Compounds, extracellular vesicles and uses thereof

Abstract

The present disclosure concerns a compound for in situ labelling and synchronously promoting the secretion of extracellular vesicles from cells. The compound comprises a sugar or glucosamine linked to a drug. The drug may be a photosensitizer. The present disclosure also concerns the extracellular vesicle thereof, and methods of treating a tumor and / or a proliferative disease.

Description

[0001] Compounds, Extracellular Vesicles and Uses Thereof

[0002] Priority

[0003] The present application claims priority to Chinese patent application CN 202410952522.5, filed on 16 July 2024, the content of which is herein incorporated in its entirety.

[0004] Technical Field

[0005] The present invention relates, in general terms, to glucose-modified compounds and their uses in therapy thereof. The present disclosure also relates to extracellular vesicles comprising the glucose-modified compounds and the methods of producing the extracellular vesicles thereof.

[0006] Background

[0007] In recent decades, with the rapid development of nanotechnology, numerous synthetic nano-scaled drug delivery systems, such as liposomes, metal nanoparticles and polymeric micelles, have been developed. These nanoformulations protect drugs from premature degradation, enhance accumulation at diseased sites, and minimize off-target effects, owing to their unique features such as high drug-loading capacity, surface functionalization with targeting ligands, and the enhanced permeability and retention effect. However, several issues associated with nano-drug delivery systems remain to be addressed, including poor biocompatibility, nonspecific distribution, high immunogenicity and rapid body clearance.

[0008] Extracellular vesicles (EVs) have emerged as a natural alternative for therapeutic cargo delivery, due to their low immunogenicity and high biocompatibility compared with synthetic nanomaterials. Cancer cell derived EVs typically exhibit enhanced immune escape and homologous targeting ability based on membrane-specific protein profiles inherited from the parent cells, making them more favorable candidates for drug delivery in cancer therapy. Currently, post-loading strategy is commonly adopted for drug loading into EVs through chemical or physical methods such as electroporation, extrusion and chemical precipitation, followed by repurification of EVs. However, these postloading approaches may compromise the biofunctions of EVs due to the adverse effects associated with physical or chemical interventions. For example, electroporation often induces EV aggregation, while extrusion can disrupt the integrity of EV membrane structures. Pre-loading is an emerging method allowing continuous and easy production of functional EVs without damaging the membrane integrity by gene transfection or co-incubation of cells with drugs. However, the current pre-loading strategies remain in their infancy as they not only suffers from low efficiency but also restricts the loading of certain biomolecules, such as RNAs and proteins, which are more suitable for transfection. Therefore, it is highly desirable to develop a simple and efficient drug loading strategy for EVs.

[0009] It would be desirable to overcome or alleviate at least one of the abovedescribed problems.

[0010] Summary

[0011] The present disclosure provides a compound of formula (I), or a salt, solvate or stereoisomer thereof: wherein

[0012] X is a heteroatom selected from 0 or NR;

[0013] R is selected from H and optionally substituted alkyl; and Ri is a moiety derived from a photosensitiser.

[0014] In some embodiments, the compound is a compound of Formula (la) : wherein

[0015] L is a linker;

[0016] X is a heteroatom selected from 0 or NR;

[0017] R is selected from H and optionally substituted alkyl; and

[0018] R2 is a moiety comprising a n conjugated system.

[0019] In some embodiments, L is a non-cleavable linker.

[0020] In some embodiments, L is a linker having a chain length of 2 to 20 atoms.

[0021] In some embodiments, L comprises C1-C20 alkyl and / or 1 to 10 monomeric units of ethylene glycol. ected from wherein n is independently an integer selected from 1 to 10; and

[0022]

[0023] In some embodiments, the compound is selected from wherein n is an integer selected from 1 to 10.

[0024] In some embodiments, the compound is characterised by a UV / vis absorption of about 400 nm to about 600 nm.

[0025] In some embodiments, the compound is characterised by UV / vis emission of about 600 nm to about 900 nm.

[0026] In some embodiments, the compound is capable of exhibiting aggregation- induced emission (AIE).

[0027] In some embodiments, the compound is capable of generating reactive oxygen species within 30 sec when irradiated with light. The present disclosure also provides an extracellular vesicle comprising a compound as disclosed herein, or a salt, solvate or stereoisomer thereof.

[0028] In some embodiments, the extracellular vesicle is derived from a cancer cell.

[0029] In some embodiments, the compound is encapsulated within the extracellular vesicle.

[0030] In some embodiments, the extracellular vesicle is characterised by a particle size of about 120 nm to about 200 nm.

[0031] In some embodiments, the extracellular vesicle is characterised by a labelling efficiency of more than about 70%.

[0032] In some embodiments, the extracellular vesicle is characterised by a drugloading capability (compound in EV) about 0.1 % to about 5 % (compound / EV, wt%).

[0033] In some embodiments, the extracellular vesicle is characterised by a zeta potential of about -7 mV to about -8.5 mV.

[0034] In some embodiments, the extracellular vesicle is characterised by a purity of more than about 90%.

[0035] The present disclosure also provides a pharmaceutical composition comprising a glucose-modified compound or an extracellular vesicle as disclosed herein, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof.

[0036] The present disclosure also provides a method of preparing an extracellular vesicle (EV), comprising : a) contacting a cell with a compound as disclosed herein, and b) culturing the cell in the dark under predetermined conditions in order to generate the EV.

[0037] In some embodiments, the cell is a cancer cell. In some embodiments, the compound is provided at a concentration of less than about 100 pM.

[0038] In some embodiments, the cell is contacted with the compound for a duration of at least 20 min.

[0039] In some embodiments, the method further comprises a step after step b) of isolating the extracellular vesicle from the cell.

[0040] In some embodiments, the method is characterized by at least a 4 fold increase in production of EV relative to a control, wherein the control is a cell uncontacted with the compound.

[0041] The present disclosure also provides a method of treating a tumor and / or a proliferative disease in a subject in need thereof, comprising administering a compound, or an extracellular vesicle as disclosed herein or a pharmaceutically acceptable salt, solvate or stereoisomer thereof to the subject.

[0042] In some embodiments, the proliferative disease is cancer or an inflammatory disease.

[0043] In some embodiments, the method is selective to tumor and / or proliferative disease compared to normal cell.

[0044] In some embodiments, the method further comprises irradiating the compound or extracellular vesicle in order to photodynamically treat the subject.

[0045] The present disclosure provides a compound, or an extracellular vesicle as disclosed herein or a pharmaceutically acceptable salt, solvate or stereoisomer thereof for use in treating a tumor and / or a proliferative disease.

[0046] The present disclosure provides a use of a compound, or an extracellular vesicle as disclosed herein or a pharmaceutically acceptable salt, solvate or stereoisomer thereof in the manufacture of a medicament for the treatment of a tumor and / or a proliferative disease. Brief description of the drawings

[0047] Embodiments of the present invention will now be described, by way of nonlimiting example, with reference to the drawings in which :

[0048] Figure 1 shows the synthesis of TBG.

[0049] Figure 2 shows 1H NMR spectrum of compound TB in methanol-d.

[0050] Figure 3 shows 13C NMR spectrum of compound TB in methanol-d.

[0051] Figure 4 shows ESI-Mass spectrum of compound TB.

[0052] Figure 5 shows 1H NMR spectrum of compound TBG in methanol-d.

[0053] Figure 6 shows 13C NMR spectrum of compound TBG in methanol-d.

[0054] Figure 7 shows ESI-Mass spectrum of compound TBG.

[0055] Figure 8 shows UV / Vis absorption and photoluminescence spectra of TB and TBG.

[0056] Figure 9 shows a) PL spectra of TBG in toluene / methanol mixtures with different toluene fractions, b) The plot of relative emission intensity (1 / 10) at 720 nm versus the composition of the toluene / methanol mixtures of TBG.

[0057] Figure 10 shows a-d) Fluorescence spectra of DCF formed from the oxidation of DCFH (10 pM) in an aqueous solution. ROS species were investigated in the presence of PBS, Ce6, TB and TBG upon white light irradiation (400-700 nm, 20 mW cm-2for 150 s), respectively, e) summary of DCF intensity at a wavelength of 525 nm.

[0058] Figure 11 shows cell viability of HeLa in TBG culture medium for 24 h under dark condition. The cell viability was analyzed by MTT assay. n = 3.

[0059] Figure 12 shows TEM images of HeLa cells after culturing with TBG (20 pM) for 30 mins (HeLa cells without TBG treatment was set as the control).

[0060] Figure 13 shows confocal images of fluorescence imaging of HeLa cells after culturing with TB or TBG for 30 mins. The fluorescence was from TB or TBG (ex = 493 nm, em = 750 nm). Scale bar = 10 pm.

[0061] Figure 14 shows fluorescence imaging of TBG in HeLa cells after culturing for different time. The fluorescence was from TBG (ex = 493 nm, em = 750 nm). Scale bar = 10 pm.

[0062] Figure 15 shows schematic diagram of EVs collection by differential ultracentrifugation. TBG were cultured with HeLa cells in a dark environment at 37°C under a humidified atmosphere with 5% CO2.

[0063] Figure 16 shows a) images of EVs, TBG and TBG-EVs observed under natural light (NL) and UV light (365 nm). b) Confocal image of extracted TBG-EVs. (ex = 493 nm, em = 750 nm).

[0064] Figure 17 shows flow cytometry data were analyzed on NanoFCM based on collected a) pure EV and b) TBG-EVs in PBS. The samples were diluted 1000 times using PBS before measurements. PC5 channel with the excitation and emission wavelengths at 488 and 670 nm was collected from TBG fluorescence signal.

[0065] Figure 18 shows a) zeta potential of TBG-EVs and EVs. b) Stability test by DLS (around 2.0 x 1010particles / mL TBG-EVs and EVs stored in PBS at 4 °C for 7 days. Inner: TEM image with negative stain of TBG-EV. Scale bar = 200 nm). Figure 19 shows western blot analysis of HeLa cell lysate, EVs and TBG-EVs.

[0066] Figure 20 shows the yield of EVs obtained from HeLa cells treated with TBG and PBS was assessed using NanoFCM.

[0067] Figure 21 shows RNA sequencing analysis on the promotion of EV yields by TBG. a) The volcano plot shows the highly differentially expressed genes between HeLa cells treated with TBG and without TBG detected by RNA sequencing analysis (n = 3). Compared with HeLa group, the TBG-treated HeLa group showed downregulated expression of 394 genes and upregulated expression of 19 genes, b) GO term functional enrichment analysis of differentially expressed RNA genes. The horizontal axis represents the value of the significance test -loglO(P), and the vertical axis represents the kinds of biological processes.

[0068] Figure 22 shows a-b) Gene set enrichment analysis (GSEA) was performed to compare the gene sets involved in the transmembrane transporter activity and energy production. (NES means normalized enrichment score, FDR means false discovery rate, and FDR < 0.05).

[0069] Figure 23 shows flow cytometry analysis of cellular median fluorescence intensity (MFI) in HeLa cells following TBG treatment, with or without prior Cyt- B pretreatment.

[0070] Figure 24 shows EV yield from HeLa cells with or without pretreating with Cyt- B for 20 min, followed by GlcN, TB, and TBG treatment, were evaluated using NanoFCM. Statistical significance was analyzed via one-way ANOVA test followed by Tukey's post hoc test, **** p < 0.0001, *** P < 0.001, ** p < 0.01.

[0071] Figure 25 shows GLUT-1 immunocytochemical staining of HeLa cells untreated or treated with GlcN, TB, and TBG. The green fluorescence (GLUT-1) was from the secondary antibody Alexa 488 (ex = 488 nm, em = 530-600 nm) and blue fluorescence was from Hoechst (ex = 405 nm, em = 420-500 nm). Scale bar = 100 pm.

[0072] Figure 26 shows ATP concentration of HeLa cells under different treatment conditions, including untreated, GIcN-treated, TB-treated, and TBG-treated groups. Data presented as mean ± SD, n = 3. Statistical significance was analyzed via one-way ANOVA test followed by Tukey's post hoc test, **** p < 0.0001, *** P < 0.001, ** P < 0.01.

[0073] Figure 27 shows schematic illustration of in situ generation process of cancer cell-derived TBG-EVs.

[0074] Figure 28 shows Fluorescence imaging of TBG and TB in (A) MDA-MB-231 and (B) A549 and corresponding EV yield of (C) MDA-MB-231 and (D) A549. The red fluorescence was from TBG or TB (ex = 493 nm, em = 720 nm). Scale bar = 10 pm. Data presented as mean ± SD, n = 3. Statistical significance was analyzed via one-way ANOVA test, P < 0.0001.

[0075] Figure 29 shows in vitro biological functions of HeLa cell-derived TBG-EVs. Confocal images of the selectivity of TBG-EVs towards HeLa, A549, MDA-MB- 231, NIH-3T3 and HEK-293T cells. TBG-EVs derived from HeLa cells were cocultured with cells at 37 °C for 6 h under dark conditions, followed by Hoechst staining. The red fluorescence was from TBG (ex = 493 nm, em = 650-800 nm) and blue fluorescence was from Hoechst (ex = 405 nm, em = 500-540 nm). Scale bar = 25 pm.

[0076] Figure 30 shows a) flow cytometry analysis and b) corresponding data analysis of selectivity of TBG-EVs to different cells. TBG-EVs were co-cultured with cells at 37 °C for 6 h under dark conditions.

[0077] Figure 31 shows intracellular R.OS generation analysis by incubating cells with TBG-EVs at 37 °C for 6 h under dark conditions, followed by incubation with DCFH-DA and subsequent light irradiation (60 mW cm-2) for 5 min. The green fluorescence was from DCFH-DA with ex = 488 nm and Zem =500-550 nm. Scale bar = 50 pm. Figure 32 shows PDT effects of TBG and TBG-EVs on HeLa cells. PDT experiments were conducted by culturing with different concentrations of TBG- EVs with cells at 37 °C for 6 h under dark conditions, followed by light irradiation (60 mW cm-2) for 10 min. The cell viability was analyzed by MTT assay. Data presented as mean ± SD, n = 3.

[0078] Figure 33 shows PDT effects of TBG and TBG-EVs on NIH-3T3 cells. PDT experiments were conducted by culturing with different concentrations of TBG- EVs with cells at 37 °C for 6 h under dark conditions, followed by light irradiation (60 mW cm-2) for 10 min. The cell viability was analyzed by MTT assay. Data presented as mean ± SD, n = 3.

[0079] Figure 34 shows in vivo PDT effect of HeLa cell-derived TBG-EVs in HeLa tumorbearing BALB / c nude mice. A-F) Tumor growth curve of G1-G6 groups at the period of 20-day treatment. Mice received i.v. injection of the following groups via the tail vein: PBS (Gl), EVs (G2), TBG (G3), TBG-EVs (G4), TBG with light irradiation (G5), TBG-EVs with light irradiation (G6). (120 mW cm-2 for 10 min at 8 h post-injection).

[0080] Figure 35 shows a summary of the tumor growth curve of G1-G6 groups at the period of 20-day treatment. Statistical significance was analyzed via one-way ANOVA test followed by Tukey's post hoc test, **** p < 0.0001, * P < 0.05.

[0081] Figure 36 shows photographs of excised tumor tissues from mice in groups Gl- G6 on day 20.

[0082] Figure 37 shows weight of excised tumor tissues from mice in groups G1-G6 on day 20. Statistical significance was analyzed via one-way ANOVA test followed by Tukey's post hoc test, **** p < 0.0001, * P < 0.05.

[0083] Figure 38 shows H&E staining of tumor tissues from G1-G6 groups after 20- day treatments, Scale bar: 50 pm. Data presented as mean ± SD, n = 4.

[0084] Figure 39 shows body weight changes of G1-G6 groups at the period of 20-day treatment. Statistical significance was analyzed via one-way ANOVA test followed by Tukey's post hoc test, **** p < 0.0001, * P < 0.05.

[0085] Figure 40 The H&E staining analysis of major organs (heart, liver, spleen, lung, and kidney) of different formulations (G1-G6), Scale bar: 200 pm.

[0086] Detailed description Glucose is one of the most widely available metabolic substrates for adenosine triphosphate (ATP) production, which plays a key role in promoting EV secretion. Besides, glucose can be uptake into cell-derived EVs via an active and energydependent mechanism mediated by glucose transporters of EVs. Hence, feeding glucose-modified therapeutic drugs into EV-secreting cells offers a feasible strategy to facilitate drug loading onto EVs through the biological metabolism process, potentially achieving a higher yield of EVs while keeping the membrane integrity of EVs intact. One option of such drugs is the photosensitizers (PS), which possess efficient reactive oxygen species (R.OS) generation ability under light irradiation but show low toxicity to the parent cells in the absence of light. Therefore, the inventors focus on PS to ensure that they do not interfere with the activity of parent cells during the generating process of PS-loaded EVs. Moreover, strong photosensitization, high fluorescence and wash-free properties of PS with aggregation-induced emission (AIE) properties make them suitable for EV labelling and tracking. This motivates the inventors to develop PS modified with glucose as a promising attempt to generate PS-engineered EVs through cell glycometabolism for image-guided photodynamic therapy (PDT).

[0087] The present disclosure is predicated on the understanding that the application of naturally occurred and automatically released EVs in cancer therapy is limited by the low secretion efficiency and unsatisfactory therapeutic effect. Therefore, it is believed that employing exogenous materials to cells in-situ enhance the generation of endogenous cancer cell-derived EVs and functionalise them with photosensitisers simultaneously would be an improved strategy for cancer therapy. The generation of EVs from cancer cells requires the consumption of adenosine triphosphate (ATP). Cancer cells are highly dependent on the process of glycolysis to generate ATP, and glucose is one of the most widely available carbohydrate sources for ATP supply by glycolytic metabolism. Derived from the mechanism on EV secretion in cancer cells, it is presumed that photosensitisers (PSs) grafted on glucose as the carbohydrate sources of cancer cells will promote the generation of PS-engineered EVs (PS-EVs).

[0088] Without wanting to be bound by theory, the inventors develop a metabolism- oriented strategy for cancer cells through the design of glucose-conjugated PS for facile generation of PS-engineered EVs in higher yield, leading to an effective and selective PDT ablation of cancer cells (Figure 27).

[0089] In some embodiments, p-D-glucose or derivative thereof was modified with a photosensitizer (PS) to form PS conjugated glucose (PSG), which was further applied as part of substrates for cancer cells. For example, p-D-glucosamine may also be used. In this process, the PS-engineered extracellular vesicles (PS- EVs) were synthesized in situ via an active, energy-dependent mechanism mediated by the transmembrane transporter of EVs. The enhanced yield of PS- engineered EVs was observed by co-culturing cancer cells with PSG as compared to that with p-D-glucose free PS. Gene expression analysis was further conducted to validate the importance of chemically modified glucose to produce cargo-loading EVs. Furthermore, it provides a method for generating cargo- loaded EVs through biological metabolism in higher yield.

[0090] In particular, / ?-D-glucose is modified with l-(4-carboxybutyl)-4-(7-(4- (diphenylamino)phenyl)benzo[c][l,2,5] thiadiazol-4-yl)pyridin-l-ium (named TB) via an amide bond, forming TBG, followed by application as a metabolic substrate for cancer cells to in situ biosynthesize TBG-engineering EVs (TBG- EVs). A higher yield of EVs is observed within 30 min when cells are cocultured with TBG, compared to / ?-D-glucose free TB and control. Moreover, TBG-EVs not only keep the pristine excellent self-recognition ability to parent cells but also exhibit efficient PDT effect. Furthermore, our findings reveal that the high yield of EVs induced by TBG is attributed to the enhanced transmembrane transporter activity and the acceleration of ATP-dependent metabolic processes, emphasizing the crucial role of chemically modified glucose in facilitating the efficient production of cargo-loaded EVs. Importantly, this metabolism-driven strategy is successfully validated in three cell lines, highlighting its broad applicability. Specifically, it led to a 12.7-fold increase in EV yield in MDA-MB- 231 cells (the triple-negative breast cancer cell line), a 5.9-fold increase in A549 cells (the lung carcinoma epithelial cell line), and a 4.3-fold increase in HeLa cells (the cervical cancer cell line).

[0091] Thus, the present disclosure presents a simple and versatile method for engineering EVs, resulting in increased yields and improved effectiveness for biomedical applications. By modifying therapeutic drugs with glucose, its subsequent uptake into EVs-secreting cells facilitate the loading of therapeutic drugs onto EVs through the biological metabolism process while keeping the membrane integrity of EVs intact. Without further modification or washing, the EVs may be used as a therapeutic agent, which simplifies the imaging process and facilitates the in-situ imaging of EVs. A high yield may also be obtained by co-culturing parent cells with PSG compared with g-D-glucose free PS and blank control.

[0092] Cell-derived EVs typically demonstrate enhanced immune evasion and precise homologous targeting capabilities due to their membrane-specific protein profiles inherited from parent cells. This inherent advantage makes them highly suitable as drug delivery carriers for cancer treatment. Photosensitizers exhibit robust fluorescence when exposed to light. Consequently, the collaborative interplay of EVs and PS facilitates the tracking and visualization of intracellular processes during the treatment.

[0093] This method also presents an alternative strategy for labelling and tracking the endogenous formation and secretion of EVs without washing procedure and generating engineered-EV in higher labelling efficiency.

[0094] Accordingly, the present disclosure provides a compound of formula (I), or a salt, solvate or stereoisomer thereof: wherein

[0095] X is a heteroatom selected from 0 or NR;

[0096] R is selected from H and optionally substituted alkyl; and

[0097] Ri is a moiety capable of providing a cancer treatment effect. The compound may be a glucose-modified compound or a glucosamine-modified compound. The compound may comprise a drug or a therapeutic compound.

[0098] The present disclosure provides a compound of formula (I), or a salt, solvate or wherein

[0099] X is a heteroatom selected from 0 or NR;

[0100] R is selected from H and optionally substituted alkyl; and Ri is a moiety derived from a photosensitiser.

[0101] A photosensitizer is a substance that, when exposed to light, becomes activated and can then induce chemical reactions, often leading to cell damage or death. In medicine, particularly in photodynamic therapy (PDT), photosensitizers may be used to target and destroy cancerous or infected cells.

[0102] In some embodiments, the compound is a compound of Formula (la), or a salt, solvate or stereoisomer thereof:

[0103] L is a linker;

[0104] X is a heteroatom selected from 0 or NR;

[0105] R is selected from H and optionally substituted alkyl; and R2 is a moiety comprising a n conjugated system.

[0106] Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and / or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. The invention additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers. "Optically-enriched," as used herein, means that the compound is made up of a significantly greater proportion of one enantiomer. In certain embodiments the compound of the present invention is made up of at least about 90% by weight of a preferred enantiomer. In other embodiments the compound is made up of at least about 95%, 98%, or 99% by weight of a preferred enantiomer. Preferred enantiomers may be isolated from racemic mixtures by any method known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts or prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33: 2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ, of Notre Dame Press, Notre Dame, IN 1972).

[0107] The present disclosure pertains to compounds and their various forms, including ionic forms, tautomers, isomers, polymorphs, pseudopolymorphs, amorphous forms, solvates, co-crystals, chelates, esters, prodrugs, and protected forms. The disclosure also encompasses methods of utilizing these compounds for various purposes. It should be noted that terms like "crystalline form," "polymorph," can be used interchangeably to include all crystalline and amorphous forms, such as polymorphs, pseudopolymorphs, solvates (including hydrates), co-crystals, unsolvated polymorphs (including anhydrates), conformational polymorphs, amorphous forms, and mixtures thereof, unless a specific crystalline or amorphous form is specified. In certain embodiments, the compounds and their subgroups include polymorphs, solvates, co-crystals, isomers, tautomers, and / or oxides. In other embodiments, they may include polymorphs, solvates, and / or co-crystals.

[0108] When X is 0, the Markush structure above shows the structure of glucose. It should be noted that other sugars and isomers thereof may also be used. For example, natural and unnatural sugars may be used. For example, D-glucose and / or L-glucose may be used. Other sugars, such as monosaccharide (hexose such as fructose, galactose) and disaccharide (sucrose, lactose) may be used. These sugars are included within the scope of the disclosure.

[0109] Similarly, the above mentioned modifications may also be made when X is NR.

[0110] In some embodiments, R is H. In some embodiments, R is optionally substituted C1-C5 alkyl .

[0111] The linker L may be an optionally substituted linear or branched (i.e., multiarmed) linker. Each chain of the linker may have a chain length of 0 to 50 atoms, such as 0 to 45 atoms, 0 to 40 atoms, 0 to 35 atoms, 0 to 30 atoms, 0 to 25 atoms, 0 to 20 atoms, 0 to 15 atoms, 0 to 10 atoms, 0 to 8 atoms, 0 to 6 atoms, 0 to 5 atoms, or 0 to 3 atoms. Branched linkers may have 2 to 50 arms.

[0112] In some embodiments, L is a linear chain with a chain length of 1 to 50 atoms. L may be a linear chain with a chain length of 1 to 45 atoms, 1 to 40 atoms, 1 to 35 atoms, 1 to 30 atoms, 1 to 25 atoms, 1 to 20 atoms, 1 to 15 atoms, 1 to 10 atoms, 1 to 8 atoms, 1 to 6 atoms, 1 to 5 atoms, or 1 to 3 atoms. In some embodiments, L is a linker having a chain length of 2 to 20 atoms.

[0113] In some embodiments, L is a branched linker with 2 to 50 branch chains, with each branch chain having a chain length of 1 to 50 atoms. In some embodiments, L is a branched linker with 2 to 8 branch chains, with each branch chain having a chain length of 1 to 50 atoms. In some embodiments, L is a branched linker with 2 to 50 branch chains, with each branch chain having a chain length of 1 to 10 atoms. In some embodiments, L is a branched linker with 2 to 8 branch chains, with each branch chain having a chain length of 1 to 10 atoms.

[0114] In some embodiments, L comprises optionally substituted C1-C20 alkyl and / or 1 to 10 monomeric units of ethylene glycol.

[0115] "Alkyl" as a group or part of a group refers to a straight or branched aliphatic hydrocarbon group, preferably a C1-C50 alkyl. Examples of suitable straight and branched alkyl substituents include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, t-butyl, hexyl, and the like. The group may be a terminal group or a bridging group.

[0116] The term "optionally substituted" herein denotes that the group may or may not be further substituted or fused (so as to form a condensed polycyclic system), with one or more non-hydrogen substituent groups. In certain embodiments the substituent groups are one or more groups independently selected from the group consisting of halogen, =0, =S, alkyl, alkenyl, alkynyl, azidoalkyl, haloalkyl, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, heteroaryl alkyl, arylalkyl, cycloalkylalkenyl, heterocycloalkylalkenyl, arylalkenyl, heteroarylalkenyl, cycloalkylheteroalkyl, heterocycloalkylheteroalkyl, arylheteroalkyl, heteroarylheteroalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkoxycycloalkyl, alkoxyheterocycloalkyl, alkoxyaryl, alkoxyheteroaryl, alkoxycarbonyl, alkylaminocarbonyl, alkenyloxy, alkynyloxy, cycloalkyloxy, cycloalkenyloxy, heterocycloalkyloxy, heterocycloalkenyloxy, aryloxy, phenoxy, benzyloxy, heteroaryloxy, arylalkyloxy, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulphonylamino, sulphinylamino, sulphonyl, alkylsulphonyl, arylsulphonyl, aminosulphonyl, sulphinyl, alkylsulphinyl, arylsulphinyl, aminosulphinylaminoalkyl, COOH, C=O, C(O)O, C(O)NH, NHC(O), NHC(O)O, NHC(O)NH, C(=NOH), SH, S, 0 and acyl.

[0117] The linker may be cleavable or non-cleavable. Cleavable linkers are linkers that can be cleaved under mild conditions, i.e. conditions under which the activity of the photosensitizer is not affected. Many known linkers fall in this category and are described below.

[0118] Disulfide containing linkers are linkers cleavable through disulfide exchange, which can occur under physiological conditions.

[0119] Acid-labile linkers are linkers cleavable at acid pH. For example, certain intracellular compartments, such as endosomes and lysosomes, have an acidic pH (pH 4-5), and provide conditions suitable to cleave acid-labile linkers.

[0120] Linkers that are photo-labile are useful at the body surface and in many body cavities that are accessible to light. Furthermore, infrared light can penetrate tissue.

[0121] Some linkers can be cleaved by peptidases. Only certain peptides are readily cleaved inside or outside cells, see e.g. Trouet et al., 79 Proc. Natl. Acad. Sci. USA, 626-629 (1982) and Umemoto et al. 43 Int. J. Cancer, 677-684 (1989). Furthermore, peptides are composed of o-amino acids and peptidic bonds, which chemically are amide bonds between the carboxylate of one amino acid and the o-amino group of a second amino acid. Other amide bonds, such as the bond between a carboxylate and the s-amino group of lysine, are understood not to be peptidic bonds and are considered non-cleavable.

[0122] Some linkers can be cleaved by esterases. Only certain esters can be cleaved by esterases present inside or outside cells. Esters are formed by the condensation of a carboxylic acid and an alcohol. Simple esters are esters produced with simple alcohols, such as aliphatic alcohols, and small cyclic and small aromatic alcohols.

[0123] A non-cleavable linker is any chemical moiety that is capable of linking a photosensitizer to glucose in a stable, covalent manner and does not fall under the categories listed above as cleavable linkers. Thus, non-cleavable linkers are substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase- induced cleavage, and disulfide bond cleavage.

[0124] "Substantially resistant" to cleavage means that the chemical bond in the linker or adjoining the linker in at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% of the compound population remains non-cleavable by an acid, a photolabile-cleaving agent, a peptidase, an esterase, or a chemical or a physiological compound that cleaves the chemical bond (such as a disulfide bond) in a cleavable linker, for within a few hours to several days of treatment with any of the agents described above.

[0125] Furthermore, "non-cleavable" refers to the ability of the chemical bond in the linker or adjoining to the linker to withstand cleavage induced by an acid, a photolabile-cleaving agent, a peptidase, an esterase, or a chemical or a physiological compound that cleaves a disulfide bond, at conditions under which the photosensitizer or the compound does not lose its activity.

[0126] Examples of cross-linking reagents that form non-cleavable linkers between the photosensitizer and glucose comprise a maleimido- or haloacetyl-based moiety. Such cross-linking reagents comprising a maleimido-based moiety include N- succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N- succinimidyl-4-(N-maleimidomethyl)-cyclohexane-l-carboxy-(6- amidocaproate), which is a "long chain" analog of SMCC (LC-SMCC), K- maleimidoundecanoic acid N-succinimidyl ester (KMUA), y-maleimidobutyric acid N-succinimidyl ester (GMBS), s-maleimidocaproic acid N- hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(a-maleimidoacetoxy)-succinimide ester [AMAS], succinimidyl- 6-(P-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4-(p- maleimidophenyl)-butyrate (SMPB), and N-(p-maleimidophenyl)isocyanate (PMPI).

[0127] In some embodiments, the linker L is cleavable. L may be cleavable at an end adjacent to R2. The linker may be hydrolytically stable, i.e., the linker is substantially stable in water and does not react with water at useful pH values, including but not limited to, under physiological conditions for an extended period of time. The linker can be hydrolytically unstable, i.e., the linker is degradable in water or in aqueous solutions, including for example, blood. Linkers can be susceptible to cleavage (cleavable linker), such as, acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active. In this regard, the linker can be a pro-charged linker (linker which will become charged after cell processing), a hydrophilic linker, or a dicarboxylic acid-based linker.

[0128] The linker can be enzymatically unstable or degradable by one or more enzymes. By way of example only, polyethylene glycol (PEG) and related polymers may include degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule. Such degradable linkages include, but are not limited to, ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biologically active agent, wherein such ester groups generally hydrolyze under physiological conditions to release the biologically active agent. Other hydrolytically degradable linkages include but are not limited to carbonate linkages; imine linkages resulting from reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; hydrazone linkages which are a reaction product of a hydrazide and an aldehyde; acetal linkages that are the reaction product of an aldehyde and an alcohol; orthoester linkages that are the reaction product of a formate and an alcohol; peptide linkages formed by an amine group, including but not limited to, at an end of a polymer such as PEG, and a carboxyl group of a peptide.

[0129] The linker may be conjugated to the drug / photosensitizer and / or sugar / glucosamine via a conjugating group. The conjugating group is a functional group capable of reacting with another functional group to form a conjugated moiety. For example, condensation reactions may occur between a carboxylate group and hydroxyl or amino group. Such a moiety may include, but is not limited to, esters, carbonates, carbamates, imines phosphate esters, oximes, hydrazones, acetals, orthoesters, amides, thioethers, succinimides, triazoles, isoxazolines, pyridazines, and peptide linkages.

[0130] In some embodiments, the conjugating group is a click functional group capable of coupling with another click functional group via a click reaction to form a click adduct. The click reaction may be, for example, an SN2 reaction, a Diels-Alder reaction, a conjugate addition reaction, or a cycloaddition reaction (which may or may not utilize a Cu(I) catalyst). Examples of click functional groups include but are not limited to alkyne groups (such as a linear alkynyl, cyclooctynyl, and dibenzocyclooctynyl), alkene groups (such as trans-cyclooctenyl and norbornyl), diene groups (such as tetrazinyl and tetrazolyl), azide, maleimide, thiol, 1,3- nitrone, aldehyde, ketone, hydrazine, and hydroxylamine.

[0131] The table below exemplifies different pairs of click conjugates that will react with each other to form a covalent bond. For example, the linker may comprise a click functional group (such as an azide moiety) at one of its ends, for coupling to an alkynyl moiety on the linkage group or on a ligand via a click cycloaddition reaction. Functional groups for conjugation (e.g., click functional groups) can be incorporated into the ligand, linkage group or linker by methods known in the art.

[0132] In some embodiments, L is selected from wherein n is independently an integer selected from 1 to 10; and bond to R.2 or amino.

[0133] In some embodiments, R.2 is a moiety derived from a photosensitiser. The photosensitiser is thus linked to glucose via a linker. As mentioned, the photosensitiser is useful for treating diseases via PDT. Photosensitizers absorb light at a specific wavelength, becoming excited. This excited state allows them to interact with oxygen, producing reactive oxygen species (ROS) like singlet oxygen. The ROS are highly reactive and can damage or destroy nearby cells and tissues.

[0134] Photosensitizers are able to absorb light at specific wavelengths due to a core of conjugated n-electrons. In many cases, this conjugation allows for the absorption of light in the visible or near-infrared (NIR) range, and the delocalization of electrons, which is crucial for energy transfer and the generation of reactive oxygen species. The extent of conjugation influences the wavelength of light absorbed by the photosensitizer. Examples of structures having conjugated n-electrons includes, but is not limited to, tetrapyrrole, porphyrin, chlorin, and phthalocyanine.

[0135] Examples of photosensitizers include, but are not limited to, methylene blue, toluidine blue, Rose Bengal, eosin, xanthene dye, erythrosine, photofrin, 5- aminolevulinic acid (ALA), methyl aminolevulinate (Metvixia), Verteporfin, benzoporphyrin, Temoporfin (Foscan), curcumin, hypericin, chlorophyllin, motexafin lutetium, padoporfin, and bacteriochlorin. The photosensitizer may be linked to the linker L at any site. Preferably, the linkage point does not significantly affect its conjugation system. For example, amidation and / or click chemistry may be used to conjugate the photosensitizer to the linker and to the glucose.

[0136] In some embodiments, R2 is capable of exhibiting aggregation-induced emission (AIE). In some embodiments, R.2 is characterised in that the cancer treatment effect is the generation of reactive oxygen under light irradiation.

[0137] In some embodiments, R2 is selected from

[0138] In some embodiments, the compound is selected from wherein n is an integer selected from 1 to 10.

[0139] In some embodiments, the compound is characterised by a UV / vis absorption of about 400 nm to about 600 nm.

[0140] In some embodiments, the compound is characterised by UV / vis emission of about 600 nm to about 900 nm.

[0141] In some embodiments, the compound is capable of exhibiting aggregation- induced emission (AIE).

[0142] In some embodiments, the compound is capable of generating reactive oxygen species when irradiated with light. In some embodiments, the compound is capable of generating reactive oxygen species when irradiated with light within 30 sec.

[0143] The present disclosure also provides a method of preparing the compound, comprising subjecting glucosamine to an amidation reaction with a reactant, wherein the reactant has a -OH moiety. The present disclosure also provides a method of preparing the compound, comprising subjecting a sugar such as glucose to a carboxylation reaction with a reactant, wherein the reactant has a -OH moiety. In other embodiments, the reactant has a -C(O)OH moiety.

[0144] The reactant may be a drug, or may be a photosensitizer. The reactant may be a linker conjugated drug, or a linker conjugated photosensitizer.

[0145] In some embodiments, the method is for preparing the compound wherein n is an integer selected from 1 to 10. In some embodiments, the method comprises subjecting glucosamine, photosensitiser, benzotriazole- N,N,N',N'-tetramethyluronium hexafluorophosphate, 4-(dimethylamino)pyridine and N,N- diisopropylethylamine to an amide reaction in a solvent, cooling to room temperature, extracting, and purifying by column chromatography to obtain the compound, wherein the photosensitizer has a structural formula of in some embodiments, the method is performed at a temperature of about 35 °C to about 40°C. In some embodiments, the method is performed for a duration of about 24 h to about 36 h. In some embodiments, the solvent is dimethylformamide.

[0146] The present disclosure also provides an extracellular vesicle comprising the compound as disclosed herein, or a salt, solvate or stereoisomer thereof.

[0147] In some embodiments, the extracellular vesicle is derived from a cancer cell.

[0148] In some embodiments, the compound is encapsulated within the extracellular vesicle.

[0149] In some embodiments, the extracellular vesicle is characterised by a particle size of about 120 nm to about 200 nm. In some embodiments, the particle size is about 120 nm to about 180 nm, about 120 nm to about 160 nm, or about 140 nm to about 160 nm.

[0150] In some embodiments, the extracellular vesicle is characterised by a labelling efficiency of more than about 70%. This means that 70 out of 100 extracellular vesicles are labelled with the compound of Formula (I). In some embodiments, the labelling efficiency is more than about 75%, about 80%, about 85%, or about 90%. In some embodiments, the extracellular vesicle is characterised by a drugloading capability (compound in EV) about 0.1 % to about 5 % (compound / EV, wt%). This means that about 0.1 g to about 5 g of compound of Formula (I) is loaded in 100 g EVs. In some embodiments, the drug-loading capability is about 0.2 % to about 4 %, about 0.2 % to about 3 %, about 0.2 % to about 2 %, about 0.2 % to about 1 %, about 0.3 % to about 1 %, about 0.4 % to about 1 %, about 0.5 % to about 1 %, about 0.6 % to about 1 %, about 0.7 % to about 1 %, or about 0.8 % to about 1 %.

[0151] In some embodiments, the extracellular vesicle is characterised by a zeta potential of about -7 mV to about -8.5 mV.

[0152] In some embodiments, the extracellular vesicle is characterised by a purity of more than about 90%. The purity may be measured relative to the presence of cell debris. A higher relative amount of cell debris means that the purity is correspondingly lower. In some embodiments, the purity is more than about 92%, about 94%, about 96% or about 98%.

[0153] The present disclosure also provides a pharmaceutical composition comprising a compound or an extracellular vesicle as disclosed herein or a pharmaceutically acceptable salt, solvate or stereoisomer thereof. The pharmaceutical composition may further comprise an excipient.

[0154] The present disclosure also provides a method of preparing an extracellular vesicle (EV), comprising : a) contacting a cell with a compound as disclosed herein, and b) culturing the cell in the dark under predetermined conditions in order to generate the EV.

[0155] In some embodiments, the compound is encapsulated within the EV.

[0156] In some embodiments, the cell is a cancer cell. In some embodiments, the cell is selected from MDA-MB-231 cell (triple-negative breast cancer cell line), A549 cell (lung carcinoma epithelial cell line), HeLa cell (cervical cancer cell line), and a combination thereof.

[0157] In some embodiments, the compound is provided at a concentration of less than about 100 pM. In some embodiments, the compound is provided at a concentration of less than about 90 pM, or about 80 pM.

[0158] In some embodiments, the cell is contacted with the compound for a duration of at least 20 min. In some embodiments, the cell is contacted with the compound for a duration of at least 30 min, 40 min, 50 min or 60 min.

[0159] In some embodiments, the method further comprises a step after step b) of isolating the extracellular vesicle from the cell. The extracellular vesicle may be obtained by lysing the cell and collected using differential ultra-centrifugation.

[0160] In some embodiments, the method is characterized by at least a 4 fold increase in production of EV relative to a control. The control may be a cell uncontacted with the compound. In other embodiments, the increase is at least 4.2 fold, 4.4 fold, 4.6 fold, 4.8 fold, 5 fold, 6 fold, 8 fold, 10 fold, or 12 fold.

[0161] The present disclosure also provides a method of treating a tumor and / or a proliferative disease in a subject in need thereof, comprising administering a therapeutically effective amount of a compound, or an extracellular vesicle as disclosed herein or a pharmaceutically acceptable salt, solvate or stereoisomer thereof to the subject.

[0162] The present disclosure provides a compound, or an extracellular vesicle as disclosed herein or a pharmaceutically acceptable salt, solvate or stereoisomer thereof for use in treating a tumor and / or a proliferative disease.

[0163] The present disclosure provides a use of a compound, or an extracellular vesicle as disclosed herein or a pharmaceutically acceptable salt, solvate or stereoisomer thereof in the manufacture of a medicament for the treatment of a tumor and / or a proliferative disease. The disease may be a disease or disorder of any cell, tissue, organ or system, such as a disease of the liver, kidney, pancreas, blood, heart, brain, lungs, bladder, skin, muscle, bones, cartilage, eye, nose, tongue, ear, vascular system, gastrointestinal system, nervous system, endocrine system, reproductive system, or immune system. In some embodiments, the proliferative disease is cancer or an inflammatory disease. In some embodiments, the proliferative disease is selected from breast cancer, cervical cancer, and lung cancer.

[0164] In some embodiments, the method is selective to tumor and / or proliferative disease compared to normal cell. The selectivity may be characterised as shown in Figure 29 and 30. As shown in the fluorescence microscopy images of Figure 29, bright fluorescence signal from TBG in HeLa cells and weak red fluorescence in A549 and MDA-MB-231 cells after TBG-EVs treatment were observed. Indiscernible red fluorescent signal was displayed in NIH-3T3 and HEK293 cells after being treated with TBG-EVs, which demonstrated the selectivity. As shown in Figure 30, TBG-EVs could be uptake by HeLa cells well, followed by decreased TBG-EVs uptake by A549 and MDA-MB-231 cells, while there is almost no uptake of TBG-EVs by NIH-3T3 and HEK293 cells, which were consistent with the results from the fluorescence microscopy images. The selectivity may be characterised by a fluorescence intensity of a target cell relative to a control cell (for example NIH-3T3 or HEK-293T). In some embodiments, the selectivity is about 1 time to about 20 times, about 1.5 times to about 20 times, about 2 times to about 20 times, about 3 times to about 20 times, about 4 times to about 20 times, about 5 times to about 20 times, about 5 times to about 18 times, about 5 times to about 16 times, about 5 times to about 14 times, about 5 times to about 12 times, about 5 times to about 10 times, about 6 times to about 10 times, about 7 times to about 10 times, or about 8 times to about 10 times.

[0165] In some embodiments, the method further comprises irradiating the compound or extracellular vesicle in order to photodynamically treat the subject. The irradiation may be at about 480 nm to about 560 nm, or preferably 488 nm.

[0166] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredient(s) to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition or formulation would be administered. Such formulations are sterile. "Pharmaceutically acceptable" carriers and excipients (vehicles, additives) are those which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed.

[0167] A "proliferative disease" refers to a disease that occurs due to abnormal growth or extension by the multiplication of cells (Walker, Cambridge Dictionary of Biology; Cambridge University Press: Cambridge, UK, 1990). A proliferative disease may be associated with : 1) the pathological proliferation of normally quiescent cells; 2) the pathological migration of cells from their normal location (e.g., metastasis of neoplastic cells); 3) the pathological expression of proteolytic enzymes such as the matrix metalloproteinases (e.g., collagenases, gelatinases, and elastases); or 4) the pathological angiogenesis as in proliferative retinopathy and tumor metastasis. Exemplary proliferative diseases include cancers (i.e., "malignant neoplasms"), benign neoplasms, angiogenesis, inflammatory diseases, autoinflammatory diseases, and autoimmune diseases.

[0168] The terms "neoplasm" and "tumor" are used herein interchangeably and refer to an abnormal mass of tissue wherein the growth of the mass surpasses and is not coordinated with the growth of a normal tissue. A neoplasm or tumor may be "benign" or "malignant," depending on the following characteristics: degree of cellular differentiation (including morphology and functionality), rate of growth, local invasion, and metastasis. A "benign neoplasm" is generally well differentiated, has characteristically slower growth than a malignant neoplasm, and remains localized to the site of origin. In addition, a benign neoplasm does not have the capacity to infiltrate, invade, or metastasize to distant sites. Exemplary benign neoplasms include, but are not limited to, lipoma, chondroma, adenomas, acrochordon, senile angiomas, seborrheic keratoses, lentigos, and sebaceous hyperplasias. In some cases, certain "benign" tumors may later give rise to malignant neoplasms, which may result from additional genetic changes in a subpopulation of the tumor's neoplastic cells, and these tumors are referred to as "pre-malignant neoplasms." An exemplary pre-malignant neoplasm is a teratoma. In contrast, a "malignant neoplasm" is generally poorly differentiated (anaplasia) and has characteristically rapid growth accompanied by progressive infiltration, invasion, and destruction of the surrounding tissue. Furthermore, a malignant neoplasm generally has the capacity to metastasize to distant sites.

[0169] As used herein, the term "cancer" refers to a malignant neoplasm (Stedman's Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990). Exemplary cancers include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma; endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarcinoma); Ewing's sarcoma; eye cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B- cell CLL, T-cell CLL)); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLiySLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenstrom's macroglobulinemia), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma); a mixture of one or more leukemia / lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease); hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis; kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer (e.g., Paget's disease of the penis and scrotum); pinealoma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget's disease of the vulva).

[0170] The term "angiogenesis" refers to the formation and the growth of new blood vessels. Normal angiogenesis occurs in the healthy body of a subject for healing wounds and for restoring blood flow to tissues after injury. The healthy body controls angiogenesis through a number of means, e.g., angiogenesisstimulating growth factors and angiogenesis inhibitors. Many disease states, such as cancer, diabetic blindness, age-related macular degeneration, rheumatoid arthritis, and psoriasis, are characterized by abnormal (i.e., increased or excessive) angiogenesis. Abnormal angiogenesis refers to angiogenesis greater than that in a normal body, especially angiogenesis in an adult not related to normal angiogenesis (e.g., menstruation or wound healing). Abnormal angiogenesis can provide new blood vessels that feed diseased tissues and / or destroy normal tissues, and in the case of cancer, the new vessels can allow tumor cells to escape into the circulation and lodge in other organs (tumor metastases).

[0171] As used herein, an "inflammatory disease" refers to a disease caused by, resulting from, or resulting in inflammation. The term "inflammatory disease" may also refer to a dysregulated inflammatory reaction that causes an exaggerated response by macrophages, granulocytes, and / or T-lymphocytes leading to abnormal tissue damage and / or cell death. An inflammatory disease can be either an acute or chronic inflammatory condition and can result from infections or non-infectious causes. Inflammatory diseases include, without limitation, atherosclerosis, arteriosclerosis, autoimmune disorders, multiple sclerosis, systemic lupus erythematosus, polymyalgia rheumatica (PMR), gouty arthritis, degenerative arthritis, tendonitis, bursitis, psoriasis, cystic fibrosis, arthrosteitis, rheumatoid arthritis, inflammatory arthritis, Sjogren's syndrome, giant cell arteritis, progressive systemic sclerosis (scleroderma), ankylosing spondylitis, polymyositis, dermatomyositis, pemphigus, pemphigoid, diabetes (e.g., Type I), myasthenia gravis, Hashimoto's thyroiditis, Graves' disease, Goodpasture's disease, mixed connective tissue disease, sclerosing cholangitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, pernicious anemia, inflammatory dermatoses, usual interstitial pneumonitis (UIP), asbestosis, silicosis, bronchiectasis, berylliosis, talcosis, pneumoconiosis, sarcoidosis, desquamative interstitial pneumonia, lymphoid interstitial pneumonia, giant cell interstitial pneumonia, cellular interstitial pneumonia, extrinsic allergic alveolitis, Wegener's granulomatosis and related forms of angiitis (temporal arteritis and polyarteritis nodosa), inflammatory dermatoses, hepatitis, delayed-type hypersensitivity reactions (e.g., poison ivy dermatitis), pneumonia, respiratory tract inflammation, Adult Respiratory Distress Syndrome (ARDS), encephalitis, immediate hypersensitivity reactions, asthma, hayfever, allergies, acute anaphylaxis, rheumatic fever, glomerulonephritis, pyelonephritis, cellulitis, cystitis, chronic cholecystitis, ischemia (ischemic injury), reperfusion injury, allograft rejection, host-versus-graft rejection, appendicitis, arteritis, blepharitis, bronchiolitis, bronchitis, cervicitis, cholangitis, chorioamnionitis, conjunctivitis, dacryoadenitis, dermatomyositis, endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis, fibrositis, gastritis, gastroenteritis, gingivitis, ileitis, iritis, laryngitis, myelitis, myocarditis, nephritis, omphalitis, oophoritis, orchitis, osteitis, otitis, pancreatitis, parotitis, pericarditis, pharyngitis, pleuritis, phlebitis, pneumonitis, proctitis, prostatitis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, testitis, tonsillitis, urethritis, urocystitis, uveitis, vaginitis, vasculitis, vulvitis, vulvovaginitis, angitis, chronic bronchitis, osteomyelitis, optic neuritis, temporal arteritis, transverse myelitis, necrotizing fasciitis, and necrotizing enterocolitis. As used herein, an "autoimmune disease" refers to a disease arising from an inappropriate immune response of the body of a subject against substances and tissues normally present in the body. In other words, the immune system mistakes some part of the body as a pathogen and attacks its own cells. This may be restricted to certain organs (e.g., in autoimmune thyroiditis) or involve a particular tissue in different places (e.g., Goodpasture's disease which may affect the basement membrane in both the lung and kidney). The treatment of autoimmune diseases is typically with immunosuppression, e.g., medications which decrease the immune response. Exemplary autoimmune diseases include, but are not limited to, glomerulonephritis, Goodpasture's syndrome, necrotizing vasculitis, lymphadenitis, peri-arteritis nodosa, systemic lupus erythematosis, rheumatoid, arthritis, psoriatic arthritis, systemic lupus erythematosis, psoriasis, ulcerative colitis, systemic sclerosis, dermatomyositis / polymyositis, antiphospholipid antibody syndrome, scleroderma, pemphigus vulgaris, ANCA- associated vasculitis (e.g., Wegener's granulomatosis, microscopic polyangiitis), uveitis, Sjogren's syndrome, Crohn's disease, Reiter's syndrome, ankylosing spondylitis, Lyme arthritis, Guillain-Barre syndrome, Hashimoto's thyroiditis, and cardiomyopathy.

[0172] The term "autoinflammatory disease" refers to a category of diseases that are similar but different from autoimmune diseases. Autoinflammatory and autoimmune diseases share common characteristics in that both groups of disorders result from the immune system attacking a subject's own tissues and result in increased inflammation. In autoinflammatory diseases, a subject's innate immune system causes inflammation for unknown reasons. The innate immune system reacts even though it has never encountered autoantibodies or antigens in the subject. Autoinflammatory disorders are characterized by intense episodes of inflammation that result in such symptoms as fever, rash, or joint swelling. These diseases also carry the risk of amyloidosis, a potentially fatal buildup of a blood protein in vital organs. Autoinflammatory diseases include, but are not limited to, familial Mediterranean fever (FMF), neonatal onset multisystem inflammatory disease (NOMID), tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS), deficiency of the interleukin-1 receptor antagonist (DIRA), and Behcet's disease. The compound of the invention can be administered to a subject as a pharmaceutically acceptable salt thereof. Suitable pharmaceutically acceptable salts include, but are not limited to salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.

[0173] Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium. In particular, the present invention includes within its scope cationic salts eg sodium or potassium salts, or alkyl esters (eg methyl, ethyl) of the phosphate group.

[0174] Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.

[0175] It will be appreciated that any compound that is a prodrug of the compound of formula (I) is also within the scope and spirit of the invention. Thus the compound of the invention can be administered to a subject in the form of a pharmaceutically acceptable pro-drug. The term "pro-drug" is used in its broadest sense and encompasses those derivatives that are converted in vivo to the compound of the invention. Such derivatives would readily occur to those skilled in the art. Other texts which generally describe prodrugs (and the preparation thereof) include: Design of Prodrugs, 1985, H. Bundgaard (Elsevier); The Practice of Medicinal Chemistry, 1996, Camille G. Wermuth et al., Chapter 31 (Academic Press); and A Textbook of Drug Design and Development, 1991, Bundgaard et al., Chapter 5, (Harwood Academic Publishers). The compound of the invention may be in crystalline form either as the free compound or as a solvate (e.g. hydrate) and it is intended that both forms are within the scope of the present invention. Methods of solvation are generally known within the art.

[0176] The term "therapeutic effect" refers to some extent of relief of one or more of the symptoms of a disorder (e.g., a neoplasia or tumor) or its associated pathology. "Therapeutically effective amount" as used herein refers to an amount of an agent which is effective, upon single or multiple dose administration to the cell or subject, in prolonging the survivability of the patient with such a disorder, reducing one or more signs or symptoms of the disorder, preventing or delaying, and the like beyond that expected in the absence of such treatment. "Therapeutically effective amount" is intended to qualify the amount required to achieve a therapeutic effect. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the "therapeutically effective amount" (e.g., ED50) of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in a pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0177] The terms "treating", "treatment" and the like include relieving, reducing, alleviating, ameliorating or otherwise inhibiting the effects of the disease for at least a period of time. It is also to be understood that terms "treating", "treatment" and the like do not imply that the disease, or a symptom thereof, is permanently relieved, reduced, alleviated, ameliorated or otherwise inhibited and therefore also encompasses the temporary relief, reduction, alleviation, amelioration or otherwise inhibition of the disease, or of a symptom thereof.

[0178] The terms "subject", "patient" or "host", used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates (e.g., humans, monkeys and apes, and includes species of monkeys such from the genus Macaca (e.g., cynomologus monkeys such as Macaca fascicularis, and / or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpanzees (Pan troglodytes)), rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards etc.), and fish.

[0179] As used herein, the term "effective amount" relates to an amount of compound which, when administered according to a desired dosing regimen, provides the desired therapeutic activity. For purposes of this disclosure, a therapeutically effective amount of a compound, conjugate or composition is an amount that is sufficient to palliate, ameliorate, stabilise, reverse, prevent, slow or delay the progression of a disease state. Dosing may occur at intervals of minutes, hours, days, weeks, months or years or continuously over any one of these periods. A therapeutically effective amount can be administered in one or more administrations. Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage may be in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage may be in the range of 1 mg to 250 mg per kg of body weight per dosage.

[0180] Suitable dosage amounts and dosing regimens can be determined by the attending physician and may depend on the severity of the condition as well as the general age, health and weight of the patient to be treated. The compound of the invention may be administered in a single dose or a series of doses. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a composition, preferably as a pharmaceutical composition. The formulation of such compositions is well known to those skilled in the art. The composition may contain any suitable carriers, diluents or excipients. These include all conventional solvents, dispersion media, fillers, solid carriers, coatings, antifungal and antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents and the like. It will be understood that the compositions of the invention may also include other supplementary physiologically active agents.

[0181] The carrier must be pharmaceutically "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the patient. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

[0182] Other modes of administration including topical or intravenous administration may also be possible. Topical application typically involves administering the compound of the invention in an amount between 0.1 ng and 10 mg.

[0183] The compound, composition or combinations of the invention may also be suitable for intravenous administration. For example, a compound or a pharmaceutically acceptable salt, solvate or prodrug thereof may be administered intravenously at a dose of up to 16 mg / m2.

[0184] The compound, composition or combinations of the invention may also be suitable for oral administration and may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water- in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. In another embodiment, the compound or a pharmaceutically acceptable salt, solvate or prodrug is orally administerable.

[0185] A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g inert diluent, preservative disintegrant (e.g. sodium starch glycolate, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

[0186] The compound of the invention may be suitable for topical administration in the mouth including lozenges comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth gum; pastilles comprising the active ingredient in an inert basis such as gelatine and glycerin, or sucrose and acacia gum; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0187] The compound of the invention may be suitable for topical administration to the skin may comprise the compounds dissolved or suspended in any suitable carrier or base and may be in the form of lotions, gel, creams, pastes, ointments and the like. Suitable carriers include mineral oil, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Transdermal patches may also be used to administer the compounds of the invention. The compound of the invention may be suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bactericides and solutes which render the compound, composition or combination isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compound, composition or combination may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

[0188] Preferred unit dosage composition or combinations are those containing a daily dose or unit, daily sub-dose, as herein above described, or an appropriate fraction thereof, of the active ingredient.

[0189] It should be understood that in addition to the active ingredients particularly mentioned above, the composition of this invention may include other agents conventional in the art having regard to the type of composition or combination in question, for example, those suitable for oral administration may include such further agents as binders, sweeteners, thickeners, flavouring agents disintegrating agents, coating agents, preservatives, lubricants and / or time delay agents. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include cornstarch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable flavouring agents include peppermint oil, oil of Wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and / or methacrylic acid and / or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

[0190] Examples

[0191] Materials and methods

[0192] Synthesis of the compound TBG. To synthesise TBG, N,N-diphenyl-4-(7- (pyridin-4-yl)benzo[c][l,2,5]thiadiazol-4-yl)aniline (named TA, 191 mg, 0.42 mol) and 5-bromovaleric acid (152 mg, 0.84 mol) were dissolved in 50 mL of acetonitrile in a 100 mL round bottom flask equipped with a condenser. The mixture was heated to reflux for 24 h. After cooling to room temperature, the solvent was removed and the residue was purified by preparative thin-layer chromatography (methanol / dichloromethane = 1 / 20, V / V) to give the desired product compound l-(4-carboxybutyl)-4-(7-(4-

[0193] (diphenylamino)phenyl)benzo[c][l,2,5] thiadiazol-4-yl)pyridin-l-ium (named TB, 56 mg, 21% yield) as dark red solid. NMR (400 MHz, MeOD) 5 9.07 (d, J = 5.8 Hz, 2H), 8.99 (d, J = 5.8 Hz, 2H), 8.47 (d, J = 7.4 Hz, 1H), 8.06 (d, J = 7.4 Hz, 3H), 7.36 (t, J = 7.3 Hz, 4H), 7.21-7.09 (m, 8H), 4.70 (s, 2H), 2.46 (s, 2H), 2.15 (s, 2H), 1.75 (s, 2H).13C NMR (101 MHz, MeOD) 5 175.23, 153.84, 153.10, 153.01, 149.25, 147.18, 144.06, 137.78, 132.07, 130.36, 129.26, 129.19, 126.47, 126.29, 125.03, 123.87, 123.72, 121.54, 60.51, 30.31, 29.37, 21.07. HR ESI-MS, m / z: [M-Br]+calcd 557.2006, found 557.2084.

[0194] Then, compound TB (20 mg, 0.03 mmol), glucosamine hydrochloride (13.2 mg, 0.06 mmol), 4-(dimethylamino)pyridine (10 mg, 0.08 mmol), N,Ndiisopropylethylamine (DIPEA) (36 pL, 0.20 mmol) and HBTU (130 mg, 0.34 mmol) were dissolved in dry DMF (10 mL) and stirred at 38°C for 36 h. The desired product was purified by HPLC to give compound TBG (10.27 mg, 41% yield) as dark red solid. HPLC conditions: Agilent ZORBAX SB-C18 (9.4 x 150 mm) column; gradient: 0-23-24-26-27-30 min, 70%-30%-0%-0%-70%-70% B (A: H2O containing 0.1% TFA, B: acetonitrile containing 0.1% TFA); flow rate is 2.0 mL min-1; UV-Vis detector: 480 nm.XH NMR (400 MHz, CD3OD) 5 9.02 (d, J = 5.8 Hz, 2H), 8.96 (d, J = 5.9 Hz, 2H), 8.44 (d, J = 7.5 Hz, 1H), 8.04 (t, J = 6.7 Hz, 3H), 7.34 (t, J = 7.4 Hz, 4H), 7.13 (dd, J = 17.1, 8.0 Hz, 8H), 4.67 (t, J = 7.0 Hz, 2H), 3.86 (d, J = 11.3 Hz, 1H), 3.79 (d, J = 10.8 Hz, 1H), 3.76 - 3.47 (m, 3H), 3.47 - 3.31 (m, 2H), 2.38 (d, J = 5.4 Hz, 2H), 2.13 (d, J = 7.2 Hz, 2H), 1.75 (s, 2H).13C NMR (101 MHz, CD3OD) 5 174.08, 153.86, 153.10, 152.99, 149.27, 147.20, 144.07, 137.81, 132.05, 130.37, 129.26, 129.20, 126.48, 126.29, 125.04, 123.90, 123.73, 121.55, 91.19, 71.73, 71.29, 71.19, 61.33, 60.38, 54.40, 47.60, 30.17, 29.39, 21.69. HR ESI-MS, m / z: [MBr]+calcd 718.2694, found 718.2904.

[0195] Cell culture. The HeLa (human cervical cancer), A549 (the lung carcinoma epithelial cell line), MDA-MB-231 (the triple-negative breast cancer cell line) cell lines, NIH-3T3 (mouse embryonic fibroblasts) and HEK293 (human embryonic kidney 293 cells) were all purchased from the American Type Culture Collection (ATCC). HeLa (human cervical cancer), A549 (lung epithelial carcinoma), MDA- MB-231 (triple-negative breast cancer), NIH-3T3 (mouse embryonic fibroblasts) and HEK293 (human embryonic kidney) cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM). The culture medium contains 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin under a humidified atmosphere containing 5% CO2 at 37°C. FBS was ultracentrifuged in the ultracentrifuge (Beckman Coulter, CA) by 110,000 x g at 4°C for 2 h, and then filtered with a 0.22 pm filter (Millipore, Merck, Germany) to obtain EV-free FBS.

[0196] Observations of in situ generation of EVs and PS-EVs. To compare the generation of extracellular vesicles (EVs) from parent cells under CLSM, 2 x 104cells were seeded in 8-well plates and cultured for 20 h. Then, HeLa cells were cultured in the presence of TB and glucose-conjugated TB (TBG) for 30 min and observed via confocal laser scanning microscopy (CLSM). To monitor the generation process of TBG-EVs under CLSM, HeLa cells were incubated with TBG for 30 mins, 8h and 24 h, respectively. To observe the secretion of EVs under TEM, cells were collected after coculture with TBG for 30 min. CLSM images were obtained with excitation at 493 nm with emission collected at 720 nm.

[0197] Extraction of EVs and PS-EVs. To extract EVs in large scale, 20 vials of HeLa cells were cultured in T175 flasks by EV-free medium with or without 10 pM PS for 48 h in a dark environment at 37°C under a humidified atmosphere with 5% CO2. The above culture medium was centrifuged at 4°C by 2000 g for 10 min and 10000 g for 30 min to remove dead cells and cell fragments, respectively. Then, the upper culture medium was further collected and centrifuged at 110,000 g for 2 h at 4°C. The upper cell culture medium was discarded, and the lower EVs and PS-EVs were collected and wash by PBS for twice and subsequently concentrated at 110,000 g for 1 h at 4°C. The EVs and TBG-EVs were dispersed in PBS and stored at -80°C for further experiments.

[0198] To extract EVs in small scale, cells were incubated in the 6-well plate at a density of 2 x 105cells / well for 20 h and then cultured with ImL 10 pM PSs for additional 24 h, the collected supernatant was centrifuged at 4 °C by 2,000xg for 10 min and 10,000xg for 30 min to remove dead cells and cell fragments, respectively. Then the supernatant was further purified by size exclusion chromatography to collect purified EVs for further analysis.

[0199] Observation of extracted EVs. The fluorescence and morphology of extracted EVs was examined by CLSM and TEM. EVs were fixed by 4% paraformaldehyde for 30 mins firstly. For the CLSM observation, 10 pL of suspended EVs were dropped onto microslide and observed. For the TEM observation, 5 pL of suspended EVs were dropped onto copper mesh coated by carbon support films and then settled for 5 min. Excess fluid was discarded and washed by water for 3 times. The EVs were negatively stained with 2% phosphotungstic acid for 60 s. Then, the samples were dried in air and captured.

[0200] Nano-Flow Cytometry Analysis (NanoFCM) Study. EVs were collected from HeLa cell line by ultracentrifugation and then preserved in 250 pL PBS. Briefly, the EVs were diluted 1000 times using PBS before analysis on the NanoAnalyzer (NanoFCM). The EVs were detected using small threshold settings (68-155 S16M-Exo), and the labelled population was examined in the PC5 channel (excitation, 488 nm / emission, 670 nm). PBS was set as the blank, while pure EVs were assigned as the control group. Subsequently, identical procedures were applied to the small EV samples for flow analysis.

[0201] GLUT-1 Inhibition Experiment. 2 x 105cells were seeded in 6-well plates and cultured for 20 h. Following this, the cells were pretreated with 10 pM cytochalasin B for 20 min to inhibit GLUT-1 activity, and then washed three times with PBS. The cells were subsequently cocultured with the corresponding molecular solutions. To assess cellular uptake of TBG, the cells were incubated with TBG for 90 min and then collected for flow cytometry study. To evaluate EV yield, the cells were cultured for an additional 24 h, and the supernatant was collected for EV isolation, followed by NanoFCM analysis.

[0202] ATP Test. 2 x 105cells were seeded in 6-well plates and cultured for 20 h. The cells were then cocultured with the corresponding molecular solutions for 24 h, followed by three washes with PBS. Afterward, the cells were lysed with 100 pL of R.IPA Lysis Buffer on ice for 30 min and collected using a cell scraper. The resulting suspension was centrifuged at 10,000 rpm for 10 min, and the supernatant was collected. Finally, the supernatant was analyzed following the protocol of the ATP Bioluminescence Assay Kit CLS II (Sigma-Aldrich).

[0203] Cell Cytotoxicity Study. For cytotoxicity study, 1 x 104cells were seeded in 96-well plates and cultured for 20 h. After that, cells were treated with TBG and TBG-EVs at various concentrations. After 6 h incubation, the culture medium was replaced by fresh medium. And then, cells were exposed to light irradiation (60 mW cm-2for 10 min) and further cultured for 24 h. Then, 100 pL of 5 mg mL'1MTT solutions were added to the 96-well plates. After 4 h incubation at 37 °C, the medium was replaced with 100 pL of DMSO. The obtained solution was measured at the wavelength of 570 nm.

[0204] Animal Experiment. All the animal procedures conformed to the guidelines of the Institutional Animal Care and Use Committee of Southern University of Science and Technology. The in vivo antitumor therapeutic efficacy of TBG-EV was evaluated by a subcutaneous tumor model using female BLAB / c nude mice bearing HeLa tumor. 100 pL of PBS containing 5 x 106HeLa cells were subcutaneously injected into the right side of the BALB / c nude mice. After seven days, the mice were randomly divided into six groups (n = 4) with the following treatments respectively: PBS (Gl), EVs (G2), TBG (G3), TBG-EVs (G4), TBG with light irradiation (G5), TBGEVs with light irradiation (G6). The mice in different groups were intravenously administrated with 100 pL of corresponding formulas. After 8 h of drug administration, the mice in the G5 and G6 groups were irradiated under the white light (120 mW cm-2) for 10 min. During the treatment, the tumor volume and body weight of mice were recorded every two days. The tumor volume was calculated following the formula: V = 0.5 x length x width2. After 20 days of observation, the mice were sacrificed, and the blood and main organs (tumor, heart, liver, spleen, lung and kidney) of the mice were harvested for further evaluation.

[0205] Statistical Analysis. Statistical Analysis Data were presented as mean ± standard deviation (SD) derived from n > 3 independent biological replicates. Statistical analyses were conducted using GraphPad Prism, and Microsoft Office Excel. Statistical significance was analyzed via one-way or two-way ANOVA test followed by Tukey's post hoc test, **** p < 0.0001, *** P < 0.001, ** p < 0.01, * P < 0.05, P > 0.05 considered as no significant. Detailed information regarding the statistical tests employed and the number of replicates was provided in the plots.

[0206] Microscopy of extracted PS-EVs

[0207] The fluorescence and morphology of extracted PS-EVs was examined by CLSM and TEM. PS-EVs were fixed using 4% paraformaldehyde for 30 mins. For the CLSM observation, 10 pL of PS-EV suspension was dropped onto microslide and observed. For the TEM observation, 5 pL of PS-EV suspension was dropped onto copper mesh coated by carbon support films and then settled for 5 min. Excess fluid was discarded and washed by water for 3 times. The PS-EVs were negatively stained with 2% phosphotungstic acid for 60 s. Then, the samples were dried in air and captured.

[0208] Western blot. Cells, PS-treated cells, EVs and PS-EVs were lysed and the concentration was evaluated by a bicinchoninic acid (BCA) Protein Assay Reagent (Thermo Scientific Pierce) according to the manufacturer's instructions. All the protein samples were separated by a Sodium Dodecyl Sulfate-PAGE (SDS-PAGE), followed by transferring to a PVDF membrane (Bio-Rad). Membranes were blocked in 5% milk dissolved in lxTBST overnight with anti- ALIX, anti-CD9, anti-Calnexin, anti-GAPDH and the anti-GLUT-1. After washing, the bound primary antibodies reacted with secondary antibodies for 2 h. A chemiluminescence (ECL) system (Thermo Fisher) was used to detect the immunoreactive bands.

[0209] Drug-loading capacity. The drug-loading capacity was determined by measuring UV-vis absorbance at 493 nm. TBG was dissolved in PBS to prepare different concentrations of TBG solutions from 0-100 pM. The fitting curve of TBG solution is Absorbance = 0.0018x + 0.0299. The drug-loading capacity was calculated according to the following formula :

[0210] Drug-loading capacity = rnii / mz, where mi and m? represent the mass of TBG and the total mass of TBG-EVs, respectively.

[0211] ROS assay. 2,7-dichlorodihydrofluorescein (DCFH, 10 pM) was utilised as a ROS indicator to detect ROS generation of TBG (10 pM) in DMSO / PBS mixtures (v / v = 1 / 99) upon illumination (400-700 nm, 20 mW cm2for 5 mins). Changes in DCFH fluorescence were monitored at 525 nm.

[0212] Intracellular ROS detection was detected via DCFH-DA. HeLa or NIH-3T3 cells (2x l05cells) were seeded in 8-well confocal chambers and cultured for 20 h. After removing the medium, the cells were treated with 10 pM TBG-EVs for 6 h and further incubated with DCFH-DA (10 pM) for 30 min. Then the cells were illuminated under 60 mW cm-2light for 5 min followed by CLSM observation.

[0213] Analysis of cellular uptake of TBG-EVs and real-time imaging of the uptake of TBG-EVs. To compare the uptake of TBG and TBG-EVs into different cell lines (HeLa, A549, MDA-MB-231, NIH-3T3 and HEK293 cells), 2x l05cells were seeded in 8-well confocal chambers and cultured for 20 hours. After that, cells were treated with 10 pM TBG or TBG-EVs for 6 h. Cell nuclei were labelled with the commercial dye Hoechst and visualised using CLSM. The signal of TBG and TBG-EVs were collected in Aem = 650-800 nm as red (Aex = 488 nm), and that of Hoechst was collected in the range of 500-540 nm as blue (Aex = 405 nm). For the real-time imaging of the uptake of TBG-EVs, the cells were incubated at 37°C in a Quad Black live cell chamber equipped with the Leica TCS SP8 CLSM system. TBG-EVs were then injected at a final concentration of 10 pM. The images in a fixed field were continuously scanned from 1 to 60 min.

[0214] Immunofluorescence staining. To determine the influence of molecular to GLUT-1 activity. HeLa cells were cultured with PBS, 20 pM glucose, 20 pM TB and 20 pM TBG for 6 h. These dyes were washed by PBS for three times and fixed in 4% paraformaldehyde at room temperature (RT) for 15 min and then washed 3 times with PBS. Next, the cells were blocked with 1% nonfat milk at RT for 30 min, and then incubated with the primary antibody anti GLUT-1 at 4 °C overnight, followed by incubation with the secondary antibody Alexa 488 at RT in the dark for 2 h. Finally, the cells were counterstained with 1 pg / mL Hoechst for 5 mins, and the CLSM were taken later.

[0215] Example 1: Synthesis and characterisation of glucose-conjugated photosensitiser

[0216] A glucose-conjugated photosensitiser (PSG), named TBG, was designed and synthesised via an amidation reaction between glucosamine and TB (Figure 1), and the structure was confirmed using NMR and ESI-mass spectroscopy (Figures. 2 to 7).

[0217] The optical properties of TBG were measured as shown in Figure 8, the optical properties of TBG were measured as shown in Figure 8, with TBG displaying a broad UV / Vis absorption from 400-600 nm in the solution of DMSO / water mixture (1 :99, v / v) with a red emission at 720 nm. The slight shifts in absorption and emission compared with TB can be attributed to glucose modification-induced changes in ?r-conjugation, solvation effects, and aggregation behavior. Moreover, as illustrated in Figure 9, TBG exhibits a typical AIE character, displaying weak emission in methanol solution (molecular state) and gradually enhanced red emission in toluene / methanol mixtures with an increased toluene fraction (aggregation state). The AIE properties allow TBG to produce minimal background fluorescence in aqueous media but show strong fluorescence in cells, eliminating the washing steps for cell imaging. The ROS production capacity of TBG was subsequently evaluated by employing 2,7- dichlorodihydrofluorescein (DCFH) as the ROS indicator. As shown in Figure 10, the fluorescence intensity of DCFH dramatically increased after being treated with TBG upon white light irradiation (400-700 nm) at a power of 20 mW'Cnr2, thus revealing the efficient ROS generation ability of TBG. Notably, the fluorescence intensity of DCFH significantly increased in the presence of TBG, surpassing that of Chlorin e6 solution, indicating its superior ROS generation capability.

[0218] Example 2: Generation of TBG-EVs via endogenous cellular pathways Design, Synthesis and Characterization of TBG

[0219] Generally, the application of naturally occurring and automatically released EVs in cancer therapy is limited by the low secretion efficiency and unsatisfactory therapeutic effect. Therefore, employing exogenous materials to simultaneously in situ enhance the generation of endogenous cancer cell-derived EVs and functionalize them with PS would be a feasible and promising strategy for cancer therapy. The generation and secretion of EVs from cancer cells require ATP consumption. Cancer cells are highly dependent on the glycolysis process to generate ATP, and glucose is one of the most widely available carbohydrate sources for ATP supply by glycolytic metabolism. Based on the mechanism of EV secretion in cancer cells, we hypothesize that PS grafted on glucose as the carbohydrate source of cancer cells will promote the generation of PS- engineered EVs. Hence, a glucose-modified PS, referred to as TBG, was synthesized via an amidation reaction between glucosamine (GlcN) and TB (Figure 1).

[0220] In Situ Generation of TBG-EVs

[0221] Next, we investigated the ability of TBG to promote EV generation in cancer cells. HeLa cells, as a model cancer cell, were chosen as parent cells to generate TBG-EVs through co-culturing with TBG. To ensure the high production efficiency of EVs, the parent cells must be alive and active during the production process. Since the generated ROS from PS would cause damage to HeLa cells upon light irradiation, the whole culture and extraction processes were carefully carried out in the dark condition. We also studied the viability of HeLa cells treated with TBG by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays as demonstrated in Figure 11. The cell growth was not inhibited by TBG after incubation in the dark for 24 h. The cell viabilities were above 90% at the concentration of TBG between 0-80 pM, demonstrating high biocompatibility of TBG during the process of TBG-EV secretion under dark conditions.

[0222] To examine the EV production yield after incubation with TBG, transmission electron microscopy (TEM) images of ultrathin cell slices were first captured. HeLa cells were co-cultured with TBG for 30 min, and the produced EVs were compared with those from untreated cells. As observed, EVs were found to be much more abundant with an average size of approximately 200 nm around the TBG-treated cells (Figure 12). To further demonstrate the roles of glucose in TBG, HeLa cells were cultured with TB for 30 min as a comparison, and observed with confocal laser scanning microscopy (CLSM). As shown in Figure 13, more abundant EVs with red fluorescence were around the TBG-treated cells in accordance with the TEM results, while few EVs were observed in TB-treated cells. Extended co-culture of HeLa cells with TBG was also monitored by CLSM revealing continuous generation of TBGEVs by the cells and their subsequent release into the extracellular space (Figure 14).

[0223] Characteristics of Extracted TBG-EVs

[0224] Subsequently, the released EVs were collected by differential centrifugation as depicted in Figure 15 and observed under natural light and UV light (Figure 16a). Comparing negligible fluorescence noted in EVs solution and TBG solution (Phosphate buffered saline (PBS): DMSO = 99: 1, v / v), strong red fluorescence was detected in TBG-EVs under UV light due to the AIE effect of TBG molecules. The fluorescence from TBG-EVs was obtained via CLSM as well (Figure 16b). Subsequently, the drug-loading capability of TBG in EVs was calculated to be 0.8% (TBG / TBG-EVs, wt%). The quantitative percentage of EVs labeled with TBG was further analyzed using Nano-flow cytometry analysis (NanoFCM). PBS was set as the blank, and pure EVs in PBS was set as the negative control. It's worth noting that the labeling efficiency of EVs by TBG was approximately 77.4% (Figure 17b), which significantly surpassed the labeling efficiency of EVs by TB (50.8%), and was consistent with the fluorescence intensity observed under UV light. This might be attributed to the metabolism-oriented active labeling of TBG rather than passive labeling.

[0225] The extracted EVs were then subjected to detailed characterization. The surface charge of the TBG-EVs was determined to be -7.69 mV, which was slightly higher than that of EVs (-9.42 mV) and TB-EVs (-8.23 mV) (Figure 18a). Sizewise, TBG-EVs displayed an average hydrodynamic diameter of 145.5 ± 4.8 nm determined by DLS, which was comparable to that of pure EVs (134.3 ± 1.7 nm) and TB-EVs (135.1 ± 3.8 nm). Next, the morphology of the TBG-EVs with negative staining was observed by TEM. The result showed that TBG-EVs were spherical, and the particle size was consistent with the result measured by DLS. Moreover, both EVs and TBG-EVs displayed excellent colloidal stability in PBS at 4 °C after 7 days storage (Figure 18b).

[0226] Considering that the purity and surface protein composition of EVs are critical to their specific recognition property, three EV marker proteins, ALG-2- interacting protein X (Alix), tumor susceptibility gene 101 (TSG101), CD9 and calnexin in TBG-EVs were examined and compared with EVs and HeLa cell lysates via western blotting with GAPDH as the protein reference (Figure 19). As calnexin is a marker of endoplasmic reticulum that mainly exists in cells but not EVs, the negligible expression of calnexin in both EVs and TBG-EVs indicates that the collected products were EVs of high purity instead of cell debris. Meanwhile, TSG101 and Alix, participating in EV biogenesis and secretion, are widely used as EV marker proteins. Alix and CD9 are also known to mediate the internalization and uptake rate of EVs. Hence, TSG101, Alix and CD9 specifically enriched in both EVs and TBG-EVs suggested that the composition and function of TBG-EVs were kept intact.

[0227] The EV yield from HeLa cells obtained through large-scale extraction was quantitatively analyzed using NanoFCM for comparison as depicted in Figure 20. The result reveals a significant improvement in EV yield upon TBG treatment, with a nearly 4.3-fold increase compared to the untreated group.

[0228] Mechanism Behind the Promotion of EV Yield by TBG To further investigate the mechanism underlying the promotion of EV yields by TBG through glucose metabolism, we initially analyzed the transcriptome of HeLa cells using RIMA sequencing. Specifically, HeLa cells were untreated or treated with TBG for 30min under dark conditions, followed by collection for RNA sequencing analysis. As illustrated in the volcano plot in Figure 21a, TBG-treated HeLa cells exhibited upregulation of 19 genes and downregulation of 394 genes compared to the control group. To better understand the relevant signaling pathways, the differentially expressed genes were subjected to Gene Ontology (GO) biological process analysis and further analyzed by Metascape (Figure 21b). The result demonstrated that the differentially expressed genes were significantly enriched in the pathways of stimulus response, metabolic process and positive regulation of biological processes. Specifically, these pathways are involved in glucose uptake and energy supply, which play key roles in the biogenesis and secretion of cancer-derived EVs. Therefore, we speculate that the addition of TBG would stimulate HeLa cells to upregulate the glucose metabolic process and energy supply for TBG-EVs generation. To further verify our conjecture, gene set enrichment analysis (GSEA), a powerful analytical method for interpreting gene expression data, was also performed to compare the gene sets that were involved in the biological process of EVs in HeLa cells untreated or treated with TBG. Consistently, TBG treated group exhibited upregulated expressions of gene sets involved in the reinforcement of active transmembrane transporter activity and ATP synthesis (Figure 22a, b).

[0229] Subsequently, cellular analysis was performed. We first verified whether the glucose transporter was involved in TBG uptake. It's well-known that GLUT-1 (Glucose Transporter 1) is a membrane protein responsible for the facilitated diffusion of glucose across the plasma membrane. Studies have also demonstrated that GLUT-1 can still recognize and transport D-glucose when the hydroxyl group at the C2 position is substituted. Meanwhile, we utilized cytochalasin B (Cyt-B), a known GLUT-1 blocker, to study its effect on the cellular uptake of TBG. As shown in Figure 23, HeLa cells pretreated with Cyt-B for 20 min exhibited a reduced fluorescence signal from TBG. Notably, the most obvious decrease in EV yield was observed in the TBG-treated group after Cyt- B pretreatment (Figure 24), confirming that GLUT-1 is important for cellular uptake of TBG.

[0230] Following recognition by GLUT-1 and uptake into HeLa cells, TBG is expected to be phosphorylated by hexokinase to form TBG-6-Phosphate. However, further metabolism by enzymes such as glucose-6-phosphate dehydrogenase and phosphohexose isomerase is inhibited due to the substitution at the C2 position of D-glucose. The disruption in glucose metabolism induces a metabolic stress response, simulating "false glucose starvation". In turn, this triggers an adaptive response, including the upregulation of GLUT-1 expression as verified by the GLUT-1 immunocytochemical staining of HeLa cells in Figure 25. Notably, the upregulation of GLUT-1 expression is closely linked to the increased ATP production (Figure 26). Notably, ATP plays a critical role in EV secretion, as it can drive ATP mediated rearrangement of the cell membrane and support submembrane actin cytoskeleton dynamics involved in EV docking, ultimately contributing to a higher EV yield.

[0231] The complete mechanism diagram is shown in Figure 27.

[0232] To demonstrate the universality of TBG to promote PS-engineered EV generation, MDA-MB-231 cells and A549 were selected to coculture with TBG as shown in Figure 28. Through fluorescence imaging, EVs with red fluorescence were more abundant around TBG-treated cells than TB-treated cells in both cell lines, consistent with the observations in HeLa cells. Additionally, EV yield analysis confirmed that TBG-treated cells produced significantly more EVs compared to untreated or TB-treated cells, with a 12.7-fold increase in MDA- MB-231 cells and a 5.9-fold increase in A549 cells. And the differences in fold change observed among different cell lines may be attributed to intrinsic variations in glycolytic activity and stimulus response levels.

[0233] In Vitro Evaluation of the Biological Function of TBG-EVs

[0234] The biological function of TBG-EVs was studied by investigating the selectivity of TBG-EVs from HeLa cells towards different cell lines, including HeLa, A549, MDA-MB-231, NIH-3T3 (mouse embryonic fibroblasts) and HEK-293T (human embryonic kidney 293 cells) using bare TBG as the control for comparison. All the cells were incubated with TBG-EVs, followed by labeling with commercial nuclear dye Hoechst, and visualized under CLSM. As shown in Figure 29, the bright red fluorescence from TBG in HeLa cells and weak red fluorescence in A549 and MDA-MB-231 cells after TBG-EVs treatment were observed. But the indiscernible red fluorescent signal was displayed in NIH-3T3 and HEK293 cells after being treated with TBG-EVs. Obviously, the bright red fluorescence intensity of HeLa cells was far superior over that of other cell lines which demonstrated the stronger internalization capability of HeLa cells to TBG-EVs compared with other cell types. In stark contrast to TBG-EVs, all cells presented red fluorescent signal without any visible difference when culturing with TBG, indicating excellent selectivity of TBG-EVs over TBG molecules. Subsequently, the selectivity of TBG-EVs was also examined with the abovementioned cell lines by flow cytometry experiments (Figure 30). Similarly, the TBG-EVs could be uptake by HeLa cells well, followed by decreased TBG-EVs uptake by A549 and MDA-MB-231 cells, while there is almost no uptake of TBG-EVs by NIH-3T3 and HEK293 cells, which were consistent with the results from the fluorescence microscopy images. Collectively, the above results demonstrate the high selfrecognition specificity of TBGEVs to their source cells (HeLa), as well as notable adhesion and recognition ability to other similar cancer cells including A549 cells and MDA-MB-231 cells, while exhibiting weak tropism to NIH-3T3 cells and HEK293 cells. The selectivity of EVs can be attributed to both the intrinsic properties of EVs and the characteristics of target cells. Specifically, the membrane proteins and lipids on the surface of EVs directly influence their binding to target cells and demonstrate superior homologous targeting ability. Meanwhile, cancer cells internalize EVs by the overexpressed intercellular adhesion molecules (e.g., CD54) on the surface of cancer cells, which plays a crucial role in regulating the cellular uptake of tumor-derived EV.

[0235] Considering TBG has an efficient ROS generation ability that can induce cell death under light irradiation, we proceed to investigate the ROS generation capacity of TBG-EVs in cancer cells (Figure 31). A green fluorescent indicator 2,7-Dichlorofluorescein diacetate (DCFH-DA) was used to evaluate the generation of total ROS. After culturing HeLa cells with TBG-EVs for 6 h, the fluorescence of DCFH-DA could not be found in HeLa cells without light irradiation but appeared distinct green fluorescence after light irradiation under 60mWcm-2for 10 min. Subsequently, the ROS generation of TBG-EVs in NIH- 3T3 has also been detected. Regardless of light or dark conditions, NIH-3T3 cells exhibited weak green fluorescence, likely due to their limited capacity for internalizing TBG-EVs. Therefore, the PS decorated with EVs may be favorable for PS to work at the desired cancer sites.

[0236] Lastly, to assess the specific cancer cell killing capability of TBG-EVs, the viability of NIH-3T3 and HeLa cells was evaluated using the typical colorimetric MTT method following treatment with various concentrations of TBG-EVs under dark or light irradiation. As shown in Figure 32, upon light illumination, a significant reduction in HeLa cell viability was observed. When the concentration of TBG in TBG-EVs reached 10 pM, almost all HeLa cells were damaged under light irradiation. However, no significant difference in cell viability was observed in NIH-3T3 cells under light or dark conditions, likely due to their poor uptake to TBG-EVs (Figure 33). As a comparison, after culturing HeLa and NIH-3T3 with bare TBG molecules, high cytotoxicity was observed in both cell lines after light irradiation. These results indicate the more promising selective killing of TBG- EVs over TBG molecules in cancer PDT therapy, which offers minimal side effects to normal cells.

[0237] In Vivo Evaluation of the Biological Function of TBG-EVs

[0238] The in vivo PDT therapeutic effects of TBG-EVs were further evaluated using a subcutaneous tumor model in HeLa tumor bearing BALB / c nude mice. The tumor accumulation capability and biodistribution of TBG-EVs were evaluated through ex vivo fluorescence imaging of tumors and major organs at different time points after intravenous (i.v.) injection. Fluorescence in the tumor site gradually increases, reaching a peak around 8 h, indicating efficient tumor targeting and retention. Fluorescence intensity was observed in the liver and kidney starting from 0.5 h. After peaking in the liver at 8 h, fluorescence signals in both the liver and kidney gradually decline. By 24 h, the overall fluorescence intensity in major organs has markedly decreased. Subsequently, the following groups were administered to mice via tail vein injection to evaluate their tumor inhibitory effects: PBS (Gl), EVs (G2), TBG (G3), TBG-EVs (G4), TBG with light irradiation (G5), TBGEVs with light irradiation (G6). Following i.v. administration of G1-G6, we monitored tumor growth during the therapeutic process (Figure 34A-F and 35). Significant inhibition of tumor growth was observed in the G6 group, which could be attributed to the increased ROS generation from TBG-EVs, resulting from the enhanced TBG accumulation facilitated by the superior targeting ability of the EVs. In contrast, the other groups (G1-G4) exhibited a similar tumor growth progression without any inhibition and the G5 showed a slightly decreased tumor volume due to the ROS generation from the small amount of TBG. On day 20, mice were sacrificed, and their tumors were dissected for further study (Figure 36). Tumor weight measurements (Figure 37) and histological analysis with hematoxylin and eosin (H&E) staining (Figure 38) further confirmed the enhanced anti-tumor activity of TBG-EVs under light irradiation, demonstrating their effective PDT capability. During the treatment process, the body weights of mice remained stable (Figure 39). Additionally, the mice serum was collected for blood biochemical analysis, and the sections of major organs were analyzed by H&E staining (Figure 40). The results showed that TBG-EVs had no significant effect on major indexes of liver and kidney functions, and there were no noticeable pathological changes in major organs, indicating minimal side effects of TBG-EVs on normal tissues. In conclusion, these results demonstrated that TBG-EVs under light irradiation could efficiently suppress tumor growth in vivo with minimal phototoxic side effects.

[0239] Discussion and Conclusion

[0240] In this study, we develop a metabolism-oriented strategy for cancer cells to generate EVs enriched with PS by feeding cells with glucose-conjugated PSs. During the cultural process, TBG serves as a substrate and would be effectively taken up by cancer cells, leading to the enhancement of transmembrane transporter activity and the acceleration of ATP-dependent metabolic processes. Notably, co-culturing three cancer cell lines with TBG results in up to a 12.7- fold increase in EV yield, fully demonstrating the high efficiency and broad applicability of our strategy. Moreover, TBG is also efficiently loaded into the EVs with 77.4% labeling efficiency. Meanwhile, our production process has been validated at multiple scales using optimized isolation techniques, ensuring efficient EV collection and purification while demonstrating scalability. Furthermore, both in vitro and in vivo studies confirm that the extracted TBG- EVs retain excellent targeting ability toward cancer cells, leading to superior PDT efficacy in tumor ablation under light irradiation. Our findings highlight the critical role of glucose-modified PS in the in situ engineering of PS-enriched EVs, and offer insights into the mechanism underlying their enhanced yield. These results support that co-culturing cancer cells with glucose conjugated PS serves as a simple and effective strategy for EV engineering.

[0241] It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

[0242] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0243] As used in this application, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an agent" includes a plurality of agents, including mixtures thereof.

[0244] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0245] Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of" will be understood to indicate that the recited element(s) is / are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

[0246] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims

1. Claims1. A compound of formula (I), or a salt, solvate or stereoisomer thereof:whereinX is a heteroatom selected from 0 or NR;R is selected from H and optionally substituted alkyl; andRi is a moiety derived from a photosensitiser.

2. The compound according to claim 1, wherein the compound is a compound of Formula (la):whereinL is a linker;X is a heteroatom selected from 0 or NR;R is selected from H and optionally substituted alkyl; and R2 is a moiety comprising a n conjugated system.

3. The compound according to claim 2, wherein L is a non-cleavable linker.

4. The compound according to claim 2 or 3, wherein L is a linker having a chain length of 2 to 20 atoms.

5. The compound according to any one of claims 2 to 4, wherein L comprises C1-C20 alkyl and / or 1 to 10 monomeric units of ethylene glycol.

6. The compound according to any one of claims 2 to 5, wherein L is selected fromwherein n is independently an integer selected from 1 to 10; andbond to R.2 or amino.

7. The compound according to any one of claims 2 to 6, wherein R.2 is selectedThe compound according to any one of claims 1 to 7, wherein the compound is selected fromwherein n is an integer selected from 1 to 10.

9. The compound according to any one of claims 1 to 8, wherein the compound is characterised by a UV / vis absorption of about 400 nm to about 600 nm.

10. The compound according to any one of claims 1 to 9, wherein the compound is characterised by UV / vis emission of about 600 nm to about 900 nm.

11. The compound according to any one of claims 1 to 10, wherein the compound is capable of exhibiting aggregation-induced emission (AIE).

12. The compound according to any one of claims 1 to 11, wherein the compound is capable of generating reactive oxygen species within 30 sec when irradiated with light.

13. An extracellular vesicle comprising a compound according to any one of claims 1 to 12, or a salt, solvate or stereoisomer thereof.

14. The extracellular vesicle according to claim 13, wherein the extracellular vesicle is derived from a cancer cell.

15. The extracellular vesicle according to claim 13 or 14, wherein the compound is encapsulated within the extracellular vesicle.

16. The extracellular vesicle according to claim 13 or 15, wherein the extracellular vesicle is characterised by a particle size of about 120 nm to about 200 nm.

17. The extracellular vesicle according to claim 13 or 16, wherein the extracellular vesicle is characterised by a labelling efficiency of more than about 70%.

18. The extracellular vesicle according to claim 13 or 17, wherein the extracellular vesicle is characterised by a drug-loading capability (compound in EV) about 0.1 % to about 5 % (compound / EV, wt%).

19. The extracellular vesicle according to claim 13 or 18, wherein the extracellular vesicle is characterised by a zeta potential of about -7 mV to about -8.5 mV.

20. The extracellular vesicle according to claim 13 or 19, wherein the extracellular vesicle is characterised by a purity of more than about 90%.

21. A pharmaceutical composition comprising a compound of any one of claims 1 to 12 or an extracellular vesicle according to any one of claims 13 to 20, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof.

22. A method of preparing an extracellular vesicle (EV), comprising: a) contacting a cell with a compound according to any one of claims 1 to 12, and b) culturing the cell in the dark under predetermined conditions in order to generate the EV.

23. The method according to claim 22, wherein the cell is a cancer cell.

24. The method according to claim 22 or 23, wherein the compound is provided at a concentration of less than about 100 pM.

25. The method according to any one of claims 22 to 24, wherein the cell is contacted with the compound for a duration of at least 20 min.

26. The method according to any one of claims 22 to 25, wherein the method further comprises a step after step b) of isolating the extracellular vesicle from the cell.

27. The method according to any one of claims 22 to 26, wherein the method is characterized by at least a 4 fold increase in production of EV relative to a control, wherein the control is a cell uncontacted with compound.

28. A method of treating a tumor and / or a proliferative disease in a subject in need thereof, comprising administering a compound according to any one of claims 1 to 12, or an extracellular vesicle according to any one of claims 13 to 20 to the subject.

29. The method according to claim 28, wherein the proliferative disease is cancer or an inflammatory disease.

30. The method according to claim 28 or 29, wherein the method is selective to tumor and / or proliferative disease compared to normal cell.

31. The method according to any one of claims 28 to 30, wherein the method further comprises irradiating the compound or extracellular vesicle in order to photodynamically treat the subject.

32. A compound according to any one of claims 1 to 12, or an extracellular vesicle according to any one of claims 13 to 20 or a pharmaceutically acceptable salt, solvate or stereoisomer thereof for use in treating a tumor and / or a proliferative disease.

33. Use of a compound according to any one of claims 1 to 12, or an extracellular vesicle according to any one of claims 13 to 20 or a pharmaceutically acceptable salt, solvate or stereoisomer thereof in the manufacture of a medicament for the treatment of a tumor and / or a proliferative disease.

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