ₂ ) ₉ - Cio lauric acid CH ₃ (CH ₂ )ioCOOH 12:0 CH ₃ (CH ₂ )io- Cii tridecylic acid CH ₃ (CH ₂ )iiCOOH 13:0 CH ₃ (CH ₂ )ii- Ci ₂ myristic acid CH ₃ (CH ₂ )i ₂ COOH 14:0 CH ₃ (CH ₂ )i ₂ - Ci ₃ pentadecanoic acid CH ₃ (CH ₂ )i ₃ COOH 15:0 CH ₃ (CH ₂ )i ₃ - Ci ₄ palmitic acid CH ₃ (CH ₂ )i ₄ COOH 16:0 CH ₃ (CH ₂ )i ₄ - Cl5 margaric acid CH ₃ (CH ₂ )i ₅ COOH 17:0 CH ₃ (CH ₂ )i5- Cl6 Common Name Chemical Formula Code Corresponding Alkyl C _x stearic acid CH ₃ (CH ₂ )i ₆ COOH 18:0 CH3(CH2)l6- Cl7 nondecylic acid CH ₃ (CH ₂ )i7COOH 19:0 CH3(CH2)l7- Cl8 arachidic acid CH ₃ (CH ₂ )i ₈ COOH 20:0 CH3(CH2)l8- Cl9

Where the term "comprises" is used herein in relation to lipids within liposomes, it is to be understood to mean that the lipid in question is one of the lipids forming the lipid bilayer of the liposome, either alone or in combination with other lipids.

By way of non-limiting example, the inventors have shown that high concentrations of sugar compounds can be encapsulated inside 1 ,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC) liposomes to produce vesicular contrast agents (Fig 2) that generate an appreciable CEST signal.

The inventors have further formulated liposomes including lipids with PEG at the headgroup (MW 2000 - 4000) or lipids with n-EG units at the head-group (MW 900 - 1500). These "shielded" lipids provide a steric coating on the surface of the membrane to hinder clearing of the particles by the reticuloendothelial system (RES). This prolongs the circulating plasma half-life of the drug. The plasma half-life of the drug can be "tuned" from several hours to days depending on the size of the PEG and fatty acids attached to the lipid anchor. In many "standard" liposomal formulations, this is achieved by including 5-10% of lipids such as PEG2000-DSPE in the formulation (N. Bertrand et al., Adv. Drug Deliv. Rev., 66, 323 (2014)).

The inventors have noted that liposomes bearing large polymeric PEG moieties often have greatly reduced cellular uptake and targeting selectivity (L. Sun et al., Coll. Surf. B Biointerfaces, 135, 56 (2015)).

Cationic lipids with smaller n-EG units at the head group have recently been shown to provide formulations with prolonged in vivo circulation and also excellent cellular uptake when they reach the tumour target (M. F. Mohd Mustapa et ai, Bioconj. Chem., 20, 518 (2009); J. B. Wong et al., Mol. BioSyst, 4, 532 (2008); N. Mitchell et al., Biomaterials, 34, 1 179 (2013); see also US 7,598,421 and US 9,399,016)

However, n-EG phospholipids have not been previously reported and their effects on liposome stability and lipoCEST signal have not been studied.

The inventors have investigated liposomes which comprises an n-EG lipid based on the DPPC structure, such as 3-({hydroxy[(17-hydroxy-4-oxo-6,9,12, 15-tetraoxa-3- azaheptadecyl)oxy]phosphoryl}oxy)propane-1 ,2-diyl dipalmitate (DPPE-EG4) (Fig 2) DPPE-EG4 has been formulated into Lipo 2DG-CEST reagents with similar CEST contrast to Lipo2DG-CEST reagents formulated with DPPC alone or with the industry standard, PEG2000-DPPC. Unexpectedly, these liposomes gave excellent CEST signals when encapsulating sugar compounds.

Thus in embodiments of the invention the liposome may comprise a phospholipid, such as a phosphocholine lipid.

In embodiments of the invention the liposome may comprise DPPC.

In embodiments of the invention the liposome may comprise 1 ,2-distearoyl-sn-glycero-3- phosphocholine (DSPC).

In some embodiments the lipid may comprise n-ethylene glycol (n-EG), polyethylene glycol (PEG) at the head group. The liposome may comprise a mixture of lipids, including n-EG lipids and unmodified lipids (i.e. lipids not comprising n-EG or PEG).

The liposome may comprise e.g. up to 50% n-EG lipid, e.g. about 10% to 40%, e.g. about 30% n-EG lipid.

The liposome may comprise e.g. 50% or more DPPC e.g. about 90% to 60%, e.g. about 70% DPPC.

As explained in more detail below, cell-targeting peptides may optionally be attached to the exterior of the liposomes, via the n-EG or PEG lipids.

Preferred n-EG lipids

One aspect of the present invention relates to phosholipids which are polyethyleneglycol- modified phosphatidylethanolamines of Formula 1 , and their uses in the methods and compositions described herein:

Formula 1 wherein:

-R ^FA1 is the hydrocarbon chain of a corresponding fatty acid R ^FA1 -C(=0)OH;

-R ^FA2 is the hydrocarbon chain of a corresponding fatty acid R ^FA2 -C(=0)OH;

n is an integer from 1 to 10;

X is -OH, -NH ₂ , or -Q;

-Q is -L ¹ -A or -L ² -Pep; -L ¹ - is a linking group;

-A is a reactive conjugating group;

-L ² - is a linking group; and

-Pep is a peptide group.

In one embodiment:

-R ^FA1 is a linear or branched saturated C9-19 alkyl group; and -R ^FA2 is a linear or branched saturated C9-19 alkyl group.

In one embodiment:

-R ^FA1 is a linear or branched saturated Cn-17 alkyl group; and -R ^FA2 is a linear or branched saturated Cn-17 alkyl group.

In one embodiment:

-R ^FA1 is a linear saturated Cn-17 alkyl group; and

-R ^FA2 is a linear saturated Cn-17 alkyl group.

In one embodiment:

-R ^FA1 is a linear saturated Cn , C13, C15, or C17 alkyl group; and -R ^FA2 is a linear saturated Cn , C13, C15, or C17 alkyl group.

In one embodiment, -R ^FA1 and -R ^FA2 are the same.

In one embodiment, -R ^FA1 and -R ^FA2 are different.

In one embodiment, n is an integer from 1 to 8.

In one embodiment, n is an integer from 1 to 6.

In one embodiment, n is an integer from 2 to 10.

In one embodiment, n is an integer from 2 to 8.

In one embodiment, n is an integer from 2 to 6.

In one embodiment, X is -OH.

For example, in one embodiment, the polyethyleneglycol-modified phosphatidylethanolamine has the following formula:

O H In a more specific embodiment, the polyethyleneglycol-modified

phosphatidylethanolamine has the following formula (wherein each of -R ^FA1 and -R ^FA2 is a linear saturated C15 alkyl group (as found in palmitic acid); n is 4; and X is -OH), conveniently denoted "DPPE-EG4-OH"):

In one embodiment, X is -NH2. In one embodiment, X is -Q.

In one embodiment, -Q, if present, is -L ¹ -A.

In one embodiment, the reactive conjugating group, -A, is suitable for reaction with a sulfhydryl group (-SH), for example, as found in peptides, for example as a cysteine residue. This is described in more detail below.

Properties of liposomes Typically the liposomes described herein are equal to or between 10 to 500 nm in diameter, and the concentration of sugar compound encapsulated in the liposome is equal to or between 5 and 100 mM, and the sugar compound is encapsulated in an aqueous solution of pH equal to or between 5 and 8. In some embodiments the liposomes described herein are equal to or between 50 and 300 nm in diameter e.g. 150 nm to 250 nm e.g. about 200 nm in diameter.

In some embodiments the liposomes described herein the concentration of sugar compound encapsulated in the liposome equal to or between 15 and 60 mM, e.g.

between 20 and 40 mM.

In some embodiments the sugar compound is encapsulated in an aqueous solution of pH between pH 5.7 to 6.2 e.g. pH 5.8 to 6.0. The inventors have shown that under physiologically relevant conditions (20% PBS, pH 5.8 (mimicking the acidic microenvironment of the tumors), 37 °C) liposomes having these properties are stable for at least 24 hr with minimal (4%) leakage of the sugar.

Furthermore, as demonstrated in the Examples, and summarised in the Table below, the liposome-encapsulated LipoGluco-CEST reagent can produce a larger CEST contrast than free glucose, and the Lipo2DG-CEST reagent can produce a larger CEST contrast than free 2-DG The table shows the CEST signal (herein defined as the standard measure of the asymmetry in the magnetization transfer ratio MTR _aS ym) from glucose and 2-DG liposomes and the free sugar controls with equal overall concentration at pH 5.8 and 20 °C or 37 °C. Signal is expressed as the average percentage water suppression caused by hydroxyl group saturation in the range 0-3.75 ppm.

Interestingly, both free 2-DG and the Lipo2DG-CEST reagent produce inherently larger CEST contrasts than free or liposome-encapsulated glucose, despite possessing one less exchangeable hydroxyl proton.

Methods and uses of imaging The CEST imaging methods and uses of the present invention may be for the purpose of metabolic imaging.

The CEST imaging methods and uses of the present invention may be for the purpose of tumour imaging.

The CEST imaging methods and uses of the present invention may be for the purpose of perfusion imaging.

The CEST imaging methods and uses of the present invention may be for the purpose of imaging of the glucose transport intracellularly.

The CEST imaging methods and uses of the present invention may be for the purpose of imaging of the glycolytic rate in an individual. Tumour imaging may be for the purpose of distinguishing tumour types, or for "dose painting", defined as the process to use the parts of tumours with the largest metabolism as targets for radiotherapeutic interventions.

The agents of the present invention have particular utility in tumour imaging and theranostics.

For example, based on the results described herein, as the extracellular tumour microenvironment is more acidic than normal tissue and blood (6.0 - 7.0, vs 7.4) when the sugar compound (e.g. 2-DG) liposomes reach the tumour, the CEST signal may be significantly enhanced. ln one embodiment the agents of the invention may be used to distinguish tumour types with differing metabolic characteristics and pathophysiologies - for example based on differential tumour glucose accumulation e.g. in colorectal cancers. Liposomal agents of the present invention may be expected to preferentially accumulate in tumor tissue over normal tissues via the enhanced permeability and retention (EPR) effect (see e.g. Maeda, Hiroshi, et al. "Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review." Journal of controlled release 65.1 (2000): 271- 284; N. Bertrand et al., Adv. Drug Deliv. Rev., 66, 323 (2014))

Enhanced tumour targeting - means for peptide conjugation

Additionally, tumour selectivity can be enhanced by the presence of cell-targeting peptides to the exterior of the liposomes (abbreviated to "pep" in some of the formulae hereinafter).

This can be conveniently achieved using the lipids including a reactive conjugating group, for example present on the n-EG or PEG group. By way of example, in the aspects described above, X may be -Q. In one

embodiment, -Q, if present, is -L ¹ -A. In one embodiment, the reactive conjugating group, -A, is suitable for reaction with a sulfhydryl group (-SH), for example, as found in peptides, for example as a Cys residue, which may optionally be at the terminus of a cell- targeting peptide.

Examples of such reactive conjugated groups include: maleimide groups; iodoacetyl groups; pyridyldithiol groups; vinylsulfone groups; and thiosulfonate groups. Examples of conjugation reactions using such groups are shown below.

iodoacetyl sulfhydryl conjugate

reagent on protein (thioether bond)

pyridylthiol sulfhydryl conjugate

pyridine-2-thione reagent on protein (disulfide bond)

thiosulfonate sulfhydryl conjugate

reagent on protein (disulfide bond)

vinyls ulfone sulfhydryl conjugate

reagent on protein (β-thiosulfonyl) For example, in one embodiment, the group -L ¹ -A comprises a maleimide group, and has the following formula: For example, in one embodiment, the polyethyleneglycol-modified

phosphatidylethanolamine has the following formula:

In a more specific embodiment, the polyethyleneglycol-modified

phosphatidylethanolamine has the following formula (wherein each of -R ^FA1 and -R ^FA2 is a linear saturated C15 alkyl group (as found in palmitic acid); n is 4; and X is -L ¹ -A as described above):

In one embodiment, the reactive conjugating group, -A, is suitable for reaction with an amino group (-NH2), for example, as found in peptides. Examples of such reactive conjugated groups include: N-hydroxysuccinimide (NHS) ester groups; Sulflo-N-hydroxysuccinimide (NHS) ester groups; pentafluorophenyl ester groups; and hydroxymethylphosphine groups. Examples of conjugation reactions using such groups are shown below.

N-hydroxysuccinimide (NHS) ester amine conjugate

reagent on protein (amide bond) + Sulfo-NHS

sulfo-A/-hydroxysuccinimide (NHS) ester amine conjugate

reagent on protein (amide bond)

pentafluorophenyl ester amine conjugate

reagent on protein (amide bond)

hydroxymethyl phosphine amine conjugate

reagent on protein (amine bond)

In one embodiment, the reactive conjugating group, -A, is suitable for reaction with a hydroxyl group (-OH), for example, as found in peptides.

Examples of such reactive conjugated groups include: isocyanate groups. An example of a conjugation reaction using such groups is shown below.

isocyanate hydroxyl conjugate

reagent on protein (carbamate)

In one embodiment, -Q, if present, is -L ² -Pep. For example, in one embodiment, the group -L ² -Pep comprises a group derived from a maleimide group, and has the following formula:

For example, in one embodiment, the polyethyleneglycol-modified

phosphatidylethanolamine has the following formula:

In a more specific embodiment, the polyethyleneglycol-modified

phosphatidylethanolamine has the following formula (wherein each of -R ^FA1 and -R ^FA2 is a linear saturated C15 alkyl group (as found in palmitic acid); n is 4; and X is -L ² -P as described above):

Tumour targeting peptides

The tumour targeting peptide may be any known in the art, and may be selected by those skilled in the art according to the required tumour target in which the invention is to be applied.

Preferred lipid-peptide conjugates are those bearing peptide sequences such as

YHWYGYTPQNVI, LARLLT and CAEYLR, which are known to bind to the EGFR receptor which is overexpressed on several types of tumor cells (see e.g. Z. Li et al., FASEB J 19, 1978 (2005); S. Song et al., FASEB J 23, 1396 (2009); C.-Y. Han et al. Int. J. Nanomedicine S, 1541 (2013)).

Purely by way of non-limiting embodiments, the peptide group (sometimes referred to as -Pep herein) may be selected from

Receptor Peptide sequence Ref

IL-1 1 Ra GCRRAGGSC 1

GRP 78 SNTRVAP 1 Bombesin receptors D-Tyr-Gln-Trp-Ala-Val-bAla-His-Phe-Nle-NH2 2,3

(bombesin and other analogues)

Somostatin receptors FCFWKTCT-ol (Octreotide and other analogues) 4

Integrin receptors C(RGDyK) (and other RGD peptides) 5

1. V. J. Rao et al., J. Cont. Rel., 240, 267 (2016)

2. P. Moreno et al., Exp. Opin. Ther. Targets, 20, 1055 (2016)

3. H. Zhang et al., Cancer Res., 64, 6707 (2004)

4. R. Mikolajczak, H. R. Maecke, Nuc. Med. Rev., 19, 126 (2016)

5. X. Wang et al., J. Cont. Rel., 193, 139 (2014)

As explained above, any of these may include an additional Cys residue at the N- terminus to facilitate conjugation to the lipid (e.g. via a maleimide).

Lipids comprising cell targeting peptides may optionally be additional to any of the phospholipids or shielded lipids described above. For example lipid-peptide conjugates may be those shown below in Formula 2 or Formula 3. Any of these conjugates may be e co-formulated with mixtures of DPPC and n-EG- or PEG- lipids to afford cell-targeted shielded liposomes.

Formula 2

Formula 3 Theranostics

Liposomal agents of the present invention may be employed as theranostic agents. As explained above liposomes can be localized at the tumour via the EPR effect, and optionally other cell targeting agents.

As explained above, 2DG is a highly effective glycolytic inhibitor agent, and has been shown to be an effective radiosensitizer in vitro, through disruption of the thiol metabolism (X. Lin et al., Cancer Res., 63, 3413 (2003). 2DG has also been proposed as a very potent, and longer-lasting, GlucoCEST agent (F. A. Nasrallah et al., J. Cereb. Blood Flow Metab., 33, 1270 (2013); M. Rivlin et al., Sci. Rep., 3, 3045 (2013). 2-DG may therefore have utility for "dose painting" in which CEST imaging of 2-DG is used to define areas within the tumour which may require a higher radiation dose, related to tumour metabolism, and the tumor is also radiosensitized by the administration of 2DG. However, 2DG itself cannot be used as a radiosensitizing agent in humans due to its toxicity: 2DG would be barely detectable by GlucoCEST at the levels needed to avoid severe secondary effects (L.E. Raez, et al., Cancer Chemother Pharmacol 71 , 523 (2013)).

The use of liposomal delivery can mitigate toxicity arising from off-target effects.

Therefore, for example, 2-DG loaded liposomes can be employed as theranostic agents for the simultaneous imaging and treatment of tumors. An example use is adjuvant therapy, in which the agents are employed for radiosensitisation prior to radiotherapy. Example cancers which usually involve radiotherapy as part of their standard of care include colorectal cancer, and head and neck squamous cell carcinoma.

Other cancers which can be effectively treated with radiation therapy can be found here: https://www.cancer.gov/about-cancer/treatment/types/radiatio n-therapy.

and include brain, chest, pelvis and so on, The combination of the characteristics of the agents of the invention thus allow for high levels of sensitivity and resolution, thereby permitting dose painting to be performed during radiotherapy planning.

In some embodiments the agents may also include other anticancer agents co- encapsulated with the sugar compound e.g. along with the 2-DG or glucose.

However in some embodiments the agents may not include any other anticancer agents co-encapsulated with the sugar compound. Typically, the agent is used in an effective amount. An "effective amount", as used herein in relation to diagnostics or theranostics, is an amount being fit for the purpose intended. Thus for diagnostics this will be sufficient to generate a CEST signal. For theranostics this will be sufficient to show benefit to the individual, optionally in combination with other therapies as described herein. The actual amount administered, and rate and time- course of administration, will depend on the subject and the intervention in hand.

Decisions on dosage etc. are within the responsibility of general practitioners and other medical doctors, and will typically take account of the purpose of the imaging and\or disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners.

Other products of the invention

In another aspect of the present invention there is provided a CEST imaging agent comprising: a liposome, encapsulating a sugar compound, which is 2-DG. Also provided is use of this 2-DG agent as a theranostic, which is optionally targeted to the tumour, as described above. For example for radiosensitization prior to radiotherapy.

Also provided are n-EG phospholipids of Formula 1 above. Examples include DPPE- EG4-OH as described herein. As shown in the Examples, these n-EG phospho lipids can provide stabilised liposomes should provide prolonged in vivo circulation and with improved tumor targeting in vivo, compared to liposomes formulated with small amounts of DSPE-PEG2000, while at the same time providing a lipoCEST signal comparable to liposomes formulated without any form of shielding (such as 100% DPPC liposomes).

This is quite unexpected since PEG or n-EG shielding might reasonably have been expected to slow down the exchange of water across the lipid membrane.

Also provided are lipid-peptide conjugates such as those shown in Formula 2 or Formula 3 above.

Also provided are liposomes comprising these n-EG phospholipids and\or lipid-peptide conjugates In another aspect of the present invention there is provided a population of agents as described herein, and use of the same in the methods of the invention.

Other aspects In another aspect of the present invention there is provided use of an agent which comprises a liposome encapsulating a sugar compound, which compound is selected from glucose or a glucose analogue, wherein: the liposome is equal to or between 10 to 500 nm in diameter, and the concentration of sugar compound encapsulated in the liposome equal to or between 5 and 100 mM, and the glucose is encapsulated in an aqueous solution of pH between 5 and 8, in a method of saturation transfer mediated imaging, such as chemical exchange saturation transfer (CEST) imaging, in a subject, which is optionally theranostic imaging, in a subject.

In another aspect of the preset invention there is provided a method for saturation transfer mediated imaging, such as chemical exchange saturation transfer (CEST) imaging, in a subject, which is optionally theranostic imaging, in a subject, which method comprises administering an agent of the invention to the subject and imaging the agent in the subject using magnetic resonance imaging. In another aspect of the present invention there is provided use of an agent of the invention in the preparation of diagnostic, which is optionally a theranostic, reagent for use in the methods described herein.

In another aspect of the present invention there is provided use of a liposome to modulate a glycaemic response in a subject to a reagent for saturation transfer mediated imaging, such as chemical exchange saturation transfer (CEST) imaging, in a subject, which reagent is a sugar compound selected from glucose or a glucose analogue, said liposome being used to encapsulate said sugar compound.

Preferably the liposome is as described herein e.g. equal to or between 10 to 500 nm in diameter, and the concentration of sugar compound encapsulated in the liposome equal to or between 5 and 100 mM, and the sugar compound is encapsulated in an aqueous solution of pH between 5 and 8.

Further description of liposomes

As explained hereinbefore, the present invention utilises liposomes to deliver sugar compounds for imaging.

Liposomes and methods of preparing them are described herein.

Further examples of liposomes and methods of preparing them are known in the art, and are summarised (by way of non-limiting example) in WO2014/124006 or WO2016036735.

Briefly, liposomes are vesicles or particles which possess a lipid bilayer enclosing an aqueous compartment. In the present invention the sugar compound is encapsulated within the aqueous compartment. The lipids may be natural or synthetic lipids.

Liposomes composed of natural phospholipids are biologically inert and weakly immunogenic, and they possess low intrinsic toxicity. Liposomes can be classified according to their lamellarity (uni-, oligo-, and multi-lamellar vesicles), size (small, intermediate, or large) and preparation method (such as reverse phase evaporation vesicles, VETs).

Unilamellar vesicles comprise one lipid bilayer and generally have diameters of 50-250 nm. They contain a large aqueous core and are the preferred type of liposome used herein. As used herein the term "SUV" refers to a small unilamellar liposome vesicle (SUV) having a single lipid bilayer.

Lipids for liposomes

Selection of the appropriate lipids for liposome composition is governed by the factors of: (1 ) liposome stability, (2) phase transition temperature, (3) charge, (4) non-toxicity to mammalian systems, (5) encapsulation efficiency, (6) lipid mixture characteristics. The vesicle-forming lipids preferably have two hydrocarbon chains, typically acyl chains, and a head group, either polar or nonpolar. The hydrocarbon chains may be saturated or have varying degrees of unsaturation. For the present invention saturated chains are preferred.

As explained hereinbefore, there are a variety of synthetic vesicle-forming lipids and naturally-occurring vesicle-forming lipids, including the sphingolipids, ether lipids, sterols, phospholipids, particularly the phosphoglycerides, and the glycolipids, such as the cerebrosides and gangliosides.

Phosphoglycerides include phospholipids such as phosphatidylcholine,

phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, phosphatidylserine phosphatidylglycerol and diphosphatidylglycerol (cardiolipin), where the two hydrocarbon chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation. Lipids containing either saturated and unsaturated fatty acids are widely available to those of skill in the art. Additionally, the two hydrocarbon chains of the lipid may be symmetrical or asymmetrical. The above-described lipids and

phospholipids whose acyl chains have varying lengths and degrees of

saturation can be obtained commercially or prepared according to published methods.

Exemplary phosphatidylcholines include dilauroyl phophatidylcholine,

dimyristoylphophatidylcholine, dipalmitoylphophatidylcholine, distearoylphophatidyl- choline, diarachidoylphophatidylcholine, dioleoylphophatidylcholine, dilinoleoyl- phophatidylcholine, dierucoylphophatidylcholine, palmitoyl-oleoyl-phophatidylcholine, egg phosphatidylcholine, myristoyl-palmitoylphosphatidylcholine, palmitoyl-myristoyl- phdsphatidylcholine, myristoyl-stearoylphosphatidylcholine, palmitoyl-stearoyl- phosphatidylcholine, stearoyl-palmitoylphosphatidylcholine, stearoyl-oleoyl- phosphatidylcholine, stearoyl-linoleoylphosphatidylcholine and palmitoyl-linoleoyl- phosphatidylcholine. Assymetric phosphatidylcholines are referred to as 1-acyl, 2-acyl-sn- glycero-3-phosphocholines, wherein the acyl groups are different from each other.

Symmetric phosphatidylcholines are referred to as l,2-diacyl-sn-glycero-3- phosphocholines.

As used herein, the abbreviation "PC" refers to phosphatidylcholine. The

phosphatidylcholine 1 ,2-dimyristoyl-sn-glycero-3-phosphocholine is abbreviated herein as "DMPC." The phosphatidylcholine l,2-dioleoyl-sn-glycero-3-phosphocholine is

abbreviated herein as "DOPC." The phosphatidylcholine l,2-dipalmitoyl-sn-glycero-3- phosphocholine is abbreviated herein as "DPPC."

In general, saturated acyl groups found in various lipids include groups having the trivial names propionyl, butanoyl, pentanoyl, caproyl, heptanoyl, capryloyl, nonanoyl, capryl, undecanoyl, lauroyl, tridecanoyl, myristoyl, pentadecanoyl, palmitoyl, phytanoyl, heptadecanoyl, stearoyl, nonadecanoyl, arachidoyl, heneicosanoyl, behenoyl, trucisanoyl and lignoceroyl. The corresponding lUPAC names for saturated acyl groups are trianoic, tetranoic, pentanoic, hexanoic, heptanoic, octanoic, nonanoic, decanoic, undecanoic, dodecanoic, tridecanoic, tetradecanoic, pentadecanoic, hexadecanoic, 3,7, 1 1 , 15- tetramethylhexadecanoic, heptadecanoic, octadecanoic, nonadecanoic, eicosanoic, heneicosanoic, docosanoic, trocosanoic and tetracosanoic. Unsaturated acyl groups found in both symmetric and asymmetric phosphatidylcholines include myristoleoyl, palmitoleyl, oleoyl, elaidoyl, linoleoyl, linolenoyl, eicosenoyl and arachidonoyl. The corresponding lUPAC names for unsaturated acyl groups are 9-cis-tetradecanoic, 9-cis- hexadecanoic, 9-cis-octadecanoic, 9-trans-octadecanoic, 9-cis-12-cis-octadecadienoic, 9- cis-12-cis-15-cis-octadecatrienoic, 1 1-cis-eicosenoic and 5~cis-8-cis-ll-cis-14-cis- eicosatetraenoic.

Exemplary phosphatidylethanolamines include dimyristoyl-phosphatidylethanolamine, dipalmitoyl-phosphatidylethanolamine, distearoyl-phosphatidylethanolamine, dioleoyl- phosphatidylethanolamine and egg phosphatidylethanolamine.

Phosphatidylethanolamines may also be referred to under lUPAC naming systems as 1, 2- diacyl-sn-glycero-3-phosphoethanolamines or 1 -acyl-2-acyl-sn-glycero-3 - phosphoethanolamine, depending on whether they are symmetric or assymetric lipids.

Exemplary phosphatidic acids include dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid and dioleoyi phosphatidic acid. Phosphatidic acids may also be referred to under lUPAC naming systems as 1 ,2-diacyl-sn-glycero-3 -phosphate or l-acyl-2-acyl- sn-glycero-3 -phosphate, depending on whether they are symmetric or assymetric lipids.

Exemplary phosphatidylserines include dimyristoyl phosphatidylserine, dipalmitoyl phosphatidylserine, dioleoylphosphatidylserine, distearoyl phosphatidylserine, palmitoyl- oleylphosphatidylserine and brain phosphatidylserine. Phosphatidylserines may also be referred to under lUPAC naming systems as 1 ,2-diacyl- sn-glycero-3-[phospho-L-serine] or 1 -acyl-2-acyl-sn-glycero-3-[phospho-L-serine], depending on whether they are symmetric or assymetric lipids. As used herein, the abbreviation "PS" refers to phosphatidylserine. Exemplary phosphatidylglycerols include dilauryloylphosphatidylglycerol,

dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoyl- phosphatidylglycerol, dimyristoylphosphatidylglycerol, palmitoyl-oleoyl- phosphatidylglycerol and egg phosphatidylglycerol. Phosphatidylglycerols may also be referred to under lUPAC naming systems as 1, 2- diacyl-sn-glycero-3-[phospho-rac-(l-glycerol)] or 1 -acyl-2-acyl-sn-glycero-3-[phospho-rac- (I -glycerol)], depending on whether they are symmetric or assymetric lipids. The phosphatidylglycerol l,2-dimyristoyl-sn-glycero-3-[phospho-rac-(l-glycerol)] is abbreviated herein as "DMPG". The phosphatidylglycerol 1 ,2-dipalmitoyl-sn-glycero-3-(phospho-rac-l - glycerol) (sodium salt) is abbreviated herein as "DPPG".

Suitable sphingomyelins might include brain sphingomyelin, egg sphingomyelin, dipalmitoyl sphingomyelin, and distearoyl sphingomyelin. Other suitable lipids include glycolipids, sphingolipids, ether lipids, glycolipids such as the cerebrosides and gangliosides, and sterols, such as cholesterol or ergosterol. As used herein, the term cholesterol is sometimes abbreviated as "Choi" or "CHOL". Additional lipids suitable for use in liposomes are known to persons of skill in the art and are cited in a variety of sources, such as 1998 McCutcheon's Detergents and Emulsifiers, 1998 McCutcheon's Functional Materials, both published by McCutcheon Publishing Co., New Jersey, and the Avanti Polar Lipids, Inc. Catalog. The overall surface charge of the liposome can affect the tissue uptake of a liposome. Anionic phospholipids such as phosphatidylserine, phosphatidylinositol, phosphatidic acid, and cardiolipin may be used. Neutral lipids such as dioleoylphosphatidyl

ethanolamine (DOPE) may be used to target uptake of liposomes by specific tissues or to increase circulation times of intravenously administered liposomes. Cationic lipids may be used for alteration of liposomal charge, where the cationic lipid can be included as a minor component of the lipid composition or as a major or sole component. Suitable cationic lipids typically have a lipophilic moiety, such as a sterol, an acyl or diacyl chain, and where the lipid has an overall net positive charge. Preferably, the head group of the lipid carries the positive charge.

Liposomes having a more rigid lipid bilayer, or a liquid crystalline bilayer, are achieved by incorporation of a relatively rigid lipid. The rigidity of the lipid bilayer correlates with the phase transition temperature of the lipids present in the bilayer. Phase transition temperature is the temperature at which the lipid changes physical state and shifts from an ordered gel phase to a disordered liquid crystalline phase. Several factors affect the phase transition temperature of a lipid including hydrocarbon chain length and degree of unsaturation, charge and headgroup species of the lipid. Lipid having a relatively high phase transition temperature will produce a more rigid bilayer. Other lipid components, such as cholesterol, are also known to contribute to membrane rigidity in lipid bilayer structures. Cholesterol is widely used by those of skill in the art to manipulate the fluidity, elasticity and permeability of the lipid bilayer. It is thought to function by filling in gaps in the lipid bilayer. In contrast, lipid fluidity is achieved by incorporation of a relatively fluid lipid, typically one having a lower phase transition temperature. Phase transition temperatures of many lipids are tabulated in a variety of sources, such as Avanti Polar Lipids catalogue and Lipidat by Martin Caffrey, CRC Press. Liposomes are preferably made from endogenous phospholipids such as dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG), phosphatidyl serine, phosphatidyl choline, dioleoyphosphatidyl choline [DOPC], cholesterol and cardiolipin, or DPPC as described hereinbefore. Surface Modification

The use of saturated phospholipids and cholesterol in the formulation of liposome delivery systems cannot fully overcome their binding with serum components, and the

consequently decreased mononuclear phagocyte system (MPS) uptake of the vesicles: saturation of the MPS with a previous administration of "empty" liposomes may be necessary.

Moreover, SUVs possess the disadvantage of low aqueous entrapment volume, and the use of charged liposomes can be toxic. These are overcome by coating the surface of the liposomes with inert molecules to form a spatial barrier. As explained above, among the different polymers investigated in attempts to improve the blood circulation time of liposomes, poly-(ethylene glycol) (PEG) has been widely used as polymeric steric stabilizer or "shield". Surface modification of liposomes with PEG can be achieved in several ways: by physically adsorbing the polymer onto the surface of the vesicles, by incorporating the PEG-lipid conjugate during liposome preparation, or by covalently attaching reactive groups onto the surface of preformed liposomes. One most widely used method at present is to anchor the polymer in the liposomal membrane via a cross-linked lipid (ie, PEG- distearoylphosphatidylethanolamine [DSPE]). Grafting PEG onto liposomes has demonstrated several biological and technological advantages. The most significant properties of PEGylated vesicles are their strongly reduced MPS uptake and their prolonged blood circulation and thus improved distribution in perfused tissues. Moreover, the PEG chains on the liposome surface avoid vesicle aggregation, improving stability of formulations.

The behaviour of PEGylated liposomes depends on the characteristics and properties of the specific PEG linked to the surface. The molecular mass of the polymer, as well as the graft density, determine the degree of surface coverage and the distance between graft sites. The most evident characteristic of PEG-grafted liposomes (PEGylated-liposomes) is their circulation longevity, regardless of surface charge or the inclusion of stabilizing agent such as cholesterol.

Preparation of liposomes The liposomes described herein can be prepared by a variety of techniques known in the art - see e.g. Liposome Technology, Vols. 1 , 2 and 3, Gregory Gregoriadis, ed., CRC Press, Inc. The method selected is dependent on a variety of factors, such as: (1 ) the physicochemical characteristics of the material to be entrapped and those of the liposomal ingredients; (2) the nature of the medium in which the lipid vesicles are dispersed; (3) the effective concentration of the entrapped substance and its potential toxicity; (4) additional processes involved during application/delivery of the vesicles; (5) optimum size, polydispersity and shelf-life of the vesicles for the intended application; and (6) batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.

For example, liposomes can be created by sonicating phosphatidylcholine rich

phospholipids in water. Low shear rates create multilamellar liposomes. Continued high- shear sonication tends to form smaller unilamellar liposomes. In this technique, the liposome contents are the same as the contents of the aqueous phase.

In some embodiments, liposomal particles are prepared using a conventional thin film hydration and extrusion method. The lipid, PEG-conjugated lipid, and stabilizer, if present, are dissolved in an organic solvent (e.g., chloroform) at pre-determined molar ratios. A small proportion of a labeled-lipid, such as rhodamine labeled PE (Rho-PE), can be added to the mixture to enable visualization of the liposomal particles via fluorescence microscopy. The mixture is placed in a rotavap with reduced atmosphere pressure to evaporate the organic solvent. The resulting lipid film is hydrated, such as with phosphate buffered saline (PBS), while agitated using a water bath sonicator to form multi-lamellar vehicles (MLV). The suspension is subsequently extruded through polycarbonate filters with pore sizes of 400 nm and 200 nm to generate unilamellar vehicles.

Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.

The invention will now be further described with reference to the following non-limiting Figures and Examples. Other embodiments of the invention will occur to those skilled in the art in the light of these.

The disclosure of all references cited herein, inasmuch as it may be used by those skilled in the art to carry out the invention, is hereby specifically incorporated herein by cross- reference.

Figures

Figures

Fig 1 - two volunteers blood glucose level changes under an hyperglycemic clamp. In a), a 15 mmol plateau was easily reached while it was impossible to get the glucose level > 10 mmol glucose in b). Fig 2 - schematic of liposomal contrast agents and the chemical structures of 2-DG, glucose, DPPC and the novel PEG lipid DPPE-EG4.

Fig 3 - this shows Z spectra (top) and MTRasym curves (bottom) for liposomal samples, L4, L5 and L6, measured at room temperature with B ₀ = 9.4 T and Bi = 1.2 μΤ. L4: 0.5 M glucose; L5: 1 M glucose; L6: 2 M glucose.

Fig 4 - this shows Z spectra (a,b) and MRTasym curves (c,d) for liposome samples with overall glucose concentrations of 55.5 mM (L9), 44.6 mM (L8) and 22.6 mM (L7). Spectra were acquired with a saturation pulse frequency of 1.5 μΤ (a,c) or 8.0 μΤ (b,d) at room temperature with a Bo of 9.4 T.

Fig 5 - this shows Z spectra (a and c) and MTRasym curves (c and d) showing the pH dependence of CEST signal magnitude produced by glucose loaded liposomes L10-L13 at 25 °C (a and b) and 37 °C (c and d). The spectra were obtained with a saturation length of 80 pulses, Bi = 1 .5 μΤ and B ₀ = 9.4 T.

Fig 6 - this shows MTRasym spectra a) controls and b) 2-DG and glucose liposomes L22 and L23 at pH 5.8 in 20% PBS measured at 20 °C and 37 °C. Spectra were acquired with a saturation length of 150 pulses, Bi = 1.5 μΤ and B ₀ = 9.4 T. Fig 7 - this shows the MTRasym spectra at 1.5 μΤ and pH 6 for 2-DG liposomes, (a): 20°C, (b): 37 °C, as described in the Examples herein.

Fig 8 - Figure 8 shows the MTRasym spectra at 1.5 μΤ and 5.0 μΤ for 2-DG liposomes at pH 7 at 20°C and 37° with various PBS concentration.

Fig 9 - this summarises the results of Fig 7 and Fig 8.

Fig 10 - shows the MTRasym spectra at pH 6 or 7 for 2-DG liposomes at 20°C and 37°C using DPPC or DSPC lipids.

Fig 1 1 - this summarises the results of Fig 10.

Fig 12 - shows the MTR _aS ym spectra obtained at 37 °C and Bi = 1 .5 μΤ and 5.0 μΤ for rhodamine labelled liposomes and controls.

Fig 13 - this summarises the results of Fig 12.

Fig 14 - summarises results obtained using various temperatures and saturation powers for varying diameter 2-DG liposomes.

Fig 15 - shows the Z- and MTR _aS ym spectra for the 2-DG liposomes and a free sugar control scanned at various temperatures with Bi = 5.0 μΤ. Figs 16A and 16B - this summarise the data in Figure 15 for the liposomes (A) and control sample (B).

Fig 17 - this shows the results of the assessment of release over time from the liposomes, as described in the Examples herein.

Fig 18 - shows the Z- and MTR _asy m spectra for four sugars and their chemical structures.

Fig 19A and 19B - Figure 19A shows the Z- and MTRasym spectra obtained from 4 differentially PEGylated liposome formulations at Bi = 1.5 μΤ (top) and Bi = 1 .5 μΤ (bottom) at 20 °C whereas Figure 19B shows the same results at 37 °C.

Fig 20 - this summarises the results of Fig 19A and 19B.

Fig 21 - Shows the CEST signal produced by glucose phantoms at various pH, scanned at room temperature and 37 °C and Bi = 1 .5 μΤ and 5.0 μΤ.

Fig 22 - CEST Z-spectra (a,b) and MTRasymmetry lineshapes (c,d) for liposomes encapsulating glucose and 2-DG and the free sugar controls for 1.5 μΤ at 20 °C (a,c) and 37 °C (b,d). Fig 23 - CEST Z-spectra (a,b) and MTRasymmetry lineshapes (c,d) for liposomes encapsulating glucose and 2-DG and the free sugar controls for 5.0 μΤ at 20 °C (a,c) and 37 °C (b,d). Fig 24: MTRasymmetry values for 2-DG liposomes at a power of 1 .5 μΤ and 5.0 μΤ and temperatures of 20 °C and 37 °C a) with varying PBS concentrations at pH 6 and pH 7 b) at pH 7 with varying liposome diameter and with overall 2-DG concentrations of 39 mM or 14 mM. Fig 25 - MTRasymmetry values of 2-DG liposomes a) formulated with DPPC or DSPC lipid bilayers at pH=6 and pH =7 for 1.5 μΤ and 5 μΤ at 20 °C and 37 °C b) formulated with DPPC bilyers and scanned in a range of temperatures for 1.5 μΤ and 5 μΤ

Fig 26 - glucose levels in rats following injection with either free glucose or glucose encapsulated inside liposomes.

Fig 27 - exposure of RKO colon cancer cell line to free 2-DG or liposomally encapsulated 2-DG (97 mol% DPPC, 3 mol% DPPE-PEG2000), with and without a radiation dose (4 Gy). Lipo-2DG caused more cell death than the same concentration of free 2-DG, both with and without a radiation dose of 4 Gy.

Fig 28 - exposure of SW1417 colon cancer cell line to free 2-DG or liposomally encapsulated 2-DG. Liposome encapsulation significantly increased cell death. Examples

Example 1 - variability of glucose response

Injection of glucose is known to trigger the insulin response. Preliminary experiments in patients have shown that the response to i.v. injection of a bolus of unlabelled D-Glucose is highly variable (Fig 1 ), making it difficult to establish a reproducible experimental set under these conditions.

It is expected that this constitutes a major problem when translating this GlucoCEST technology into clinical practice, as:

1 ) individual responses from individual patients may vary widely, impacting on the robustness and reproducibility of the technique;

2) many patients might be under adjunct therapies, such as e.g. steroids, itself affecting the blood glucose level and the patient's insulin response;

3) as cancer is more prevalent ² in elderly patients, they will often suffer from comorbidities such as diabetes (type II), making the technique contraindicated for them.

Example 2- optimisation of parameters for CEST detection of lipo-qlucose 2. 1 Formulation parameters and measurements for DPPC liposomes encapsulating glucose Table 1 below shows the formulation parameters and measurements for DPPC liposomes encapsulating glucose, L4-L13. Formulation procedure: The correct molar ratios of lipids were dissolved in a mixture of CHC iMeOH (3:1 , v/v) to yield a homogeneous mixture of lipids. The solvent was removed under reduced pressure to form a thin lipid film on the inside of a round bottom flask. The thin lipid film was freeze dried overnight to remove any residual organic solvent. Hydration of the lipid film was accomplished by addition of the correct volume and concentration of sugar solution to yield the desired liposome concentration.

Sonication at a temperature above the T _m of the lipids (DPPC has a T _m of 41 °C) caused the assembly of polydisperse liposomes. During extrusion, 2 mL liposome samples were passed through 400 nm and 200 nm pore membranes at 45-50 °C until the diameters measured below 200 nm and the Pdl values approached 0.1. The liposomes were then dialysed into 0.25 M NaCI solution over a 36 h period.

Table 1 Table showing the formulation parameters and measurements for DPPC liposomes L4-L13. Characterisation: the liposomes were sized by DLS before, during and after extrusion.

Samples for DLS were prepared by taking a 5-20 μί aliquot of the stock liposome solution and diluting with H2O to give a final volume of 1 mL and a final concentration of approximately 0.2 mM. DLS measurements were taken at 25 °C in triplicate using clear 1 mL zeta potential cuvettes. The mean and standard deviation of three measurements is reported for each liposomal sample in Table 1 .

All exterior and overall glucose concentrations for glucose liposome formulations that are reported in Table 1 were obtained using the Glucose HK Assay ^® (supplied by Sigma- Aldrich). Prior to use of the assay an accuracy test was carried out using the supplied 1.0 mg/mL D-glucose standard solution. Five measurements of absorption at 340 nm (A340) were taken during each liposomal characterisation; a sample blank with intact liposomes in distilled water (SB1 ), a sample blank after disruption of the liposomes with Triton (SB2), a reagent blank (RB) and two measurements of unknown glucose concentration, one with the liposomes intact (T1 ) and one following disruption with Triton-X100 (T2). The volume of the various substituents required for these five measurements are given for a typical glucose liposome sample (30 mM DPPC encapsulating 0.5 M glucose) in Table 2.

Liposome sample volume was slightly increased for more concentrated formulations. For SB1 , SB2 and RB measurement of A340 could be carried out immediately after mixing of the components in a measuring cuvette. However for T1 and T2, 15 minutes was allowed to ensure completion of the enzymatic cascade. The mg glucose/mL was calculated using Equation 1 and converted into glucose concentration (mM).

Table 2. Table showing the volume of glucose assay reagent, liposome sample, deionised water and Triton used for each measurement of A340 taken during the Glucose HK Assay ^® . Volumes stated were used for 30 mM liposome samples encapsulating 0.5 M glucose.

Liposome Volume of

Glucose assay Volume of

Measurement sample volume deionised water

reagent (ml_) Triton (μΙ_)

(ML) (ml_)

SB1 7 1.0

SB2 7 1.0 10 RB 1.0

T1 1 .0 7

T2 1 .0 7 10

For T1 , Atotalblank - AsB1 ⁺ ARB, For T2 Atotalblank - AsB2 ⁺ ARB

ΔΑ = Ατΐ/Τ2 - Atotalblank

, (AA)(total volume,mL)(0.029) . . .

mq per mL qlucose = ( 1 )

sample volume (mL)

2.2 Investigating encapsulating higher concentrations than 0.5 M glucose

Figure 3 shows Z spectra (top) and MTR _asy m curves (bottom) for liposomal samples, L4, L5 and L6, measured at room temperature with Bo = 9.4 T and Bi = 1 .2 μΤ. L4: 0.5 M glucose; L5: 1 M glucose; L6: 2 M glucose. This experiment showed us that glucose solutions as concentrated as 1 M or more concentrated cannot be successfully encapsulated inside the DPPC lipid bilayer (see the exterior glucose concentrations reported in Table 1 , exterior concentration values higher than 5 mM are considered significant). Therefore all further formulations were carried out encapsulating 0.5 M glucose.

2.3 Investigation to effect of glucose concentration

Figure 4 shows Z spectra (a,b) and MRTasym curves (c,d) for liposome samples with overall glucose concentrations of 55.5 mM (L9), 44.6 mM (L8) and 22.6 mM (L7). Spectra were acquired with a saturation pulse frequency of 1.5 μΤ (a,c) or 8.0 μΤ (b,d) at room temperature with a Bo of 9.4 T. The pH and glucose concentration of each liposomal sample is shown in Table 1 .

Table 3 shows the CEST suppression of the water peak for 10% PBS, liposome samples L7-L9 in 10% PBS measured with Bi = 1.5 μΤ and 8.0 μΤ, at 25°C and B ₀ = 9.4 T. CEST suppression is expressed as an average of the percentage reduction in water signal caused by presaturation across the range 0-4.5 ppm.

1.5 μΤ 8.0 μΤ

power power

10% PBS -0.15 % -0.03 %

L7 1.51 % 2.1 1 %

L8 2.33 % 3.99 %

L9 2.62 % 5.16 %

Table 3

The results show that appreciable CEST signal can be obtained from glucose liposomes at both saturation powers and in a concentration dependent manor. The higher RF saturation field of 8.0 μΤ produced a larger CEST mediated reduction of the water signal. This is in agreement with evidence that higher saturation fields allow greater extents of saturation preceding exchange. Across the samples tested, an average 69.3% increase in water suppression was induced by increasing Bi from 1 .5 μΤ to 8.0 μΤ.

2.4 pH and temperature dependency of glucose loaded liposomes Figure 5 shows Z spectra (a and c) and MTRasym curves (c and d) showing the pH dependence of CEST signal magnitude produced by glucose loaded liposomes L10-L13 at 25 °C (a and b) and 37 °C (c and d). The spectra were obtained with a saturation length of 80 pulses, Bi = 1 .5 μΤ and B ₀ = 9.4 T. Table 4 shows the CEST suppression of the water peak for glucose liposomes L10-L13 measured at both 25°C and 37 °C. The spectra were acquired with a saturation length of 80 pulses, Bi = 1.5 μΤ and B ₀ = 9.4 T. CEST suppression is expressed as an average of the percentage reduction in water signal caused by presaturation across the range 0-3.75 ppm.

Table 4 For glucose liposomes, pH 6 and 37 °C is optimal for CEST signal. This makes them particularly suitable for the imaging of tumors in vivo.

Example 3 - comparison of glucose with 2-DG

3. 1 Formulation parameters and measurements for DPPC liposomes encapsulating 2- DG

Measurement of exterior and overall 2-DG concentration: The Glucose GO Assay ^® (supplied by Sigma-Aldrich) was used to measure the exterior and overall 2-DG concentration of 2-DG encapsulating liposome samples. As the assay was designed to measure glucose concentration, a calibration curve must be constructed with 2-DG. An intact liposome sample (T1 ) was measured to obtain an exterior 2-DG concentration and a disrupted liposome sample with Triton-X100 (T2) was used to measure the overall 2-DG concentration in the same way that glucose liposomes are measured with The Glucose HK Assay ^® .

Table 5 Formulation parameters are shown in table 5. As the DPPC liposomes encapsulated glucose more efficiently than 2-DG, L22 was diluted with 0.25 M NaCI in 20 % PBS at pH 5.8 to reduce the overall sugar concentration to be comparable to that of the 2-DG liposomes (measured using the Glucose HK Assay ^® following dilution as 21.6 mM overall glucose concentration).

Figure 6 shows MTRasym spectra for a) controls (23 mM glucose or 2-DG solution in 20% PBS at pH 5.8) and b) 2-DG and glucose liposomes L22 and L23 at pH 5.8 in 20% PBS. All samples were measured at both 20 °C and 37 °C. Spectra were acquired with a saturation length of 150 pulses, Bi = 1.5 μΤ and Bo = 9.4 T.

The results are summarised in Table 6:

Table 6 At pH 5.8, the same concentration of 2-DG gives a greater signal than glucose both as free sugars despite 2-DG possessing one less hydroxyl proton. Free or encapsulated 2- DG produced greater CEST contrast than free or encapsulated glucose.

Glucose and 2-DG liposomes both gave better signal than the corresponding controls. Glucose and 2-DG liposomes give greater signal at 37 °C than at room temp. The best signal in this experiment was from 2-DG liposomes at 37 °C.

Example 4 - optimisation of parameters for CEST detection of 2-DG liposomes 4. 1 Varying PBS at pH 6 and pH 7

Table 7 shows the formulation parameters and measurements for DPPC liposomes (30 mM) at pH 6 encapsulating 2-DG with varying PBS concentration.

Table 7

Figure 7 shows the MTR _asy m spectra at 1.5 μΤ and pH 6 for 2-DG liposomes, (a): 20°C, (b): 37 °C.

Table 8 shows the formulation parameters and measurements for DPPC liposomes (30 mM) at pH 7 encapsulating 2-DG with varying PBS concentration.

Table 8 Figure 8 shows the MTRasym spectra at 1.5 μΤ and 5.0 μΤ for 2-DG liposomes at pH 7 at 20°C and 37°C.

Figure 9 summarises the results at pH 6 and pH 7. CEST suppression is expressed as an average of the percentage reduction in water signal caused by presaturation across the range 0-3.5 ppm.

As can be seen, free 2-DG and 2-DG liposomes at give best signal at 37 °C and 5.0 μΤ for pH 6 and pH 7. The results show that varying PBS up to 20% does not greatly affect the CEST signal. Therefore further formulations may be done in 20% PBS to give the most stable pH.

4.2 Choice of lipid bilayer for encapsulating 2-DG

Table 9 shows the formulation parameters and measurements for DPPC and DSPC liposomes (30 mM) at pH 6 or 7 encapsulating 2-DG with 20% PBS.

Table 9 Figure 10 shows the MTRasym spectra at pH 6 or 7 for 2-DG liposomes at 20°C and 37°C using DPPC or DSPC lipids.

Figure 1 1 summarises the results. As can be seen, DPPC liposomes were generally better than DSPC liposomes across different temperatures and powers.

The best CEST signal was from DPPC liposomes at pH 7, at 37 degrees and 5.0 μΤ using an instrument having B ₀ = 9.4 T.

Further work (results not shown) confirmed this observation that liposome encapsulation with DPPC of both glucose and 2-DG gives higher CEST signals compared with the free sugar solutions. This measurement was verified at both pH 6 and 7. The increase in signal for the liposomal is believed to occur due to a reduction in the exchange rate (which is more pronounced for Lipo-2DG compared to Lipo-glucose). ln respect of the choice of liposomes, we also observe a 10-fold reduction in exchange rate when we used the DSPC lipid. DSPC is a more rigid lipid which quenches the intermembrane exchange across the lipid bilayer. Using DSPC is thus expected to have advantages when utilising scanners having lower, clinical field strengths (< 7T, typically around 3T). At these field strengths the greater reduction in exchange rate can provide a more suitable range for the CEST effect to take place.

Furthermore the greater rigidity of DSPC liposomes may provide more stable and robust encapsulation.

4.3 Incorporation of a rhodamine-lipid into DP PC lipid bilayer for encapsulating 2-DG

Table 10 shows the formulation parameters and measurements for DPPC and DSPC liposomes (30 mM) at pH 6 or 7 encapsulating 2-DG with 20% PBS. A control was made up with 35 mM free 2-DG in 0.25 M NaCI and 20% PBS at pH 7.

Table 10

Figure 12 shows the MTRasym spectra obtained at 37 oC and both 1 .5 μΤ and 5.0 μΤ. Figure 13 summarises the results.

Fig 14 shows that 0.5 Mol% Rh-DHPE lipid can be incorporated into the liposomes for future fluorescent labelling studies without detrimental effects on 2-DG encapsulation or resultant CEST signal. Example 5 - investigation of liposome diameter for CEST detection of 2-DG liposomes

Diameter varied by extrusion

Table 1 1 shows the formulation parameters and measurements for liposomes of different diameters.

Table 1 1 Liposomes with various diameters were formulated to investigate whether the curvature of the liposome effects water exchange across the bilayer in such a way that alters CEST signal.

Results are described in Figure 14 (discussed below). As before, the best CEST signal from 2-DG liposomes was at 37 °C and 5.0 uT.

5.2 Dilution to match concentration of probe sonicated liposomes

The liposomes were differentially diluted down to have comparable overall 2-DG concentrations. The parameters are shown in Table 12. Measurements taken post diluted are shown in bold.

Table 12

Figure 14 summarises the CEST results from before and after dilution of the 2-DG liposomes at various temperature and saturation power. CEST suppression is expressed as an average of the percentage reduction in water signal caused by presaturation across the range 0-3.5 ppm.

Results shows how the diameter of 2-DG encapsulating liposomes (bilayer comprising 100% DPPC, in 20% PBS at pH 7) affects the CEST signal for diameters of 120 nm, 150 nm and 200 nm. Liposomes were formulated as described in Section 2.1 and the slightly enhanced CEST signal seen for the 200 nm liposomes is assumed to be due to more efficient 2-DG encapsulation in the larger aqueous interior volume. Liposomes were differentially diluted to give overall 2-DG concentrations of 14 mM (to offset differences in encapsulation) and this trend of larger diameter giving larger CEST signal is no longer apparent. We concluded no large difference in CEST was caused by varying liposome diameter in the range 120-200 nm. Liposomes can therefore be kept in this range, as this is also a good size for the EPR effect (passive targeting of liposomes to tumors).

Example 6 - effect of temperature

DPPC liposomes (30 mM) encapsulating 2-DG (0.5 M) at pH 7 were sized to 200 nm and dialysed into 0.25 M NaCI solution with 20% PBS. The formulation parameters are shown in Table 13. The liposomes were scanned at 25, 28, 31 , 34 and 37 °C. A separate aliquot of liposomes was used for each temperature as some leakage occurs during scanning for the higher temperatures. A control was made up of 35 mM free 2-DG at pH 7 in 20% PBS. Bilayer Z-Ave Pdl Exterior [2-DG] Overall [2-DG] pH compositon (mM) (mM)

100% DPPC 178 nm 0.14 35.6 mM 0.25 mM 7

Table 13

Figure 15 shows the Z- and MTR _asy m spectra for the liposomes and the control scanned at various temperatures with Bi = 5.0 μΤ. The effect of temperature on CEST results is more pronounced for the liposomes compared to the free sugar.

Figure 16A summaries the liposome data. CEST suppression is expressed as an average of the percentage reduction in water signal caused by presaturation across the range 0- 3.5 ppm. Increasing the temperature in the range 25-37 oC causes an increase in CEST signal for 2-DG liposomes. This is more pronounced with Bi = 5.0 μΤ.

Figure 16B summaries the control data. The effect of temperature on CEST signal magnitude is more pronounced for liposome encapsulated 2-DG. This may be due to the induced change in lipid bilayer permeability/flexibility.

Example 7 - release over time: scanning times

2-DG encapsulating liposomes were incubated at 37 °C for 72 h, aliquots were removed at various time points and the exterior 2-DG concentration was tested as previously described using the Glucose GO Assay ^® . This experiment was done to predict leakage in vivo.

Figure 17 shows the release over time profile of 2-DG from DPPC liposomes at 37 degrees.

Results show that 21.3% of the encapsulated 2-DG leaks out of DPPC bilayers during the first 4 h which is how long CEST scanning takes. A measurement based on the first 6.5 h with a linear plot indicated 18.8% leakage after 4 h.

The results show that 2-DG leaks out relatively slowly at 37 degrees thus the liposomes are suitable for in vivo use.

Example 8 - CEST spectra of other sugar compounds

Overlay of CEST spectra produced by 35 mM solutions of various clinically interesting sugars at pH 6 and saturation frequency 1.5 μΤ. Glucose and 2-DG have been encapsulated inside liposomes and scanned for CEST signal. Figure 18 shows the Z- and MTR _asy m spectra for the four sugars and their chemical structures. Example 9 - PEGylation of liposomes

Polyethylene glycol (PEG) coating of liposomes is often carried out resulting in several advantages. The neutral coating reduces colloidal instability by discouraging aggregation and supressing immunological responses. Importantly, PEG coating liposomes decreases their rate of removal from the blood by reducing uptake into the reticuloendothelial system (RES) and thereby lengthening their biological half-life. Conventional PEG coating is achieved by formulating liposomes with a small molar percentage (typically 2-8 mol %) of 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine N-[carboxy(polyethyleneglycol)2000] (DSPE-PEG2000). The PEG-2000 chain is long, neutral and hydrophilic causing decreased absorption of serum proteins and opsonins as well as masking the charge of the liposome. Cells have been shown to preferentially take up charged particles and the absorption of serum proteins and opsonins to the liposome surface stimulates interaction with cell walls. Thus, PEG-2000 coating of liposomes inhibits cellular internalisation. In 2013, Mitchell et al. demonstrated that replacing DSPE-PEG2000 with lipids exhibiting short n-EG shielding units significantly enhanced liposome internalisation whilst maintaining similar clearance rates to PEG-2000-stabilised liposomes.

We synthesised a series of novel short chain PEG lipids; DPPE-EG4 (-OH moiety at polar terminus), DPPE-EG4-NH2 (-NH2 moiety at the polar terminus) and DPPE-EG4-OMe (- OMe moiety at polar terminus). Constituting 30% of the lipid bilayer with one of these lipids could create a more uniform PEG coating of the liposome surface than

incorporating 3% DSPE-PEG2000. We formulated liposomes with 30% of each short chain PEG lipid and 70% DPPC encapsulating 0.5 M 2-DG to test whether these novel lipids are capable of forming liposomes, effectively encapsulating 2-DG and whether the altered bilayer composition has any detrimental effect on generated CEST contrast. 100% DPPC liposomes and 3% DPPE-PEG2000 liposomes were also formulated for comparison. Table 14 shows the formulation parameters for the various PEGylated liposomes and the DPPC control at pH 7 and 20% PBS. The sample containing 30% of novel lipid DPPE- EG4-OMe was not able to form liposomes encapsulating 2-DG. Instead a gel was formed.

Table 14

Figure 19A shows the Z- and MTRasym spectra obtained from the 4 liposome

formulations shown in Table 14 at Bi = 1.5 μΤ (top) and Bi = 1.5 μΤ (bottom) at 20 °C whereas Figure 19B shows the same results at 37 °C Overall [2-DG] Exterior [2-DG]

Bilayer compositon Z-Ave (d.nm) Pdl

(mM) (mM)

100% DPPC 184 0.1 1 33.1 1.5 (4%)

3% DPPE-PEG2000 163 0.14 34.9 4.9 (14%)

30% DPPE-PEG4-OH 172 0.16 40.8 7.1 (17%)

30% DPPE-PEG4-NH ₂ 221 0.19 12.5 2.2 (18%)

Table 15

In between scanning at room temperature and 37 °C the exterior 2-DG concentration was measured to check that significant amounts of 2-DG had not leaked out. We noted

slightly higher but still acceptable levels of leakage from PEGylated liposomes compared to pure DPPC liposomes.

Figure 20 summarises all CEST data from these liposomes. CEST suppression is

expressed as an average of the percentage reduction in water signal caused by

presaturation across the range 0-6 ppm.

Liposomes with 30% DPPE-EG4-NH2 exhibited lower CEST signal due to lower

encapsulation than the other formulations (overall 2-DG concentration of 12.5 mM cf. 33- 40 mM).

Liposomes formulated with 30% DPPE-EG4-OMe formed a gel thus this lipid was

unsuitable under the conditions tested. The DPPE-EG4 lipid performed best; liposomes with 30% DPPE-EG4 encapsulated

similar (and slightly higher) quantities of 2-DG and the alteration to the lipid bilayer

produced no decrease in CEST contrast. No significant differences in CEST signal were seen for 100% DPPC, 3% DPPE-PEG2000 and 30% DPPE-EG4 liposomes at either temperature or saturation power. DPPE-EG4 will be the lipid we use for pending in vivo experiments.

Results show that at various temperature and saturation power our novel short chain

PEG lipid (DPPE-EG4) can be used to form liposomes that entrap 2-DG and enable

appreciable CEST contrast from 2-DG as effectively as the commercially available lipids, DPPC and DPPE-PEG2000.

We tested whether formulation of liposomes with 30% DPPE-EG-NH2 would be more effective at higher or lower pH due to change alteration of the terminal amino group. The formulation parameters and measurements from these liposomes are shown in Table 16. Bilayer compositon Z-Ave Overall [2- Exterior [2-

Pdl

(d.nm) DG] (mM) DG] (mM)

30% DPPE-PEG4-NH ₂

195 0.23 20 mM 0.2 mM

pH 6

30% DPPE-PEG4-NH ₂

193 0.17 18 mM 0.5 mM

pH 8

Table 16

The maximum overall 2-DG concentration achieved using 30% DPPE-EG-NH2 was 20 mM when formulated at pH 6 which is still significantly lower than encapsulatin with DPPC, 3% DPPE-PEG2000 or 30% DPPE-EG4.

Example 10 - use of glucose phantoms

Solutions of 35 mM glucose in 20% PBS at various pH were made up to further investigate the pH dependency of CEST signal magnitude arising from glucose. At room temperature the largest CEST signal is produced at pH 7-7.4 however at physiological temperature the optimum pH for glucose CEST contrast is shifted lower, to pH 6.75.

Summary results are shown in Figure 21 , based on the use of free glucose at varying pH.

The results show the utility of using the liposomes of the invention to image tumors because the contrast produced once the imaging agent enters the acidic tumor microenvironment will be greater than when it is circulating in the blood at pH 7.4. Example 1 1 - use of Lipo2-DG-CEST as a theranostic agent for simultaneous tumour imaging and chemotherapy

Introduction 2-Deoxy-D-glucose (2-DG) is a well-characterized glycolytic inhibitor and has been shown to inhibit tumor growth in vivo [4]. However its use has not been promoted any further due to its inherent toxicity. In this Example we investigated whether we could use the sensitivity of GlucoCEST [1] to detect liposome-encapsulated 2-DG. Such a method would allow for high-concentration targeting of the drug to cancer cells via both active and passive targeting, thereby making it less toxic and potentially applicable for

chemotherapy.

Methods 1 ,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes containing 0.5 M glucose or 0.5 M 2-DG were formulated in 20% phosphate buffer saline (PBS) at pH 5.8 via sonication, extruded to achieve an average diameter of -200 nm and dialysed into 0.25 M NaCI solution. The overall sugar concentrations for both liposome samples were enzymatically measured and adjusted to 23 mM. Free sugar controls were made up at 23 mM in 20% PBS at pH 5.8. To further examine the effect of the buffer concentration on the measured CEST signal, 2-DG-liposomes with an overall 2-DG concentration of 40 mM and at pH varying between 6 and 7 were prepared at different PBS concentrations of 0%, 5%, 10% and 20 %.

To further investigate the effect of lipid bilayer composition 2-DG liposomes were formulated using two different types of lipid (i.e. DPPC, DSPC) at pH 6 and 7. Finally, to examine the effect of liposome size on the CEST signal, liposomes were prepared with three different diameters: 200 nm, 150 nm and 120 nm, with overall 2-DG concentrations of 39 mM and 14 mM at pH=7.

All samples were imaged at 20 °C and 37 °C on a 9.4T Agilent scanner using a transmit/receive RF coil with 33mm inner diameter (Rapid Biomedical). CEST

measurements were acquired using a single-shot single-slice spin-echo (SE) echo planar imaging (EPI) sequence (TR=65.3ms, TE=4.07ms, FOV=20x20mm ² , slice

thickness=5mm, matrix size=64x64) with a saturation train prior to the readout consisting of 150 Gaussian pulses (pulse length = 50 ms, 99% duty cycle) at two different power levels, 1.5μΤ (FA=982°) and 5.0 μΤ (FA=3000°). Results

Figures 22 and 23 show the Z- and MTRasymmetry spectra for the glucose and 2-DG liposomes and their free sugar controls at power levels of 1 .5 μΤ and 5.0 μΤ, respectively. The MTRasymmetry was greater for the liposomal sugar formulations than the respective controls. Additionally, 2-DG gave a greater CEST contrast than glucose in both liposomal and control samples.

These results were consistent across both temperatures and powers (Figures 22,23c-d). Table 12 summarizes the average percentage signal enhancement between 0 and 3.5 ppm for all sample measurements.

Table 12: MTRasymmetry from 0.2-3.5 ppm for glucose, 2-DG, glucose and 2-DG- loaded liposomes with overall sugar concentrations of 23 mM at pH 5.8.

MTRasymmetry Glucose 2-DG 2-DG control Glucose 0-3.5ppm Liposomes Liposomes samples control

samples

MTRasymmetry

at 20 °C, 1.5 μΤ 3.6 % 6.0 % 4.8 % 2.9 %

MTRasymmetry

at 37 °C, 1.5 μΤ 5.0 % 7.3 % 5.9 % 3.9 %

MTRasymmetry

at 20 °C, 5 μΤ 5.5 % 10.0 % 8.0 % 5.2 %

MTRasymmetry

at 37 °C, 5 μΤ 14.0 % 15.5 % 12.0 % 10.6 % Figure 24 displays bar charts of 2-DG liposomes prepared at different PBS concentrations (Fig 24a) and with different diameters (Fig 24b). The CEST signal was found to be larger for a power level of 5 μΤ at 37 °C when compared to the signal at both 1.5 μΤ at 20 °C and 5 μΤ at 20 °C in both experiments. Subtle variations were detected in the MTRasymmetry with varying PBS concentration and liposome diameter especially for in-vivo conditions (i.e. pH=7, 37 °C) that were deemed to be due to human error during formulation since there is no obvious trend.

We concluded that 20% PBS and liposomes having an average diameter of 200nm gave the best compromise in terms of CEST signal. . I.e. 200 nm allows slightly higher encapsulation whereas 20% PBS assures stable pH without quenching any CEST signal. Figure 25 shows that 2-DG encapsulated by DSPC bilayers produced a less prominent CEST signal than DPPC bilayers. The greatest signal was acquired at 37 °C, 5 uT, pH 7 and with a bilayer comprised of DPPC. The composition of the phospholipid bilayer and the temperature affect the CEST signal because they alter the permeability of the lipid membrane and hence affect chemical exchange through intramembrane exchange.

Conclusion from Example 11

This work demonstrates for the first time that liposomes loaded with 2-DG or glucose can be used to obtain CEST contrast. The enhanced CEST detectability of the 2-DG liposomes may be i) due to a difference in the exchange rate of the hydroxyl protons on 2-DG compared to glucose at pH 5.8 and ii) through modification of the global exchange rate, by a combination of exchange through the lipid layer and the chemical exchange itself. References

(1 ) S. Walker-Samuel et al., Nature Medicine, 19, 1067 (2013);

(2) R. Siegel et al., CA: A Cancer Journal for Clinicians, 66, 7 (2016);

(3) X. Lin et al., Cancer Res., 63, 3413 (2003);

(4) F. A. Nasrallah et al., J. Cereb. Blood Flow Metab., 33, 1270 (2013);

(5) M. Rivlin et al., Sci. Rep., 3, 3045 (2013);

(6) L.E. Raez, et al., Cancer Chemother Pharmacol 71 , 523 (2013);

(7) P. Ronner, Gen. Comp. Endocrinol., 81 , 276 (1991 );

(8) M. F. Mohd Mustapa et al., Bioconj. Chem., 20, 518 (2009);

(9) N. Mitchell et al., Biomaterials, 34, 1 179 (2013).

Example 12 - liposomal encapsulation of glucose can avoid an insulin response.

Experiments were performed in vivo which verified that encapsulation of glucose in liposomes shields the glucose and avoids it triggering an insulin response. Experimental

4 fat rats (500 grams each) were injected with free glucose and glucose encapsulated inside liposomes (Lipo-glucose) at the same overall concentration.

Prior to the injection of glucose or Lipo-glucose baseline blood glucose levels were monitored. 1 ml. of either glucose or Lipo-glucose was injected via IV root (tail injection) and measurements were taken up to 1 hour post injection with a glucose meter. Animals were fasted for 20 hours prior the experiment and they were kept under the effect of 2% isofluorane in air throughout the experiment.

As shown in Figure 26, both glucose and lipo-glucose resulted in a rapid increase in blood glucose. Within the timeframe observed no insulin response (resulting in sharp drop in measured glucose to near baseline) was observed for either glucose or Lipo-glucose, suggesting that the fat rats are insulin resistant (either pro-diabetic or have already developed type II diabetes).

Generally, injection of Lipo-glucose led overall to a lower blood glucose level. Although the potential for leakage from the liposomes cannot be excluded in these experiments, nevertheless the lower peak value of blood glucose, and the fact that it was reached more quickly, and reduced more quickly, indicates the potential benefits of using Lipo-glucose for human imaging in vivo.

For all the rats glucose blood levels dropped to baseline levels after 2 hours from the end of the experiment.

In separate experiments we verified that the reading of the glucose meter was zero in the case of Lipo-glucose but read glucose normally indicating that the liposomes did not leak under the conditions tested and the encapsulated glucose was not detectable by the enzymes forming the basis of the glucose detection kit. This supports the hypothesis that encapsulated glucose may be used more safely in diabetic patients.

Conclusion from Example 12 Old, fat rats were used as a mammalian model of older, obese patients who are at risk of developing type II diabetes (or have already developed this).

Additionally, generally, injection of Lipo-glucose led overall to a lower blood glucose level. This confirms that the liposomal encapsulation may be used to make GlucoCEST more accessible to older/obese patients with type II diabetes. Furthermore liposomal encapsulation may be used to make GlucoCEST imaging results more robust and reproducible, as it reduces fluctuations in glucose response seen when injecting glucose directly into different patients.

Example 13 - further evidence for the use of Lipo-2DG as a theranostic 10 colon and rectal cancer cell lines were screened and their response to free 2DG and liposomally encapsulated 2-DG (Lipo-2DG) was measured. In these experiments neither Lipo-2DG nor free 2-DG appear to act consistently as radiosensitisers in these cell lines.

Importantly, however, Lipo-2DG was shown to be much more effective at killing RKO colon cancer cells than free 2-DG (see Figure 27). This supports the utility of Lipo-2DG as a theragnostic agent to image cancer cells and deliver a therapeutic dose of an effective anticancer drug.

Moreover, the results Example 12 indicate that lipo-2DG will shield this cytotoxic drug from toxic off-target effects and also from triggering an immune response, further supporting this utility.

A significant increase in cell death was also observed using Lipo-2DG versus free 2-DG in the SW1417 colon cancer cell line (5 mM 2-DG concentration) - See Figure 28. Two empty liposome controls were used to check that the liposomes themselves do not cause cell death, the empty liposomes were made up in either PBS or the cell media that was used for the clonogenic assays. More cell death was observed for 2-DG liposomes dialysed into in 0.25 M NaCI than 1.6 * PBS, although (without wishing to be bound by theory) it is believed the high salt content may itself have contributed to the cell death in that instance.