Conjugate and composition for treating cancer

Abstract

The disclosure relates to a conjugate comprising a nanocarrier to which the following are bound: (i) a targeting moiety which binds to a receptor on a target cell, such as folic acid, and (ii) compounds from at least one plant extract derived from a plant selected from the group consisting of Tulbaghia violacea, Annona muricata, Dicoma capensis and Dodonaea viscosa. A composition including the conjugate, a method for preparing the conjugate, uses of the conjugate and composition for treating cancer, and a kit are also provided. The nanocarrier can be a carbon nanotube, such as a single walled carbon nanotube (SWCNT), and may be functionalised with polyethylene glycol.

Description

[0001] CONJUGATE AND COMPOSITION FOR TREATING CANCER

[0002] FIELD OF THE INVENTION

[0003] This invention relates to a conjugate and composition, in particular for use in treating cancer, and to a method of manufacture of the conjugate and composition.

[0004] BACKGROUND TO THE INVENTION

[0005] Treatment for cancer includes chemotherapy with an anticancer drug or a combination of a few drugs, as well as other therapies, such as radiation therapy and surgery. Chemotherapy poses several challenges, including hypersensitivity, multidrug resistance (MDR), and metabolic transformations into toxic molecules. These result in various life-threatening side effects such as nephrotoxicity and cardiotoxicity. In addition, there is a lack of target specificity and normal cells are damaged, leading to nausea, vomiting, fever, hair loss, fatigue and weight loss. Many drugs are also ineffective at providing long lasting protection against cancer and recurrence of tumours is commonly observed after treatment has been discontinued.

[0006] There is therefore a need for alternative cancer therapies which do not have the disadvantages described above.

[0007] SUMMARY OF THE INVENTION

[0008] According to a first aspect of the invention, there is provided a conjugate including:

[0009] - a nanocarrier;

[0010] - a targeting moiety which binds to a receptor on a cancer cell; and

[0011] - compounds from at least one plant extract derived from a plant selected from the group consisting of Tulbaghia violacea, Annona muricata, Dicoma capensis and Dodonaea viscosa; wherein the targeting moiety and compounds from the at least one plant extract are conjugated to the nanocarrier.

[0012] The nanocarrier may be a carbon nanotube, such as a single walled carbon nanotube (SWCNT).

[0013] The carbon nanotube may be functionalised with polyethylene glycol. The targeting moiety may be folic acid.

[0014] The conjugate may include compounds from extracts of at least two or at least three plants selected from the group consisting of Tulbaghia violacea, Annona muricata, Dicoma capensis and Dodonaea viscosa. Alternatively, the conjugate may include compounds from extracts of all four of Tulbaghia violacea, Annona muricata, Dicoma capensis and Dodonaea viscosa.

[0015] Compounds from the Annona muricata and Dicoma capensis extracts may be substantially endohedrally conjugated to the carbon nanotube.

[0016] Compounds from the Tulbaghia violacea and Dodonaea viscosa extracts may be substantially exohedrally conjugated to the carbon nanotube.

[0017] According to a second aspect of the invention, there is provided a composition including conjugates as defined above. The composition may further include a pharmaceutically acceptable excipient.

[0018] According to a third aspect of the invention, there is provided a conjugate or composition as defined above for use in a method of treating cancer. The cancer may be breast cancer or colorectal cancer.

[0019] According to a further aspect of the invention, there is provided a use of a conjugate or composition as defined above in the manufacture of a medicament for use in a method of treating cancer. The cancer may be breast cancer or colorectal cancer.

[0020] According to a further aspect of the invention, there is provided a method of treating cancer in a subject, the method including the step of administering a therapeutically effective amount of a conjugate or composition to a subject in need thereof. The cancer may be breast cancer or colorectal cancer.

[0021] According to a further aspect of the invention, there is provided a method of producing a conjugate as defined above, the method including the steps of: conjugating a targeting moiety which binds to a receptor on a cancer cell to a nanocarrier; and contacting the nanocarrier with at least one plant extract derived from a plant selected from Tulbaghia violacea, Annona muricata, Dicoma capensis and Dodonaea viscosa and allowing compounds from the at least one plant extract to bind to the nanocarrier. The nanocarrier may be a carbon nanotube, such as a SWCNT.

[0022] The targeting moiety may be folic acid.

[0023] The method may comprise the step of functionalising the nanocarrier with polyethylene glycol before the nanocarrier is contacted with the plant extract(s).

[0024] The conjugate may be contacted with extracts of at least two or at least three plants selected from the group consisting of Tulbaghia violacea, Annona muricata, Dicoma capensis and Dodonaea viscosa. Alternatively, the conjugate may be contacted with extracts of all four of Tulbaghia violacea, Annona muricata, Dicoma capensis and Dodonaea viscosa.

[0025] Compounds from the Annona muricata and Dicoma capensis extracts may be allowed to conjugate substantially to an inner surface of the carbon nanotube, such as by bringing the carbon nanotube into contact with these extracts before bringing the carbon nanotube into contact with the Tulbaghia violacea and Dodonaea viscosa extracts.

[0026] Compounds from the Tulbaghia violacea and Dodonaea viscosa extracts may be conjugated substantially to an outer surface of the carbon nanotube, such as by only bringing the carbon nanotube into contact with these extracts after having contacted the carbon nanotube with the Annona muricata and Dicoma capensis extracts.

[0027] The plant extracts may be brought into contact with the carbon nanotubes in the following order: Annona muricata, Dicoma capensis, Dodonaea viscosa and lastly Tulbaghia violacea.

[0028] The plant extracts may be obtained by closed loop extraction.

[0029] According to a further aspect of the invention, there is provided a kit including plant extracts of at least one of Tulbaghia violacea, Annona muricata, Dicoma capensis and Dodonaea viscosa and instructions for producing a conjugate as defined above.

[0030] The kit may further include PEG and / or folic acid.

[0031] BRIEF DESCRIPTION OF THE FIGURES

[0032] Figure 1 : Diagram showing the orientation of compounds from plant extracts on a SWCNT- PEG-FA nanotube, with AM and DC inside the nanotube and DV and TV on the outer surface.

[0033] Figure 2: LC-MS chromatogram of Annona muricata by solvent extraction.

[0034] Figure 3: LC-MS chromatogram of Annona muricata by closed loop extraction.

[0035] Figure 4: LC-MS chromatogram of Dicoma capensis by solvent extraction.

[0036] Figure 5: LC-MS chromatogram of Dicoma capensis by closed loop extraction.

[0037] Figure 6: LC-MS chromatogram of Dodonaea viscosa by solvent extraction.

[0038] Figure 7: LC-MS chromatogram of Dodonaea viscosa by closed loop extraction.

[0039] Figure 8: LC-MS chromatogram of Tulbaghia violacea by solvent extraction.

[0040] Figure 9: LC-MS chromatogram of Tulbaghia violacea by closed loop extraction.

[0041] Figure 10: LC-MS chromatogram of Annona muricata, Dicoma capensis, Dodonaea viscosa and Tulbaghia violacea mixture by closed loop extraction.

[0042] Figure 1 1 : UV-Vis spectra of (a) SWCNT-PEG-FA, (b) Annona muricata bio-conjugated SWCNT-PEG-FA stirred for 6 hours, (c) Annona muricata bio-conjugated SWCNT-PEG-FA stirred for 12 hours and (d) Annona muricata bio-conjugated SWCNT-PEG-FA stirred for 24 hours.

[0043] Figure 12: UV-Vis spectra showing bio-conjugates for (a) SWCNT-PEG-FA-AM, (b) SWCNT- PEG-FA-DC, (c) SWCNT-PEG-FA-DV, (d) SWCNT-PEG-FA-TV and (e) SWCNT-PEG-FA-AM- DC-DV-TV at a reaction time of 24 hours.

[0044] Figure 13: Cytotoxicity of crude extracts after 72 hours in MCF-12A cells, HT-29 cells, MDA- MB-231 cells, and MCF-7 cells. Cells were seeded and treated with different crude extract concentrations (1 , 10, 25, 100 and 500 pg / ml) over a period of 24 hours, whereafter a WST-1 assay was performed. The percentage viable cells after the treatment period were compared with the percentage untreated viable cells.

[0045] Figure 14: Cytotoxicity of crude extracts after 72 hours in MCF-12A cells, HT-29 cells, MDA- MB-231 cells, and MCF-7 cells. Cells were seeded and treated with different crude extract concentrations (1 , 10, 25, 100 and 500 pg / ml) over a period of 48 hours, whereafter a WST-1 assay was performed. The percentage viable cells after the treatment period were compared with the percentage untreated viable cells.

[0046] Figure 15: Cytotoxicity of crude extracts after 72 hours in MCF-12A cells, HT-29 cells, MDA- MB-231 cells, and MCF-7 cells. Cells were seeded and treated with different crude extract concentrations (1 , 10, 25, 100 and 500 pg / ml) over a period of 72 hours, whereafter a WST-1 assay was performed. The percentage viable cells after the treatment period were compared with the percentage untreated viable cells.

[0047] Figure 16: Cytotoxicity of a crude extract mixture with AM-DC-DV-TV in (A) MCF-12A, (B) M DA- MB- 231 , (C) MCF-7 and (D) HT-29 cells after 72 hours of treatment. Cells were seeded and treated with a crude extract mixture at 10 pg / ml over a period of 72 hours, whereafter a WST-1 assay was performed. Results are presented as means ± SEM (n=3). The asterisk denotes a significant decrease in cell viability when compared to the control (***p<0.001 and ****p<0.0001 ). The percentage viable cells after the treatment period were compared with the percentage untreated viable cells.

[0048] Figure 17: Cytotoxicity of carbon nanotubes at 100 pg / ml in (A) MCF-12A, (B) MDA-MB-231 , (C) MCF-7 and (D) HT-29 cells after 72 hours of treatment. Cells were seeded and treated with SWCNT and SWCNT-PEG-FA nanotubes at 100 pg / ml over a period of 72 hours, whereafter a WST-1 assay was performed. Results are presented as means ± SEM (n=3). The asterisk denotes a significant decrease in cell viability when compared to the control (*p<0.05; **p<0.01 ; ***p<0.001 and ****p<0.0001 ). The percentage viable cells after the treatment period were compared with the percentage untreated viable cells.

[0049] Figure 18: Cytotoxicity of bio-conjugated carbon nanotubes in (A) MCF-12A, (B) MDA-MB- 231 , (C) MCF-7 and (D) HT-29 cells after 72 hours of treatment. Cells were seeded and treated with different bio-conjugates at a concentration of 100 pg / ml for carbon nanotubes and 10 pg / ml for crude extracts over a period of 72 hours. Whereafter, a WST-1 assay was performed. Results are presented as means ± SEM (n=3). The asterisk denotes a significant decrease in cell viability when compared to the control (*p<0.05; **p<0.01 ; ***p<0.001 and ****p<0.0001 ). The percentage viable cells after the treatment period were compared with the percentage untreated viable cells.

[0050] Figure 19: Demonstrates the mechanism of extrinsic interactions between SWCNT -PEG-FA- AM-DC-DV-TV (black) and (A) MCF-7, (B) MDA-MB-231 and (C) HT-29 cancer cell lines. Cancer cells were treated over a period of 24 hours and images were taken using a 4X objective on Zeiss Olympus inverted microscope. Figure 20: Live cell imaging of bio-conjugated carbon nanotubes stained, nucleus, cell membrane in MDA-MB-231 breast cancer cells.

[0051] Figure 21 : Live cell imaging of bio-conjugated carbon nanotubes stained, nucleus, cell membrane on MCF-7 breast cancer cell line.

[0052] Figure 22: Live cell imaging of bio-conjugated carbon nanotubes stained, nucleus, cell membrane on HT-29 colorectal cancer cell line.

[0053] Figure 23: Fluorescence microscopy images of A) MDA-MB-231 , B) MCF-7, and C) HT-29 cancer cells after treatment with SWCNT-PEG-FA-AM-DC-DV-TV for 72 hours.

[0054] Figure 24: Flow cytometry images showing the cytotoxicity of bio-conjugated carbon nanotubes in MDA-MB-231 cells after 72 hours of treatment. Cells were seeded and treated with bio-conjugates, whereafter the cells were stained with Annexin V and propidium iodide. Results are presented as means ± SEM (n=3). The asterisk denotes a significant decrease in cell viability when compared to the control (*p<0.05; **p<0.01 ; ***p<0.001 and ****p<0.0001 ).

[0055] Figure 25: Flow cytometry images showing the cytotoxicity of bio-conjugated carbon nanotubes in MCF-7 cells after 72 hours of treatment. Cells were seeded and treated with bioconjugates, whereafter the cells were stained with Annexin V and propidium iodide. Results are presented as means ± SEM (n=3). The asterisk denotes a significant decrease in cell viability when compared to the control (*p<0.05; **p<0.01 ; ***p<0.001 and ****p<0.0001 ).

[0056] Figure 26: Flow cytometry images showing the cytotoxicity of bio-conjugated carbon nanotubes in HT-29 cells after 72 hours of treatment. Cells were seeded and treated with bioconjugates, whereafter the cells were stained with Annexin V and propidium iodide. Results are presented as means ± SEM (n=3). The asterisk denotes a significant decrease in cell viability when compared to the control (*p<0.05; **p<0.01 ; ***p<0.001 and ****p<0.0001 ).

[0057] DETAILED DESCRIPTION OF THE INVENTION

[0058] A conjugate including a nanocarrier to which (i) a targeting moiety which binds to a receptor on a target cell and (ii) compounds from at least one plant extract derived from a plant selected from the group consisting of Tulbaghia violacea, Annona muricata, Dicoma capensis and Dodonaea viscosa are bound is described herein. A composition including the conjugate, a method for preparing the conjugate and uses of the conjugate and composition for treating cancer are also described.

[0059] The nanocarrier can be a nanoparticle, such as silver, gold, palladium, titanium, zinc or copper nanoparticles, nanotubes, nanomicelles or liposomes. Typically, the nanocarrier is a carbon nanotube, and more particularly is a single walled carbon nanotube (SWCNT).

[0060] The carbon nanotube can be functionalised with polyethylene glycol.

[0061] In one embodiment, the targeting moiety is folic acid, which can target folate receptors that are overexpressed in certain cancers.

[0062] The conjugate can include compounds from extracts of at least two, at least three or all four of Tulbaghia violacea, Annona muricata, Dicoma capensis and Dodonaea viscosa.

[0063] For example: compounds from a Tulbaghia violacea extract can be conjugated to the nanocarrier; compounds from an Annona muricata extract can be conjugated to the nanocarrier; compounds from a Dicoma capensis extract can be conjugated to the nanocarrier; compounds from a Dodonaea viscosa extract can be conjugated to the nanocarrier; compounds from Tulbaghia violacea and Annona muricata extracts can be conjugated to the nanocarrier; compounds from Tulbaghia violacea and Dicoma capensis extracts can be conjugated to the nanocarrier; compounds from Tulbaghia violacea and Dodonaea viscosa extracts can be conjugated to the nanocarrier; compounds from Annona muricata and Dicoma capensis extracts can be conjugated to the nanocarrier; compounds from Annona muricata and Dodonaea viscosa extracts can be conjugated to the nanocarrier; compounds from Dicoma capensis and Dodonaea viscosa extracts can be conjugated to the nanocarrier; compounds from Tulbaghia violacea, Annona muricata and Dicoma capensis extracts can be conjugated to the nanocarrier; compounds from Tulbaghia violacea, Annona muricata and Dodonaea viscosa extracts can be conjugated to the nanocarrier; compounds from Annona muricata, Dicoma capensis and Dodonaea viscosa extracts can be conjugated to the nanocarrier; compounds from Tulbaghia violacea, Dicoma capensis and Dodonaea viscosa extracts can be conjugated to the nanocarrier; or compounds from Tulbaghia violacea, Annona muricata, Dicoma capensis and Dodonaea viscosa extracts can be conjugated to the nanocarrier.

[0064] In one embodiment, more compounds from the Annona muricata and Dicoma capensis extracts are conjugated to the inner surface of the carbon nanotube (i.e. endohedrally) than to the outer surface, while more compounds from the Tulbaghia violacea and Dodonaea viscosa extracts are conjugated to the outer surface (i.e. exohedrally) than to the inner surface.

[0065] A composition can be provided which includes a plurality of these conjugates and optionally a pharmaceutically acceptable excipient. This composition can be used for treating cancer. The cancer can be any cancer and is typically a solid tumour such as gynaecological cancers (e.g. cervical cancer), oesophageal cancer, breast cancer, ovarian cancer, gastro-intestinal cancers (e.g. gastric cancer), prostate cancer, pancreatic cancer, lung cancer, liver cancer, colorectal cancer, stomach cancer, neuroblastoma and gliomas. In one embodiment the cancer is breast cancer. In another embodiment the cancer is colorectal cancer.

[0066] The composition can be formulated for administration to a patient, typically by intravenous administration.

[0067] The conjugate can be prepared by: binding a targeting moiety which binds to a receptor on a cancer cell to a nanocarrier; and contacting the nanocarrier with at least one plant extract derived from a plant selected from Tulbaghia violacea, Annona muricata, Dicoma capensis and Dodonaea viscosa and allowing compounds from the at least one plant extract to bind to the nanocarrier.

[0068] The nanocarrier is typically functionalised with polyethylene glycol before being brought into contact with the plant extract(s).

[0069] The plant extracts can be obtained by closed loop extraction or solvent extraction. Preferably, however, the plant extracts are obtained by closed loop extraction.

[0070] Compounds from the Annona muricata and Dicoma capensis extracts can be conjugated substantially to the inner surface of the carbon nanotubes by, for example, bringing the carbon nanotubes into contact with these extracts before bringing the carbon nanotubes into contact with the Tulbaghia violacea and Dodonaea viscosa extracts. Similarly, compounds from the Tulbaghia violacea and Dodonaea viscosa extracts can be conjugated substantially to the outer surface of the carbon nanotubes by, for example, only bringing the carbon nanotubes into contact with these extracts after already having contacted the carbon nanotube with the Annona muricata and Dicoma capensis extracts (Figure 1 ).

[0071] In one embodiment, the plant extracts are brought into contact with the carbon nanotubes in the following order: Annona muricata, then Dicoma capensis, then Dodonaea viscosa and then Tulbaghia violacea. This order of conjugation ensures that the folate moiety on the nanotubes is unbound and free to contact the folate receptor on target cancer cells and also ensures that the compounds that might affect healthy cells are loaded inside the nanotubes and are thus only delivered once inside the target cancer cell. The compounds can therefore exert their full activity on the subject while reducing the incidence of side effects.

[0072] A kit can also be provided for producing the conjugates. The kit can include plant extracts of at least one of, two of, three of or all four of Tulbaghia violacea, Annona muricata, Dicoma capensis and Dodonaea viscosa and instructions for producing the conjugates as described above. The kit can also include PEG and / or folic acid.

[0073] Carbon nanotubes

[0074] A carbon nanotube (CNT) is a cylindrical needle-like structure that acts as a carrier for various molecules, drugs, proteins, genes, and the like. CNTs are sheets of graphite in the form of tubes, and the tubes can be rolled up in a variety of ways, most commonly to form either single-walled carbon nanotubes (SWCNTs) or multi-walled carbon nanotubes (MWCNTs), as shown below. SWCNTs are cylindrical, 1 D structures characterized by large length to diameter ratios. MWCNTs are a collection of cone-shaped SWCNTs, although their diameter and length differ from that of the SWCNTs, as well as their properties. The most important feature that distinguishes SWCNTs is that the wall of the nanotube consists of only one graphene layer.

[0075]

[0076] Purification of SWC NTs

[0077] Various techniques have been developed for the synthesis of carbon nanotubes, including the arc discharge method, laser ablation method and chemical vapor deposition (CVD) method. These methods produce CNTs containing impurities such as amorphous carbon, graphite, and metal particles. One of the first steps in this study was therefore the purification of carbon nanotubes, using a chemical method. With this method, defects are normally introduced along the tubes and at the apex of the tubes, resulting in significant damage to the structure and morphology of CNTs. When the reaction conditions are properly controlled, purification of CNTs by removing metal catalyst particles may result in a higher degree of purity and an increased opening of the tips. Wet oxidation was conducted in this study with nitric acid. The impurities such as amorphous carbon normally have a higher oxidation rate than CNT and they are eliminated by the selective oxidative etching process used in this approach. It has been shown that amorphous carbon exhibits a high oxidative activity due to the presence of hanging bonds which can be easily oxidized. Nanotube tips may open due to oxidation, and the pentagon-shaped carbon ring tips of nanotubes are less chemically stable than the hexagonal-shaped carbon ring tips of tube cylinders. During CNT purification with nitric acid, the seven-membered rings at the tips, which are constructed from double bonds connecting two pentagons, render this region susceptible to oxidative reactions.

[0078] Both the duration of the oxidation as well as the temperature at which the catalyst particles are refluxed are important factors that determine the effectiveness of the removal of the catalyst particles. Therefore, this study investigated temperatures of 60°C and 80°C for refluxing and a 3 hour reaction time. Degradation of the CNT structure occurs if they are exposed to excessive refluxing, longer periods, high temperatures and acid concentrations. The exposure of CNTs to a short period of acid refluxing and low acid concentrations and temperatures results in a partial removal of the metal particles from the CNT structures. Nitric acid attacks the defective sites during the purification process and intercalates into the CNTs allowing the walls of the tubes to be unzipped by further oxidative etching, which in turn results in the increase of the distance between the layers of the tubes.

[0079] In order to avoid the high risk of oxidative damage from oxidation with nitric acid, hydrochloric acid was investigated as an alternative. The reaction of hydrochloric acid with carbon nanotubes displaces HCI into H+ and CI-. These in turn react with impurities in the nanotubular structure. Hydrochloric acid was found to be a less aggressive agent compared to nitric acid.

[0080] Functionalisation of SWCNT with PEG and folic acid

[0081] CNT toxicity has been reported during in vivo applications, which is a concern for their clinical applicability and a barrier to their use. During circulation in the bloodstream, it is also difficult to avoid rapid filtration of CNTs in the kidney or liver and clearance through the reticula-endothelial system (RES). In order to overcome these problems, CNTs can be functionalized, for example with proteins, nucleic acids, polyethylene glycol (PEG), polysaccharides, aptamers or polymers. They not only give the SWCNTs solubility in water (i.e. they make them hydrophilic) and compatibility with biological systems, but they also provide them with resistance to elimination from the RES and help them to extend the circulation period once they are in the blood. In this study, SWCNTs were functionalised with PEG by PEGylation (a covalent method).

[0082] Several human cancers express the folate receptor protein (PR) on their surfaces, and folic acid (FA, Vitamin B9) can bind to the folate receptor protein. A folate conjugate was therefore formed by attaching it to the CNTs to trigger cellular uptake via endocytosis. Folate conjugates tend to bind tightly to the folate receptor and trigger cellular uptake. The FA molecule is composed of two carboxyl groups as well as a large, conjugated structure. The large, conjugated molecule can slide easily between two surfaces of CNTs by means of TT-TT stacking mechanism, and the methoxy-PEG-OH (mPEG-OH) molecule can be easily attached to the surface of the FA through a condensation reaction. Folic acid conjugates poorly to the surface of the polymer because of the low reactivity of the carboxylic acid groups in PEG. Therefore, the carboxyl group of folic acid was first activated with dicyclohexylcarbodiimide (DCC) and DMAP was added to neutralize FA acidity. The combination of PEG and FA resulted in an ester bond.

[0083] Dispersion analysis of SWCNT-PEG-FA

[0084] The purification with hydrochloric acid generated radicals that opened C=C bonds for SWCNT. The oxygen containing groups in folic acid and polyethylene glycol all contributed to forming bonds on the carbon nanotube surface. As a result, the treated carbon nanotubes became hydrophilic and could be easily dispersed in water. The results showed that the functionalised carbon nanotubes can retain >90% of dispersibility over 21 day, which makes them suitable for application in cancer therapeutics.

[0085] Analysis of plant extracts

[0086] Annona muricata (AM extract)

[0087] A high abundance of Astragalin-7-rhamnoside was found in the extracts of Annona muricata (AM). Astragalin-7-rhamnoside acts by inhibiting cancer proliferation and this is mediated through arrest of the different phases of cell cycle. Epicatechins were also identified, and these are known to inhibit carcinogen activity, tumorigenesis, proliferation, angiogenesis, and induction of cell death. Their mechanism is associated with modulation of reactive oxygen species (ROS) production. Although epicatechins have a dual function of antioxidant and pro-oxidant potential, epicatechin- mediated modulation of ROS production is responsible for its anticancer effects. They act by inhibiting the nuclear factor-KB signaling. In addition, they are known to induce epigenetic modification by inhibition of DNA methyltransferase activity and regulation of acetylation of histones, leading to an upregulation of apoptosis. The other flavonoids found in the plant also offered a wide variety of anticancer effects like participating in arresting the cell cycle, inducing apoptosis, autophagy, as well as suppressing cancer cell proliferation and invasiveness. In closed loop extraction, protocatechuic acid was identified. This compound down-regulates the Ras / Akt / NF-KB pathway by targeting RhoB activation, which in turn leads to a reduction of matrix metalloproteinases-mediated cellular events in cancer cells.

[0088] Dicoma capensis (DC extract)

[0089] Solvent extraction obtained Gamma keto acids, which are known to act by inhibiting histone deacetylase activity (HDAC). The HDAC inhibitors induce cancer cell cycle arrest, differentiation, cell death, reduce angiogenesis and modulate immune response. Some antioxidant compounds were identified such as gallic acids, germacranolides and quinic acids. These compounds show anticancer activity through apoptosis-mediated cytotoxicity. Hydroxybenzoic acid was obtained, which enhances the sensitivity of human breast cancer cells to adriamycin as a specific HDAC6 inhibitor by promoting the HIPK2 / p53 pathway. The phenylpropanoic acids, abscisic acids and prenylated flavanones identified in the extract exert anti-proliferative effects, S-phase cell cycle arrest and apoptosis. Under closed loop extraction, one of the major compounds identified was a glucuronide derivative. These exhibit unique anticancer activities which include inducing apoptosis, inhibiting proliferation and invasion of tumours by suppressing a variety of cellular signalling pathways. The schizanthine alkaloids, saccharolipids and germacranolides also target DNA replication or protein synthesis, resulting in apoptosis of the neoplastic cells. The dihydropyranone compounds identified exert anti-proliferative activity. These compounds inhibit nuclear translocation of NF-KB protein. Apart from acting on N F-KB, they also regulate class I Histone deacetylase proteins such as HDAC-3 and 8 that have a crucial and regulatory role in cell-proliferation. The luteolin compounds identified are commonly known to induce cell cycle arrest, induce apoptosis, inhibit migration and epithelial mesenchymal trans-differentiation of breast cancer cells. Luteolin-induced apoptosis involves reactive oxygen species (ROS) generation, DNA damage, activation of the ATR— >Chk2— >p53 signalling pathway, inhibition of the nuclear factor (NF)-KB signalling pathway, activation of the p38 pathway and depletion of antiapoptotic proteins. The compound also inhibits breast cancer intravasation through the lymphatic barrier.

[0090] Dodonaea viscosa (DV extract)

[0091] In the solvent extraction, the coumarin derivatives obtained are potent compounds that trigger cell cycle arrest, angiogenesis inhibition, kinase inhibition, telomerase inhibition and suppression of carbonic anhydrase activity. The sesquiterpenoids inhibit the signalling pathway of Nuclear Factor-kB. Nuclear Factor-kB in cancer cells has a major role in inhibiting apoptosis, induction of metastasis, cell proliferation, cell transformation, invasion, metastasis, resistance to chemotherapy, and resistance to radiotherapy. The kaempferol identified in the extract can act by inducing the activation of cysteine proteases involved in apoptosis initiation and execution. It also induces caspases-3,-7,-9, and Poly (ADP-ribose) polymerase (PARP), therefore preventing the accumulation of reactive oxygen species (ROS) involved in cancer development. Other properties of kaempferol are the inhibition of angiogenesis and the capacity to preserve normal cell viability. Under closed loop extraction, chaicone derivatives were unique to this extraction technique. These bioactive compounds show activity against cancers via multiple mechanisms, including cell cycle disruption, autophagy regulation, apoptosis induction and immunomodulatory and inflammatory mediators.

[0092] Tulbaghia violacea (TV extract)

[0093] Under solvent extraction, procyanidins were identified and these are known to affect several key cancer-associated genes. In particular, the genes involved in cell cycle and DNA replication are altered by procyanidins. Another potent compound whose ontology belongs to the colchicine derivatives was obtained. The exact structure of the compound was unidentified in the LC-MS database. The molecular weight of the compound suggested that it is a novel congener of colchicoside. Colchicine derivatives are known to severely damage the internal scaffolding of cancer cells. The therapeutic mechanism of action of colchicine is its capacity to bind to tubulins. It binds to soluble tubulin to form tubulin-colchicine complexes in a poorly reversible manner, which then binds to the ends of microtubules to prevent the elongation of the microtubule polymer. Since tubulin plays a vital role in helping cells to divide in two, colchicine effectively stops cell division. Colchicine is a classical anti-mitotic drug which blocks mitotic cells in metaphase. Other effects of colchicine on malignancy include inhibition of cancer cell migration and metastatic potential, cell blebbing via a Rho / Rho effector kinase (ROCK) / myosin light chain kinase (MLCK) pathway, inhibition of angiogenesis, limitation of adenosine triphosphate (ATP) influx into mitochondria and release of cysteine-dependent aspartate-directed proteases (caspases) and cytochrome-c, leading to apoptotic cell death. The rutin and jasmonic acid compounds identified play multifaceted roles in suppressing the proliferation of cancer cells and inducing their death. Under closed loop extraction, tetramic acids and terniflorins were identified and their anticancer activity is shown in cell cycle arrest and as antioxidants. Colchicines were also the most abundant compounds in the crude extract.

[0094] Mixture of Annona muricata, Dicoma capensis, Dodonaea viscosa and Tulbaghia violacea by closed loop extraction

[0095] Closed loop extraction yielded more bioactive compounds compared to solvent extraction and was chosen as the extraction method. 30 bioactive compounds were found in the combined extract. One of the compounds identified was thymol glucoside. It is known to exert anticancer activity by utilizing mechanisms such as caspase-dependent apoptosis and cellular growth suppression. In cancer cells, it causes cellular damage such as lipid degeneration, mitochondrial damage, and nucleolar segregation. In addition, antioxidant and cytoprotective effects along with the antiapoptotic, anti-inflammatory / immunomodulatory, and antigenotoxic effects in normal cells may be the key mechanisms of thymol's anticancer activity in different test systems.

[0096] Bioconjugation of SWCNTs

[0097] Optimization studies were performed to determine the amount of time it takes to load SWCNT with plant compounds. The UV-Vis spectra showed the peak for SWCNT at 225 nm. This peak shifted after plant compounds were added to it. The shift in peak position was due to the conjugated pi systems becoming larger. This resulted in the energy gap for a TT - TT* transition becoming increasingly narrow, and the wavelength of light absorbed correspondingly became longer. The UV-Vis spectra of most flavonoids consist of two major absorption maxima, one of which occurs at lower wavelength range 240-285 nm and the other in the range 300-400 nm. The peak for folic acid also appeared in the range 270-290 nm. In the bioconjugates, the band for folic acid and the lower wavelength band for flavonoids overlapped causing a broader peak. The higher wavelength band for flavonoids was observed at -335 nm. The higher wavelength band originated from the A-ring benzoyl system and the lower wavelength band from the B-ring cinnamoyl system, typical of a flavonoid skeleton. The functionalization of carbon nanotubes with polyethylene glycol and folic acid allowed more reaction sites for bio-compounds to bind to the carbon nanotube framework. The UV-Vis spectrum with all extracts combined (Figure 12) showed a higher loading of bioactive compounds as the absorbance of flavonoids and SWCNT-PEG-FA was closer to each other compared to the other extracts. Flavones absorb between 304-350 nm while flavonols absorb in the range 352-385 nm, so the position of the peak absorption provided a guide to the type of flavonoid which was examined. In addition, highly oxygenated flavones and flavonols tend to absorb at longer wavelengths than those with fewer oxygen substituents. Increasing oxygenation generally leads to bathochromic shifts, and the slight shifts observed in the flavonoid peak was due to the different flavonoids in each extract as confirmed by LC-MS analysis.

[0098] In vitro cytotoxicity studies

[0099] In vitro cytotoxicity of AM, DC, DV and TV plant extracts on MCF- 12A, MDA-MB-231, MCF-7 and HT-29 cells

[0100] The in vitro cytotoxicity for the 4 cell lines was conducted over a period of 24, 48 and 72 hours (Figure 13, Figure 14 and Figure 15). For MCF-12A cells, higher concentrations of 100 and 500 pg / ml had lower cell viabilities. At these concentrations more normal cells were killed. Although higher concentrations showed a lower cell viability in cancer cells, it was important to choose a concentration which preserves more normal cells and is at the same time potent enough to kill cancer cells. A concentration of 10 pg / ml was chosen and cell viabilities down to 20% were observed in cancer cells after a 72 hour treatment period (Figure 15).

[0101] At 10 pg / ml, the MCF-12A cells had a cell viability over 70% in the extracts except for the DC extract with 65% and p<0.0001. This could have been contributed by the high presence of alkaloids in the DC extract as observed in the LC-MS chromatogram. The lowest cell viability in MDA-MB-231 and MCF-7 cells at a concentration of 10 pg / ml over a 72 hour period was found in the AM extract at 25% and 20% respectively (p<0.0001 ). In HT-29 cells, the lowest cell viability was found in the DV extract. To quantitatively determine the potency and selectivity of the plant extracts the IC50 and SI values were determined.

[0102] Determination of IC50 values and selective index

[0103] The criteria of cytotoxicity activity for crude extracts, as established by the American National Cancer Institute (NCI), is an IC50 < 30 pg / mL after 72 hours of treatment. The IC50 values calculated in Table 10 shows that all extracts induced selective cytotoxicity towards HT-29, MDA- MB-231 and MCF-7 cancer cell lines and their values were within regulatory standards. The IC50 value determined the extract concentration required to reduce a population of viable cells by 50%, when compared to the cells grown with no exposure to the extract. This change in cell population size could be a result of increased cell death or suppressed cell proliferation. A low IC50 value means that the extract is potent at low concentrations, and it will show lower systemic toxicity when administered to the patient. For the HT-29 colorectal cancer cell line, the increase of the IC50 value was in the order, DV<TV<AM<DC. The MCF-7 breast cancer cell line had an order of TV<DV<AM<DC whilst the MDA-MB-231 had an order of DV<TV<AM<DC. From this data, it can be concluded that DV was the most potent extract in HT-29 and MDA-MB-231 cells and the TV extract was the most potent in MCF-7 cells.

[0104] The selectivity index (SI) reflects the differential cytotoxicity of a compound against tumour and normal cells. The greater the SI value of a compound, the more selective it is. An SI value above 2 indicates cytotoxic selectivity. All extracts exhibited an SI value above 2 and the order of increase was DC<TV<AM<DV, TV<DC<AM<DV and TV<DC<DV<AM for HT-29, MDA-MB-231 and MCF-7 respectively. From the data, it was concluded that the DV extract offered higher selectivity in MDA-MB-231 and MCF-7 cells. In HT-29 cells, the AM extract offered the highest selectivity.

[0105] In vitro cytotoxicity of a crude extract mixture with AM-DC-DV-TV

[0106] Due to the potency of all the individual extracts used, the four plant extracts were combined so as to make use of the different properties from each (Figure 16). In Figure 16A, the cell viability of the MCF-12A cells for the crude extract mixture was about 65% (p<0.0001 ) and on cancer cell lines (Figure 16B-D) the cell viability was lower than 15% (p<0.0001 ). This showed an improvement from the individual extracts, indicating a synergistic effect. The next step was to analyse the cytotoxicity of carbon nanotubes. The volume ratio for the crude extract mixture was based on the performance of each individual extract. The AM and DV extracts had higher volumes and DC had lower volumes due to its influence on normal cells. This ratio created a balance and ensured value addition from each extract. The sequence of conjugating the extracts with CNTs were also a factor. The AM-DC-DV-TV sequence was more effective.

[0107] In vitro cytotoxicity of carbon nanotubes

[0108] The property of CNTs that cause toxicity mainly comes from their structural similarity with asbestos fibres. The cytotoxicity of pure and functionalised carbon nanotubes was analysed in Figure 17 A-D. To ensure minimal cytotoxicity to normal cells, a low nanotube concentration of 100 pg / mL was used, ensuring a cell viability of over 90% across cell lines. A concentration of 100 pg / mL was sufficient to offer low cytotoxicity and to also load high amounts of bioactive compounds from the plant extract.

[0109] In vitro cytotoxicity of bio-conjugated carbon nanotubes

[0110] The functionalised carbon nanotubes were bio-conjugated with the plant extracts and their cytotoxicity was evaluated across all four cell lines (Figure 18 A-D). In MCF-12A cells, the cell viability increased up to 85% and less than 5% cell viability was recorded in cancer cell lines. This showed a significant improvement compared to the crude mixture without nanotubes. This shows that the functionalised single walled carbon nanotubes in this study are effective nanocarriers of drug compounds with excellent target specificity.

[0111] Surface interaction of bio-conjugate with cancer cells

[0112] Carbon nanotubes can interact with cancer cells in various ways on a surface level. This is dependent on the functionalisation of carbon nanotubes and the type of cancer cell. The mechanism of interaction in Figure 19A demonstrates an engulfing technique in which the SWCNT-PEG-FA-AM-DC-DV-TV bio-conjugate engulfs the MCF-7 breast cancer cells. This ensures that the bioactive compounds are released, and apoptosis is initiated. In Figure 19B, the image demonstrates an engulfing of MDA-MB-231 cells and cell shrinkage. A “halo” effect was also observed in one of the cells, indicating the first stages of apoptosis. In Figure 19C, attractive Van der Waals forces were experienced between the bio-conjugate and the HT-29 cancer cells, and this caused the cancer cells to surround the bio-conjugate and clump together. Since folates interact with cancer cells by folate receptor a and p, the folate part of the SWCNT-PEG-FA-AM- DC-DV-TV bio-conjugate acted as the tumour-targeting ligand. A strong attractive bond to a or folate receptors on the HT-29 cancer cells resulted in the subsequent interaction observed in the image.

[0113] Fluorescence microscopy of bio-conjugated carbon nanotubes

[0114] The fluorescence microscopy images in Figures 20, 21 and 22 demonstrated carbon nanotube internalization by cancer cells. For the SWCNT-PEG-FA without the plant extract, the nanotubes entered the cell membrane and some of the nanotubes went further and encompassed the nuclear membrane. The bio-conjugated nanotubes loaded with bioactive compounds from the crude extracts also showed cellular uptake. In comparison with the SWCNT-PEG-FA conjugate, the cellular uptake in the bio-conjugates was higher as observed by the intensity of green stains inside the cells. From the images, it was concluded that the bio-conjugates can be internalized by the cancer cells, and they can also interact around the cell membrane. Furthermore, the number of bio-conjugates which were internalized varied across cell lines. The difference might have been due to the mechanism of cellular uptake. The cellular uptake mechanism for the SWCNT-PEG- FA conjugate was folate receptor mediated endocytosis. It involved the binding of the SWCNT- PEG-FA conjugate to membrane bound FRs and the immediate invagination of the plasma membrane around the receptor-ligand complex to form an intracellular vesicle or endosome. The proposed mechanism was that this vesicle acidified to a lower pH through the action of proton pumps and induced a conformational change that released the conjugate from the folate delivery system. This process was non-destructive to the SWCNT-PEG-FA conjugate. In addition, the folate receptor could regularly recycle between the cytosol and cell surface, leading to higher cellular uptake of the conjugate. Apart from folate receptor mediated endocytosis, an additional mechanism for cellular uptake in the bio-conjugates might have occurred due to a change in morphology and increase in binding sites introduced by the plant extracts. Caveolar endocytosis is a clathrin-independent endocytic process which involves bulb-shaped, 50-60nm plasma membrane invaginations called caveolae and this type of mechanism could have happened in the bio-conjugates. It involves the bio-conjugates getting transported to caveolae invaginations on the cell membrane which then internalize the particles via caveola mediated endocytosis.

[0115] As a result, two mechanisms might have been experienced by the bio-conjugates for cellular uptake, which were folate receptor mediated endocytosis and caveola mediated endocytosis, whilst the SWCNT-PEG-FA conjugate had one cellular uptake mechanism only. Passive diffusion did not occur in this case because the conjugate and bio-conjugate sizes were large. This is advantageous as it limits random entry into cells (which reduces selectivity to cancer cells). The bio-conjugates showed evident signs of apoptosis across all cell lines, which included loss of the normal sheet-like growth, cells with shrunken cytoplasm, condensed chromatin, loss of normal shape, fragmentation, and formation of apoptotic bodies. Necrosis was also observed, with the leaking of cell contents, especially in the crude extract mixture (Figure 23).

[0116] Flow cytometry of carbon nanotube bio-conjugates

[0117] Figures 24, 25 and 26 show the flow cytometry graphs for the bio-conjugates across cancer cell lines. In MDA-MB-231 cancer cells, early apoptosis >50% was observed in AM, DV and AM-DC- DV-TV bio-conjugates and late apoptosis >50% was observed in DC and TV bio-conjugates. The bio-conjugate with all four plant extracts showed the lowest percentage of healthy cells at 0.9%. The highest percentages of necrotic cells were found in the TV and AM-DC-DV-TV bioconjugates. In MCF-7 cancer cells, about 75% of the cells in the AM-DC-DV-TV bio-conjugate were at a late apoptotic stage. Cells treated with the AM bio-conjugate had the highest percentage of necrotic cells in this cell line at 20%. The bio-conjugate with all plant extracts showed the lowest percentage of healthy cells, at 2,4%. In HT-29 cancer cells, the late apoptosis percentage was more compared to early apoptosis. The bio-conjugate with all plant extracts showed 81 % of late apoptotic cells and 1 ,5% of healthy cells. The TV bio-conjugate showed the highest percentage of necrotic cells, at 30%. The results elucidated on the type of cell death which occurred. Both apoptosis and necrosis occurred, and the quantitative data differed in each cell line. The extracts which contributed to more necrosis as a type of cell death were TV and AM. This was due to the nature of bioactive compounds contained in these extracts and their interactions in the cancer cells.

[0118] Order of conjugation of compounds from plant extracts

[0119] From the LC-MS data, the AM extract had the most relative abundance of Astragalin 7- rhamnoside. This bioactive compound has low aqueous solubility and undergoes rapid metabolism by phase I and II metabolic enzymes and this causes them to be rapidly removed from circulating blood. In addition, it is prone to degradation by temperature, pH, and oxygen exposure which results in poor chemical stability. As a result, the AM extract was conjugated first and loaded inside the carbon nanotube to offer a shield. Inside the nanotube, the Astragalin 7- rhamnoside bonding was via hydrogen bonding, TT-TT interactions, and van der Waals forces.

[0120] From the Selectivity Index data, the DC extract had lower selectivity compared to the other extracts. As a result, an endohedral functionalization was chosen to enable a less direct interaction with the cell membrane and enable its potency to be exerted once the bio-conjugate has established an interaction with the cancer cells.

[0121] The DV extract was the most potent extract in HT-29 and MDA-MB-231 cells and the TV extract was the most potent in MCF-7 cells. As a result, it was decided to non-covalently functionalize these extracts on the outside walls of the nanotubes, to enable the compounds from these extracts to interact quickly with the cancer cells and exert their potency. The DV extract had a higher selectivity than TV and so it was conjugated first exohedrally.

[0122] The sequence for the plant extracts bio-conjugation was therefore established as AM-DC-DV-TV.

[0123] The invention will now be described in more detail by way of the following non-limiting examples.

[0124] Examples

[0125] Synthesis and characterisation of SWCNTs functionalised with polyethylene glycol and folic acid

[0126] Reagents and Materials

[0127] SWCNTs (purity >95% carbon basis of which >99% as carbon nanotubes and 0.84 nm average diameter), N,N'-dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), polyethylene glycol (PEG-5000), folic acid (FA) purity >97%, hydrochloric acid 37% (HCI), nitric acid 60% (HNO3), anhydrous dimethyl sulfoxide (DMSO) and ethyl ether were all purchased from MERCK.

[0128] Non oxidative purification of SWCNT with HCI

[0129] The purification process was made up of two steps of filtration. At first, about 1 g of SWCNTs were annealed in a furnace at 250°C for two hours and left to cool. This was followed by refluxing of the sample in 500 ml of 1 M HCI at 60°C and another batch at 80°C for about 3 hours. Filtration was done using a 2.5 pm pore size Whatman filter paper and the filtrate was washed with deionized water until a neutral pH. The second step of filtration was done by annealing at 200°C for 30 mins. The sample was left to cool, and it was refluxed and filtered with the same procedure as the first round. The SWCNTs were dried at 70°C for 2 hours and later stored in a cool dry place.

[0130] Oxidative purification of SWC NT with HNO3

[0131] The procedure for the oxidative purification of SWCNTs was similar to that of non-oxidative purification but in this case, 3M HNO3 was used instead of 1 M HCL

[0132] Synthesis of SWCNT-PEG conjugates

[0133] About 25 mg of purified SWCNTs in HCI at 60°C were pre-sonicated for 1 hour and dispersed in 100 ml of deionized water. A 10 ml aqueous solution of PEG at 5 mg / ml was then added into the SWCNT suspension in a dropwise manner. This was followed by a 12-hour magnetic stirring at room temperature. The product formed was centrifuged at 5 000 rpm for 10 min. The centrifugation process was done three times by dispersing in 20 mL deionized water and then the SWCNT-PEG sample was stored.

[0134] Synthesis of SWCNT-PEG-FA conjugates

[0135] About 10 mmol of folic acid and 50 mg of SWCNT-PEG were mixed in 250 mL of anhydrous dimethyl sulfoxide (DMSO) under magnetic stirring for 2 hours. Approximately 5 mmol of N,N'- dicyclohexylcarbodiimide (DCC) was added to activate the carboxyl group and start the reaction. After 30 mins, a solution of 5 mmol 4-Dimethylaminopyridine (DMAP) was added to neutralize the acidity of folic acid. The mixture was allowed to react for a further 72 hours at room temperature under magnetic stirring. Centrifugation at 5000 rpm was used to collect the SWCNT-PEG-FA sample and it was washed three times in DMSO and twice in ethyl ether. Finally, the sample was washed three times in deionized water to remove any residual DMSO.

[0136] Results

[0137] The purity of the untreated SWCNT was >95% carbon basis and >99% as carbon nanotubes. This meant that about 5 % of the SWCNT sample was impure. There are two types of impurities common in SWCNT. These are transition metal catalyst particles and carbonaceous species. The carbonaceous species include unconverted / amorphous carbon, fullerenes, multi-shell carbon nanocapsules, and nanocrystalline graphites. The two-step process used in the purification process described herein played a role in refining the SWCNT and producing a pure compound. The annealing process assisted in removing some of the unconverted carbon and the acid reflux process dissolved soluble impurities and metal. The results of the characterization are described below. FTIR characterization

[0138] FTIR spectroscopy was used to assess the different purification methods and confirm the formation of conjugates. The selection of an oxidative and non-oxidative treatment is a balance between the carbon nanotube structure being retained and obtaining a purified product. Both parameters are important as the carbon nanotube structure needs to carry enough bioactive compounds and the purified form offers less toxicity. In this case, both the hydrochloric acid treatment at 60°C and the nitric acid treatment at 80°C showed high purity. However, the extent of the damage to the carbon nanotube structure was more in the nitric acid treatment. In further experiments, SWCNT were therefore purified in HCI at 60°C.

[0139] XRD characterization

[0140] X-ray diffraction studies were used to analyse the structure of the purified SWCNT particles. X- Rays have a wavelength between 0.01 nm to 10nm and they can provide data on the degree of crystallinity by penetrating inside the crystal structure of carbon nanotubes. FTIR results (not shown) confirmed that a temperature at 60°C with HCI was more favourable for treatment as the carbon nanotube walls and ends open more and allow metal impurities to be taken off. Hydrochloric acid was found to be less aggressive and posed less damage to the tubular structure as it maintained the crystallinity of the SWCNT.

[0141] The SWCNT purified in HCI at 60°C were therefore used to make SWCNT-PEG-FA. The functionalization of SWCNT with polyethylene glycol and folic acid was successful without damaging the SWCNT backbone.

[0142] Dispersion characterization

[0143] Single walled carbon nanotubes are frequently entangled during their synthesis and they normally re-aggregate quickly if left without functionalisation. The SWCNT in nature are hydrophobic and for use in cancer therapy, they need to maintain hydrophilic properties for at least 7 days. This is based on the estimation of how long they will stay in the body before they are excreted by the renal system.

[0144] The purified nanotubes in HCI and HNO3 showed hydrophobic properties. The hydrophobic properties of carbon nanotubes were neutralized by the presence of the OH group in PEG and this caused them to be hydrophilic. The carbon nanotubes remained in suspension and did not aggregate. This also confirmed that the functionalization of the SWCNT was successful.

[0145] After 21 days, over 90% of the carbon nanotubes were still in suspension. The PEG and FA functional groups were able to attach on SWCNT and in the process counterbalance the attracting Van der Waals forces between the SWCNT bundles. Plant extraction and bio-conjugation of single walled carbon nanotubes

[0146] Materials and reagents

[0147] Ethanol (purity > 99.9 %) and butane (purity > 99.9 %) were all purchased from MERCK. A blender was used for pulverizing. Deionized water was used for aqueous solution preparation. Tulbaghia violacea (TV), Dodonaea viscosa (D\ / )and Dicoma capensis (DC) were obtained from Western Cape, South Africa. Annona muricata (AM) was collected from Luanda, Angola.

[0148] Preparation of plant leaves

[0149] Dried plant leaves were pulverised using a commercial blender. The samples were weighed and kept in an amber flask at room temperature.

[0150] Organic solvent extraction

[0151] Following the grinding process, extraction was done by using an ethanol to plant material ratio of 1 :4 and an incubation of 72 hours. After 72 hours, the extracts were filtered through a sieve with a pore size of 150 pm, a cotton plug and then filter paper. The extracts were concentrated by using a rotary evaporator at 45eC until a viscous extract was obtained. The solvent-free extracts were kept at 4°C for further use.

[0152] Closed loop extraction

[0153] In closed loop extraction, the butane solvent was loaded into a pressurized tank. The plant leaves were put into an extraction tube and butane was passed through the plant to extract compounds. The butane was looped through the system, washing over the plant material repeatedly. The compounds were then purged of all solvent in a separate vacuum oven. The extraction method occurred entirely within a closed vessel. The obtained product was stored at -20°C and reconstituted in ethanol prior to use.

[0154] Bioconjugation of carbon nanotubes with plant extracts

[0155] In one experiment, homogenous solutions of 50 mg crude plant extracts and 10 mg of SWCNT functionalized with PEG and folic acid (SWCNT-PEG-FA) were stirred at room temperature for 24 hours. The obtained dispersion was allowed to stand for 4 hours to attain equilibrium. The bioconjugated carbon nanotubes were centrifuged at 3000 rpm for 5 mins to remove any unentrapped plant compounds. The product was left to dry at room temperature for 24 hours and then stored at 8°C for further use.

[0156] In another experiment, the plant extracts were sequentially mixed with the SWCNT-PEG-FA. The order of mixing the plant extracts was: Annona muricata, Dicoma capensis, Dodonaea viscosa and then Tulbaghia violacea. This order produced the most entrapment of plant compounds on the carbon nanotubes, as both the inner and outer tube were loaded. The 4 crude extracts were mixed at a concentration of 10 pg / ml for each. The volume ratio was 1 ,0: 0,5: 1 ,0: 0,8 for AM, DC, DV and TV extracts, respectively.

[0157] Loading protocols for each extract

[0158] Endohedral Functionalization - AM and DC

[0159] About 5 g of SWCNT-PEG-FA were annealed in a furnace at 150°C for three hours and left to cool. About 100 pg of annealed SWCNT-PEG-FA were mixed with 1 ml of 10 pg / ml AM extract under continuous stirring for 3 hours. The product was centrifuged at 1500 rpm to collect the bioconjugate SWCNT-PEG-FA-AM. This was followed by addition of 0,5 ml DC extract at 10 pg / ml with a further stirring for 3 hours. The product was centrifuged at 1500 rpm to collect the bioconjugate SWCNT-PEG-FA-AM-DC. The product was left to dry at room temperature for 24 hours.

[0160] Exohedral non-covalent functionalization - DV and TV

[0161] The SWCNT-PEG-FA-AM-DC bio-conjugate was mixed with 1 ml DV extract at 10 pg / ml for 5 hours under continuous stirring. The product was centrifuged at 1500 rpm to collect the bioconjugate SWCNT-PEG-FA-AM-DC-DV. The product was left to dry at room temperature for 24 hours.

[0162] The SWCNT-PEG-FA-AM-DC-DV bio-conjugate was mixed with 0,8 ml TV extract at 10 pg / ml for 5 hours under continuous stirring. The product was centrifuged at 1500 rpm to collect the bioconjugate SWCNT-PEG-FA-AM-DC-DV-TV. The product was left to dry at room temperature for 24 hours and stored at 8 °C for further use.

[0163] Characterization of plant extracts and bioconjugates

[0164] LC-MS analysis

[0165] Profiling of bioactive compounds was done using LC-MS analysis. A Waters Synapt G2 Quadrupole time-of-flight (QTOF) mass spectrometer (MS) connected to a Waters Acquity ultraperformance liquid chromatograph (UPLC) (Waters, Milford, MA, USA) was used for high- resolution UPLC-MS analysis. Column eluate first passed through a Photodiode Array (PDA) detector before going to the mass spectrometer, allowing simultaneous collection of UV and MS spectra. Electrospray ionization was used in negative mode with a cone voltage of 15V, desolvation temperature of 275°C, desolvation gas at 650 L / h, and the rest of the MS settings optimized for best resolution and sensitivity. Data were acquired by scanning from 150 m / z to 1500 m / z in resolution mode as well as in MSE mode. In MSE mode, two channels of MS data were acquired, one at a low collision energy (4V) and the second using a collision energy ramp (40-100V) to obtain fragmentation data as well. Leucine enkephalin was used as a lock mass (reference mass) for accurate mass determination and the instrument was calibrated with sodium formate.

[0166] Separation was achieved on a Waters HSS T3, 2.1 x 100 mm, 1.7 pm column. An injection volume of 2 pL was used and the mobile phase consisted of 0.1 % formic acid (solvent A) and acetonitrile containing 0.1 % formic acid as solvent B. The gradient started at 100% solvent A for 1 min and changed to 28% solvent B over 22 min in a linear way. It then went to 40% B over 50 s and a wash step of 1 .5 min at 100% solvent B, followed by re-equilibration to initial conditions for 4 min. The flow rate was 0.3 mL / min and the column temperature was maintained at 55°C. Compounds were quantified in a relative manner against a calibration curve established by injecting a range of catechin standards from 0.5 to 100 mg / L catechin. Data was processed using MSDIAL and MSFINDER.

[0167] Functions 1 (unfragmented channel) and 2 (fragmented channel) of the Waters MSe data were processed by MSDial to produce MS1 and MS2 spectra as well as extracted ion chromatograms with associated peak height intensity data. Since calibration standards are not available for most of these compounds, the peak height intensity was converted to concentration in a semi- quantitative manner by interpolation off a calibration curve for catechin acquired under the same instrumental conditions.

[0168] Each deconvoluted feature (alignment in MSDial), together with its associated MS1 and MS spectra was exported from MSDial to MSFinder. Databases were searched by MSFinder to attempt identification of the compounds. Based on the accurate mass elemental compositions, possible compounds were identified from the listed databases and then subjected to in-silico fragmentation. According to the spectral match between the in-silico and measured spectra, a score (out of 10) was assigned to each of the possible compound matches with the highest score being accepted as the most likely (assuming a score of at least 4).

[0169] Organic solvent extraction and closed loop extraction were performed to obtain bioactive compounds from the plants and LC-MS chromatograms (not shown) were used to compare the results of the two techniques.

[0170] The major compounds identified in Annona muricata after solvent extraction are shown in Figure 2 and Table 1. Seven bioactive compounds were identified and there was a high abundance of flavonoids. The most abundant flavonoid in the extract was Astragalin 7-rhamnoside.

[0171] Table 1 : Compound identification for Annona muricata LC-MS chromatogram by solvent extraction.

[0172] Average Rt Structure rank Ontology

[0173] (min) 15.81 Epicatechin-(4beta->6j Bifiavonoids

[0174] 16.92 Kaempferol3-gentiobioside7-rhamnoside Flavonoid-7-O-glycosides

[0175] 17.00 Kaempferol3-(6"-rhamnosylsophoroside) Flavonoid-3-O-giycosides

[0176] 18.20 Rutin Flavonoid-3-O-glycosides

[0177] 19.49 Astragalin 7-rhamnoside Fiavonoid-7-O-glycosides

[0178] 19.59 Kaempferol 3-O-rutinoside Flavonoid-7-O-glycosides

[0179] 22.17 Drummondol Oxepanes

[0180] A closed loop extraction technique was used to extract the bioactive compounds. Ten bioactive compounds were obtained from Annona muricata using a closed loop extraction technique (Table 2, Figure 3). Astragalin 7-rhamnoside was the most abundant bioactive compound identified, similar to the solvent extraction method.

[0181] Table 2: Compound identification for Annona muricata LC-MS chromatogram by closed loop extraction.

[0182] Average Rt Structure rank Ontology

[0183] (min)

[0184] 4.37 Gallic acid Gallic acids

[0185] 6.19 1 ,3-Diferu loylglycerol Coumaric acids and derivatives

[0186] 7.46 Gentesic acid 5-O-glucoside Phenolic giycosides

[0187] 8.21 Protocatechuic acid Hydroxybenzoic acid derivatives

[0188] 16.75 Kaempferol 3-gentiobioside 7-rhamnoside Flavonoid-7-O-glycosides

[0189] 16.83 Kaempferol 7-galactoside 3-rutinoside Flavonoid-7-O-glycosides

[0190] 18.07 Rutin Flavonoid-3-O-glycosides

[0191] 19.38 Astragalin 7-rhamnoside Flavonoid-7-O-glycosides

[0192] 19.57 Lonicerin Flavonoid-7-O-glycosides

[0193] 21.99 Fusarentin 2-benzopyrans

[0194] Twenty bioactive compounds were obtained from Dicoma capensis using the solvent extraction method (Table 3, Figure 4). Quinic acids showed the highest relative abundance in the crude extract.

[0195] Table 3: Compound identification for Dicoma capensis LC-MS chromatogram by solvent extraction.

[0196] Average Structure rank 1 Ontology

[0197] Rt (min)

[0198] 5.13 2-Methylene-4-oxopentanedioic acid Gamma-keto acids and derivatives 6.21 Gallic acid Gallic acids

[0199] 8.72 Gentesic acid 5-O-glucoside Phenolic giycosides

[0200] 9.75 Protocatec u ic acid Hydroxybenzoic acid derivatives

[0201] 12.83 3-O-p-Coumaroyiquinic acid Quinic acids and derivatives

[0202] 13.51 Chlorogenic acid Caffeoylquinic acid

[0203] 13.64 (iS,6R)-2-succinyi-6-hydroxycyclohexa-2,4- Gamma-keto acids and derivatives diene-1 -carboxylic acid

[0204] 13.72 Eucomic acid Phenylpropanoic acids

[0205] 15.39 3-O-p-Coumaroylquinic acid Quinic acids and derivatives

[0206] 15.47 (2S)-6-(gamma,gamma-dimethylallyl)-3’,4’- 6-prenylated flavanones dimethoxy-6”,6”- dimethylpyran[2”,3”:7,8]flavanone

[0207] 16.97 3-O-Caffeoyl-4-O-methylquinic acid Quinic acids and derivatives

[0208] 17.93 5-hydroxy-2-(4-hydroxyphenyl)-7-{[3,4,5- Flavonoid-7-O-glycosides trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-3,4- dihydro-2H-1 -benzopyran-4-one

[0209] 18.35 Phaseic acid Abscisic acids and derivatives

[0210] 18.98 Cichorioside C Germacranolides and derivatives

[0211] 19.78 Glochidacuminoside C Saccharolipids

[0212] 21.16 Thymol glucoside;Thymol-beta-D-glucoside Terpene glycosides

[0213] 21.51 Morusyunnansins 2-arylbenzofuran flavonoids

[0214] 22.02 Drummondol Oxepanes

[0215] 22.10 Drummondol Oxepanes

[0216] 23.65 Luteolin Flavonoid-7-O-glucuronides

[0217] Fifteen bioactive compounds were obtained from Dicoma capensis using the closed loop system extraction method and the most abundant compounds were alkaloids (Table 4, Figure 5). The quinic acids which had a relative abundance of over 90% in the solvent extraction, showed a 60% reduction using closed loop extraction. This meant that the closed loop system favoured the extraction of alkaloids. However, no alkaloids were obtained on the solvent extraction method, and this showed that the closed loop extraction technique was more favourable.

[0218] Table 4: Compound identification for Dicoma capensis LC-MS chromatogram by closed loop extraction.

[0219] Average Structure rank Ontology

[0220] Rt (min)

[0221] 5.59 Protocatechu ic acid Hydroxybenzoic acid derivatives

[0222] 9.47 O-Desmethylvenlafaxine glucuronide Glucuronide derivatives

[0223] 12.87 Chlorogenic acid Quinic acids and derivatives

[0224] 13.05 (1 S,6R)-2-succinyl-6-hydroxycyclohexa-2,4-diene-1 - Gamma-keto acids and derivatives carboxylic acid

[0225] 14.59 Schizanthine A Alkaloids

[0226] 14.60 Schizanthine N Alkaloids

[0227] 15.00 Vomifoliol 9-[xylosyl-(1 ->6)-glucoside] Fatty acyl glycosides of mono- and disaccharides

[0228] 16.37 3-O-Caffeoyl-4-O-methylquinic acid Quinic acids and derivatives

[0229] 17.31 Epiryanodine Alkaloid

[0230] 18.09 Cichorioside M Germacranolides and derivatives 20.59 Fusarentin 2-benzopyrans

[0231] 20.84 Thymol glucoside Terpene glycosides

[0232] 20.86 Pestalotheol A Dihydropyranones

[0233] 21.95 Glochidacuminoside C Saccharolipids

[0234] 23.06 Luteolin Flavonoid-7-O-glucuronides

[0235] Fifteen bioactive compounds were obtained from Dodonaea viscosa using the solvent extraction method (Table 5, Figure 6). A flavonoid-3-O-glycoside compound called keioside was the most abundant in the crude extract.

[0236] Table 5: Compound identification for Dodonaea viscosa LC-MS chromatogram by solvent extraction.

[0237] Average Rt Structure rank Ontology

[0238] (min)

[0239] 9.43 Gentesic acid Phenolic glycosides

[0240] 11.12 Chiorogenic acid Caffeoylquinic acid

[0241] 12.91 3-O-p-Coumaroylquinic acid Quinic acids and derivatives

[0242] 13.84 Cryptochiorogenic acid Quinic acids and derivatives

[0243] 15.73 4-methylumbelliferyl alpha-D-galactoside Coumarin glycosides

[0244] 16.73 Dihydroferulic acid 4-O-glucuronide Phenolic glycosides

[0245] 18.37 Robinin Flavonoid-7-O-glycosides

[0246] 19.06 Rutin Flavonoid O-giycosides

[0247] 19.45 Astragalin 7-rhamnoside Flavonoid-7-O-glycosides

[0248] 19.57 Lonicerin;Luteolin 7-neohesperidoside Flavonoid-7-O-glycosides

[0249] 20.97 Keioside Flavonoid-3-O-glycosides

[0250] 21.11 Sexangularetin 3-glucoside 7-rhamnoside Flavonoid-7-O-glycosides

[0251] 21.53 2-oxohept-3-enedioate Medium-chain keto acids and derivatives

[0252] 21.77 Morusyunnansins 2-arylbenzofuran flavonoids

[0253] 23.51 Cinncassiol C3 Sesquiterpenoids

[0254] Twenty bioactive compounds were obtained from Dodonaea viscosa using closed loop system extraction (Table 6, Figure 7). The solvent extraction method only yielded 15 bioactive compounds and was therefore less favourable. This could have been due to some thermolabile bioactive compounds being denatured by the heat in the solvent extraction process. Keiosides also showed the most relative abundance in the crude extract. Table 6: Compound identification for Dodonaea viscosa LC-MS chromatogram by closed loop extraction.

[0255] Average Structure rank Ontology

[0256] Rt (min)

[0257] 6.25 N-(3-benzo[e][1 ,3]benzoxazol-2-ylphenyl)-2- Chaicone derivative naphthalen-2-yloxyacetamide

[0258] 6.80 Gallic acid Gallic acids

[0259] 8.93 Gentesic acid 5-O-glucoside Phenolic glycosides 9.29 Protocatechuic acid Hydroxybenzoic acid derivatives

[0260] 10.59 3-O-caffeoylquinic acids Quinic acids and derivatives

[0261] 11.05 Periplobiose O-glycosyl compounds

[0262] 12.32 3-O-p-Coumaroyiquinic acid Quinic acids and derivatives

[0263] 13.25 Chlorogenic acid Quinic acids and derivatives

[0264] 13.84 Cryptochlorogenic acid Quinic acids and derivatives

[0265] 15.73 4-methylumbelliferyl alpha-D-galactoside Coumarin glycosides

[0266] 16.73 Dihydroferuiic acid 4-O-glucuronide Phenolic glycosides

[0267] 18.37 Robinin Flavonoid-7-O-glycosides

[0268] 19.06 Rutin Flavonoid 6-glycosides

[0269] 19.45 Astragalin 7-rhamnoside Flavonoid-7-O-glycosides

[0270] 19.57 Luteoiin 7-neohesperidoside Flavonoid-7-d-giycosides

[0271] 20.97 Keioside Flavonoid-3-O-glycosides

[0272] 21.11 Sexanguiaretin 3-glucoside 7-rhamnoside Flavonoid-7-O-glycosides

[0273] 21.53 2-oxohept-3-enedioate Medium-chain keto acids and derivatives

[0274] 21.77 Morusyunnansins 2-arylbenzofuran flavonoids

[0275] 23.51 Cinncassiol C3 Sesquiterpenoids

[0276] One of the major compounds identified in this extraction which was absent in the solvent extraction method was the chaicone derivative. Six bioactive compounds were obtained from Tulbaghia violacea using the solvent extraction method (Table 7, Figure 8). Tulbaghia violacea showed unique compounds not found in other extracts and the most abundant one was a colchicine compound.

[0277] Table 7: Compound identification for Tulbaghia violacea LC-MS chromatogram by solvent extraction.

[0278] Average Rt Structure rank Ontology

[0279] (min)

[0280] 13.21 Epicatechin Catechins

[0281] 14.42 Procyanidin Flavonoid 8-C-glycosides

[0282] 15.19 Unidentified colchicine derivatives

[0283] 19.06 Rutin Flavonoid O-glycosides

[0284] 20.72 Astragalin 7-rhamnoside Flavonoid-7-O-glycosides

[0285] 22.06 Methyl tuberonate Jasmonic acids

[0286] Ten bioactive compounds were obtained from Tulbaghia violacea using closed loop extraction (Table 8, Figure 9). The solvent extraction only yielded six bioactive compounds, and this demonstrated the preservation of thermolabile compounds by the closed loop extraction technique. Table 8: Compound identification for Tulbaghia violacea LC-MS chromatogram by closed loop extraction.

[0287] Average Rt Structure rank Ontology

[0288] (min)

[0289] 9.03 Tirandamycin b Tetramic acid

[0290] 9.68 Colchicoside colchicine derivatives

[0291] 11.71 Procyanidin Flavonoid 8-C-glycosides

[0292] 12.74 Epicatechin Catechins

[0293] 13.92 Terniflorin Flavonoid-7-O-glycosides

[0294] 14.69 Unidentified colchicine derivatives

[0295] 18.39 Rutin Flavonoid-3-O-glycosides

[0296] 18.43 4'-O-Methylcarthamidin 7-(2-p-coumaroylglucoside) Flavonoid-7-O-glycosides

[0297] 20.10 Astragalin 7-rhamnoside Flavonoid-7-O-glycosides

[0298] 20.80 2-isocapryloyl-3R-hydroxymethyl-Y-butyrolactone Gamma butyrolactones

[0299] The LC-MS chromatogram for Annona muricata, Dicoma capensis, Dodonaea viscosa and Tulbaghia violacea mixture using the closed loop extraction is shown in Figure 10. The four plants were mixed in equal amounts with the objective of obtaining a more potent extract. The synergistic effect of the combinations was evidently seen in the chromatogram as a total of thirty bioactive compounds were obtained (Table 9). Table 9: Compound identification for Annona muricata, Dicoma capensis, Dodonaea viscosa and Tulbaghia violacea mixture by closed loop extraction.

[0300] Average Structure rank 1 Ontology

[0301] Rt (min)

[0302] 6.25 N-(3-benzo[e][1 ,3]benzoxazol-2-ylphenyl)-2- Chaicone derivative naphthalen-2-yloxyacetamide

[0303] 6.80 Gallic acid Gallic acids

[0304] 8.93 Gentesic acid 5-O-glucoside Phenolic glycosides

[0305] 9.29 Protocatechu ic acid Hydroxybenzoic acid derivatives

[0306] 10.59 3-O-caffeoylquinic acids Quinic acids and derivatives

[0307] 11.05 Periplobiose O-glycosyl compounds

[0308] 12.32 3-O-p-Coumaroylquinic acid Quinic acids and derivatives

[0309] 13.25 Chlorogenic acid Quinic acids and derivatives

[0310] 14.84 Epicatechin Catechins

[0311] 15.12 4-methylumbelliferyl alpha-D-galactoside Coumarin glycosides

[0312] 15.16 alpha-Hydrojuglone 4-O-b-D-glucoside Phenolic glycosides

[0313] 16.11 Veranisatin C Terpene lactones

[0314] 17.65 3",6"-Di-d-p-coumaroyltrifolin Fiavonoid 3-O-p-coumaroyi glycosides

[0315] 17.66 Robinin Flavonoid-7-O-glycosides

[0316] 18.32 Rutin Flavonoid-3-O-glycosides

[0317] 18.40 Panasenoside Flavonoid-3-O-glycosides

[0318] 18.81 Astragalin 7-rhamnoside Flavonoid-7-O-giycosides

[0319] 18.84 Neosaponarin Flavonoid-7-O-glycosides

[0320] 18.86 Kaempferol 3-O-rutinoside Flavonoid-7-O-glycosides 19.97 Glochidacuminoside C Saccharolipids

[0321] 20.20 Keioside Fiavonoid-3-O-glycosides

[0322] 20.28 Calendoflavoside Flavonoid-3-O-glycosides

[0323] 20.33 Sexanguiaretin 3-giucoside 7-rhamnoside Flavonoid-7-O-giycosides

[0324] 20.43 Quercetin 3'-methyl ether 3-galactoside-7-rhamnoside Flavonoid-7-O-glycosides

[0325] 20.48 Quercetin 3-glucosyi-(1 ->6)-galactoside Fiavonoid-3-O-glycosicies

[0326] 20.56 Quercetin 3-galactoside-7-glucoside Flavonoid-7-O-glycosides

[0327] 20.86 Thymol glucoside Terpene giycosides

[0328] 22.92 Cinncassiol C3 Sesquiterpenoids

[0329] 23.35 (-j-Epigailocatechin 3-p-coumaroate Epigailocatechins

[0330] 23.97 Luteolin Flavonoid-7-O-glucuronides

[0331] UV-Vis characterization

[0332] Polyethylene glycol and folic acid functionalised with SWCNTs provide negatively and positively charged functional groups on the surface of SWCNT-polymer complexes. The binding specificity of bioactive compounds from plants is due to the combination of many molecular properties such as hydrophobicity, electrostatic interactions, hydrogen bonding, and steric properties which form a coat on the SWCNT surface. This bonding mechanism was utilised for bio-conjugating carbon nanotubes with bioactive compounds from plants and the results were analysed by UV-Vis spectrophotometry.

[0333] UV-visible absorption studies were performed with a Cary dual-beam spectrophotometer using 1 cm quartz cuvettes in the wavelength range of 200-900 nm. This was done to determine the bioconjugation of the nanotubes.

[0334] The bioconjugation of plant compounds with carbon nanotubes was optimized as shown by the UV-Vis spectra in Figure 11 .

[0335] Optimization for the bioconjugates was conducted to obtain the reaction time required to load the maximum plant compounds into the SWCNT framework (Figure 11 ). The peak for SWCNT appeared at 225 nm for SWCNT-PEG-FA and it exhibited a hypsochromic shift to 220 nm after Annona muricata extract was added. The peak for folic acid was at 275 nm and it showed a bathochromic shift after bioconjugation.

[0336] Annona muricata extract and SWCNT-PEG-FA were stirred for 6, 12, and 24 hours. After 6 hours, the peak for flavonoids was not distinct meaning that the extract compounds were not entrapped fully. After an increase of the reaction time to 12 hours, the peak for flavonoids started emerging, however, bioconjugation had not reached its maximum. At a reaction time of 24 hours, a distinct peak appeared, and these conditions were chosen as the optimum reaction time for bioconjugation. The UV-Vis spectra of the bioconjugates are shown in Figure 12. The peak for the presence of flavonoids at -335 nm was chosen to confirm bio-conjugation since flavonoids were present in all four extracts as confirmed by LC-MS analysis. All spectra showed this peak confirming the successful bioconjugation of carbon nanotubes with plant extracts.

[0337] In vitro cytotoxicity studies of carbon nanotube bioconjugates on MCF-12A, MCF-7, MDA- MB-231 and HT-29 cell lines

[0338] The performance of the carbon nanotubes and the plant extracts with their bio-conjugates in killing cancer cells and preserving normal cells was evaluated.

[0339] Cell lines

[0340] Cytotoxicity studies of the crude extracts and bioconjugates used HT-29, a human colorectal adenocarcinoma cell line, MCF-7 and MDA-MB-231 , human breast cancer cell lines (American Type Culture Collection, Rockville, USA) and MCF12A, a human, non-tumorigenic breast epithelial cell line (University of Cape Town). For HT-29, MDA-MB-231 , and MCF-7, cell lines were cultured at 37°C and 5% CO2 in sterile Dulbecco's Modified Eagle's Medium (DMEM) (Sigma Aldrich, D5796), supplemented with 10% fetal bovine serum (Invitrogen Gibco, 10270-106) and 1 % PenStrep (invitrogen Gibco, 15140-122). Cells were aliquoted to 50 mL falcon tubes. The MCF-12A cell line was cultured in 50 / 50 DMEM and Nutrient Ham~F12 (Invitrogen Gibco, 21785- 029), 500 ng / mL hydrocortisone (Sigma-Aidrich, H0888), 10 ug / mL insulin (pharmacy), 100 ng / mL cholera toxin (Sigma Aldrich, C8052) and 20 ng / mL epidermal growth factor (EGF) (Invitrogen Gibco, 10450-013). About 10 ml growth medium was used to initially grow the cell lines in T75 flasks. When the cells were 70-80% confluent, they were passaged by incubation with TrypLE™ Express (Invitrogen Gibco, 12604-013).

[0341] Splitting and seeding of cells

[0342] Splitting was done after the cells were 80% confluent. TrypLE™ Express and growth medium were warmed to 37°C in a water bath. The old medium was removed and 4 ml TrypLE™ Express was added. The cells were placed in the shaker incubator for 4 min. To neutralise the trypsin, 8 ml of media was added. The loosened cells were transferred to a 15 ml tube and centrifuged for 3 min at 1500 rpm. The supernatant was decanted, and a pellet of cells remained. About 3 ml of medium was added to the pellet and a pipette was used to gently resuspend it. A haemocytometer was used to count resuspended cells. The cells were seeded and left to adhere for 48 hours after which the medium was changed. Treatment of cell lines

[0343] The crude extracts and carbon nanotube bioconjugates were prepared at various concentrations for the treatment of cell lines. The closed loop extraction method was chosen for the evaluation of cytotoxicity since it extracted more bioactive compounds. For the IC50 determination of crude extracts, cells were treated with various concentrations (1 , 10, 25, 100 and 500 pg / ml), and IC50 values were calculated using the AAT Bioquest software. Cell incubation was done at 24, 48, and 72 hours at 37°C. The ethanol in the crude extracts from closed loop extraction was at a final concentration of 0.07 %, and this percentage was used for the vehicle. For cytotoxicity studies on carbon nanotube bioconjugates, crude extracts at various concentrations were mixed with carbon nanotubes at 100 pg / ml. The bio-conjugated carbon nanotubes were centrifuged at 3000 rpm for 5 mins to remove any unentrapped plant compounds, this was done 3 times after resuspending them in deionized water. The product was left to dry at room temperature for 24 hours. Cells were then treated with the bio-conjugates and incubated for 72 hours at 37°C.

[0344] WST- 1 cell viability assay

[0345] The HT-29, MDA-MB-231 , MCF-7 and MCF-12A cell lines were cultured in 96 well plates with 100 pL growth medium at a seeding density of 5 x 103, 7 x 103, 8 x 103’ and 5 x 103cells / cm2respectively. The old medium was discarded once they reached confluency and treatment was conducted. After the treatment period, 10 pL of cell proliferation reagent WST-1 was added and plates were incubated for 4 h at 37°C and 5% CO2. The plates were shaken thoroughly for 1 min on a shaker. Absorbance was determined at 450 nm using a microtiter plate reader and cell viability was calculated as the fraction of viable, treated cells relative to untreated (control) cells. Each experiment was repeated three times.

[0346] Annexin V assay

[0347] Alexa fluor 488 was used to stain apoptotic cells and propidium iodide (PI) was a dead cell indicator. The cells treated with crude extracts and bio-conjugates used the procedure mentioned previously and the staining protocol was established as highlighted in Annexure F. After treatment with crude extracts and bioconjugates, cells were trypsinized, washed twice with cold PBS and re-suspended in 100 pl of binding buffer at a concentration of 500 000 cells / ml. Cells were stained with 5 pl of Annexin V and 10 pl PI for 30 min at room temperature. Data acquisition and analysis were performed with a flow cytometer (BD FACSMelody) and BD FACSChorus software v1 .1 .

[0348] Imaging

[0349] Fluorescence microscopy imaging

[0350] Cells were grown in a 6-well plate with a cover slip at the bottom. After treatment, they were stained with cell mask (orange) for the cell membrane, Hoechst (blue) for the nucleus and Alexa fluor 488 (green) for the bio-conjugates. Live cell imaging of the treatment groups was conducted, and fluorescent and bright field images were acquired using an Invitrogen EVOS™ FL Cell Imaging System (Thermo Fisher Scientific, CA).

[0351] Zeiss microscopy images

[0352] A 4X objective on a Zeiss Olympus CKX41 inverted microscope (Olympus, GMBH Japan) using the Zeiss Laboscope software (Carl Zeiss, Germany) was used.

[0353] Statistical Analysis

[0354] The visual representation and statistical analyses of the data was done using GraphPad Prism® Version 7 for Mac Os X (GraphPad Software, San Diego, CA). Values were represented as a percentage of the control and mean ± standard error of the mean (SEM). One-way ANOVAs and / or t-tests were used where applicable, using Bonferroni’s or Fisher’s LSD post-hoc tests. A p-value < 0.05 was considered to be statistically significant.

[0355] In vitro cytotoxicity results

[0356] In vitro cytotoxicity studies were carried out using the colorimetric WST-1 assay to measure the relative proliferation rates of cells in culture. The assay principle was based on the conversion of the tetrazolium salt WST-1 into a coloured dye by mitochondrial dehydrogenase enzymes. The soluble salt is released into the media. Within a given time, the reaction produces a colour change which is directly proportional to the amount of mitochondrial dehydrogenase in each culture.

[0357] In Figure 13, lower and higher concentration ranges for the crude extracts were chosen to establish the best linear fit for IC50 determination. The cell viability was expressed as a percentage of the control and all crude extracts showed varying cytotoxicity towards the cell lines. The cell viability at lower concentrations (1 ,10 and 25 pg / ml) for MCF-12A cells over a 24 hour treatment period was above 85% for all extracts (p<0.0001 ). The Annona muricata extract had the highest cell viability compared to the control. The cytotoxic effect of the extracts was evident in MDA-MB-231 , MCF-7 and HT-29 cancer cells as they showed a decrease in cell viability down to 45% at lower concentrations (Figure 13).

[0358] In Figure 14, the cell viability at lower concentrations (1 ,10 and 25 pg / ml) for MCF-12A cells over a 48-hour treatment period was above 75% for all extracts (p<0.0001 ). Furthermore, these concentrations exhibited a cell viability of 35 % in cancer cells (Figure 14).

[0359] In Figure 15, the cell viability at lower concentrations (1 ,10 and 25 pg / ml) for MCF-12A cells over a 72-hour treatment period was above 60% for all extracts (p<0.0001 ). For cancer cells, a cell viability down to 25 % was observed (Figure 15). The cell viability was significantly decreased between the vehicle and the control group. As a result, the different concentrations were compared to both the vehicle and control. For example, the cell viability for HT-29 cells was significantly decreased following AM treatment at the different concentrations (1 ,10 and 25, 100 and 500 pg / ml) when compared to the control and the vehicle (p<0.0001 ).

[0360] Determination of IC50 values and selective index

[0361] Table 10: Values for IC50 and selectivity index (SI) obtained for MCF-12A, HT-29, MDA-MB- 231 and MCF-7 cell lines treated with plant extracts using the closed loop extraction after a 72-hour period.

[0362] Compound MCF-12A HT-29 MDA-MB- MCF-7 HT-29 MDA-MB- MCF-7

[0363] IC50 IC50 231 IC50 SI 231 SI

[0364] IC50 SI

[0365] The efficacy of the extracts was expressed using the half maximal inhibitory concentration (IC50) in Table 10 taken after a 72-hour period of treatment.

[0366] The IC50 determination was calculated using the AAT Bioquest software and the selectivity index was calculated as the IC50 of normal cells over the IC50 of cancer cells. A lower cytotoxicity indicated higher potency of the extract and a higher SI indicated higher selectivity to cancer cells.

[0367] The efficacy of the crude extract mixture in cell lines was assessed in Figure 16 (A-D). The 4 crude extracts were mixed at a concentration of 10 pg / ml for each. The volume ratio was 1 :0, 5:1 :0, 8 for AM, DC, DV, and TV extracts. The viability of cancer cells was significantly decreased in the crude extract mixture with AM-DC-DV-TV groups when compared to the control (p<0.0001 ). For MCF-12A cells, cell viability was higher at 65 % and in cancer cells, they were all below 15% (p<0.0001).

[0368] The cytotoxicity of pure and functionalised carbon nanotubes was analysed in Figure 17 A-D. The cell viability results in Figure 17 (A-D) demonstrated that the carbon nanotubes alone and the functionalised carbon nanotubes did not cause a significant decrease in cell viability. The nanotubes were not toxic at a concentration of 100 pg / ml. A low concentration of 100 pg / mL was sufficient to offer low cytotoxicity and to also load high amounts of bioactive compounds from the plant extract. This ensured a cell viability of over 90% across cell lines. The functionalised carbon nanotubes were bio-conjugated with the plant extracts and their cytotoxicity was evaluated across all 4 cell lines (Figure 18 (A-D). The SWCNT-PEG-FA at a concentration of 100 pg / ml were bio-conjugated with plant extracts at a concentration of 10 pg / ml.

[0369] The viability in cancer cells was significantly decreased in the SWCNT-PEG-FA-TV, SWCNT- PEG-FA-DC, SWCNT-PEG-FA-AM, SWCNT-PEG-FA-DV, and SWCNT-PEG-FA-AM-DC-DV-TV groups when compared to the control (p<0.0001 ). Furthermore, cell viability was significantly decreased when comparing the combination SWCNT-PEG-FA-AM-DC-DV-TV group to the SWCNT-PEG-FA-TV, SWCNT-PEG-FA-DC, SWCNT-PEG-FA-AM, and SWCNT-PEG-FA-DV groups (p<0.0001 ). In MCF-12A cells, the cell viability increased up to 85%, and less than 5% cell viability was recorded in cancer cell lines. This was significant improvement compared to the crude mixture without nanotubes. Therefore, the functionalised single walled carbon nanotubes in this study were effective nanocarriers of drug compounds with excellent target specificity.

[0370] Inverted microscopy imaging

[0371] Inverted microscopy was used to assess interactions of bio-conjugates with cancer cells.

[0372] The surface interactions of the SWCNT-PEG-FA-AM-DC-DV-TV bio-conjugate with cancer cell lines was analyzed in Figure 19 (A-C). The image in Figure 19A demonstrates cancer cells engulfed by the bio-conjugate. The image in Figure 19B demonstrates engulfing, cell shrinkage and a “halo” effect. Figure 19C shows a phenomenon of attractive Van der Waals forces on cancer cells.

[0373] Fluorescence microscopy

[0374] Fluorescence microscopy was used to demonstrate whether the bio-conjugates are internalized by the cancer cells or surround the cell membrane.

[0375] The fluorescence microscopy images for the bio-conjugates are shown in Figures 20, 21 and 22 for MDA-MB-231 , MCF-7 and HT-29 cells respectively. Untreated cancer cells made up the control group and they demonstrated normal distribution and morphology across all cancer cell lines. The cell membrane and nucleus were all intact. The cancer cell lines were treated with SWCNT-PEG-FA conjugate for 72 hours and the green stain inside the cells resembled cellular internalization of the conjugate. High cellular internalization for the bio-conjugates was demonstrated after 72 hours of treatment.

[0376] Figure 23 shows the fluorescence microscopy images of three cancer cell lines mostly undergoing necrosis. The cancer cells were treated with the bio-conjugate containing four plant extracts (SWCNT-PEG-FA-AM-DC-DV-TV). The cells went through a series of morphologically distinct stages in their pathway to death. In the initial stages, the cells began to swell, the chromatin was digested, and the cell membrane was ruptured. This was followed by vacuolization of the endoplasmic reticulum. In the final stages, the organelles completely broke down and cell lysis occurred resulting in the spewing of intracellular content. All the fluorescence microscopy images for the cancer cell lines resembled this process. These results were in line with the cell viability assay which showed a higher potency for the bio-conjugate with four plant extracts.

[0377] Flow cytometry

[0378] Flow cytometry was used to provide a quantitative and qualitative analysis of the state of cancer cells after treatment. The technique enabled measurement of single-cell properties through visible and fluorescent light in a high-throughput manner.

[0379] The flow cytometry images of the bio-conjugates are shown in Figures 24 to 26. The control groups all showed >98% healthy cells across cell lines. The cells treated with the bio-conjugates over a 72-hour period all showed a significant reduction in healthy cells compared to the control. The percentage of reduction and the type of cell death varied in all bio-conjugates and across cancer cells. In MDA-MB-231 and HT-29 cells, the highest percentage of cells undergoing necrosis was seen in the Tulbaghia violacea bio-conjugate (p<0.0001 ), whereas in the MCF-7 it was the Annona muricata bio-conjugate (p<0.001 ). The bio-conjugate with all the plant extracts combined showed the lowest percentage of healthy cells <5%. Therefore, it can be concluded that there was a synergistic effect from all plant extracts leading to a higher efficacy compound.

[0380] The foregoing description has been presented for the purpose of illustration; it is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Persons skilled in the relevant art can appreciate that many modifications and variations are possible in light of the above disclosure.

[0381] The language used in the specification has been principally selected for readability and instructional purposes, and it may not have been selected to delineate or circumscribe the inventive subject matter. It is therefore intended that the scope of the invention be limited not by this detailed description, but rather by any claims that issue on an application based hereon. Accordingly, the disclosure of the embodiments of the invention is intended to be illustrative, but not limiting, of the scope of the invention set forth in any accompanying claims.

[0382] Finally, throughout the specification and any accompanying claims, unless the context requires otherwise, the word ‘comprise’ or variations such as ‘comprises’ or ‘comprising’ will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Claims

CLAIMS1. A conjugate comprising:- a nanocarrier;- a targeting moiety which binds to a receptor on a cancer cell; and- compounds from at least one plant extract derived from a plant selected from the group consisting of Tulbaghia violacea, Annona muricata, Dicoma capensis and Dodonaea viscosa, wherein the targeting moiety and compounds from the at least one plant extract are conjugated to the nanocarrier.

2. The conjugate as claimed in claim 1 , wherein the nanocarrier is a carbon nanotube, preferably a single walled carbon nanotube (SWCNT).

3. The conjugate as claimed in claim 2, wherein the carbon nanotube is functionalised with polyethylene glycol (PEG).

4. The conjugate as claimed in any one of claims 1 to 3, wherein the targeting moiety is folic acid.

5. The conjugate as claimed in any one of claims 1 to 4, wherein the compounds are from extracts of at least two plants selected from the group consisting of Tulbaghia violacea, Annona muricata, Dicoma capensis and Dodonaea viscosa.

6. The conjugate as claimed in any one of claims 1 to 4, wherein the compounds are from extracts of at least three plants selected from the group consisting of Tulbaghia violacea, Annona muricata, Dicoma capensis and Dodonaea viscosa.

7. The conjugate as claimed in any one of claims 1 to 6, wherein the compounds are from extracts of all four of Tulbaghia violacea, Annona muricata, Dicoma capensis and Dodonaea viscosa.

8. The conjugate as claimed in any one of claims 2 to 7, wherein compounds from the Annona muricata and / or Dicoma capensis extracts are substantially endohedrally conjugated to the carbon nanotube.

9. The conjugate as claimed in any one of claims 2 to 8, wherein compounds from the Tulbaghia violacea and / or Dodonaea viscosa extracts are substantially exohedrally conjugated to the carbon nanotube.

10. A conjugate comprising a single walled carbon nanotube (SWCNT) to which the following are conjugated: compounds from plant extracts of at least two of Tulbaghia violacea, Annona muricata, Dicoma capensis and Dodonaea viscosa; and folic acid.

11. A composition including conjugates as claimed in any one of claims 1 to 10.

12. The composition as claimed in claim 11 , including a pharmaceutically acceptable excipient.

13. A conjugate as claimed in any one of claims 1 to 10 or a composition as claimed in claim 11 or 12, for use in a method of treating cancer.

14. The conjugate or composition for use as claimed in claim 13, wherein the cancer is breast cancer or colorectal cancer.

15. Use of a conjugate as claimed in any one of claims 1 to 10 or a composition as claimed in claim 11 or 12 in the manufacture of a medicament for use in a method of treating cancer.

16. The use as claimed in claim 15, wherein the cancer is breast cancer or colorectal cancer.

17. A method of producing a conjugate as claimed in any one of claims 1 to 10, the method comprising the steps of:- conjugating a targeting moiety which binds to a receptor on a cancer cell to a nanocarrier; and- contacting the nanocarrier with at least one plant extract derived from a plant selected from Tulbaghia violacea, Annona muricata, Dicoma capensis and Dodonaea viscosa and allowing compounds from the at least one plant extract to bind to the nanocarrier.

18. The method as claimed in claim 17, wherein the nanocarrier is a carbon nanotube, preferably a SWCNT.

19. The method as claimed in claim 17 or 18, wherein the targeting moiety is folic acid.

20. The method as claimed in any one of claims 17 to 19, further comprising a step of functionalising the nanocarrier with polyethylene glycol before the nanocarrier is contacted with the plant extract(s).21 . The method as claimed in any one of claims 17 to 20, wherein the conjugate is contacted with extracts of at least two or at least three plants selected from the group consisting ofTulbaghia violacea, Annona muricata, Dicoma capensis and Dodonaea viscosa.

22. The method as claimed in any one of claims 17 to 21 , wherein the conjugate is contacted with extracts of all four of Tulbaghia violacea, Annona muricata, Dicoma capensis andDodonaea viscosa.

23. The method as claimed in any one of claims 17 to 22, wherein compounds from the Annona muricata and / or Dicoma capensis extracts are allowed to conjugate substantially to an inner surface of the carbon nanotube, such as by bringing the carbon nanotube into contact with compounds from the Annona muricata and / or Dicoma capensis extracts before bringing the carbon nanotube into contact with the Tulbaghia violacea and / or Dodonaea viscosa extracts.

24. The method as claimed in any one of claims 17 to 23, wherein compounds from the Tulbaghia violacea and / or Dodonaea viscosa extracts are conjugated substantially to an outer surface of the carbon nanotube, such as by bringing the carbon nanotube into contact with compounds from the Tulbaghia violacea and / or Dodonaea viscosa extracts after having brought the carbon nanotube into contact with the Annona muricata and / or Dicoma capensis extracts.

25. The method as claimed in any one of claims 17 to 24, wherein the plant extracts are brought into contact with the carbon nanotubes in the following order: Annona muricata, Dicoma capensis, Dodonaea viscosa and lastly Tulbaghia violacea.

26. A kit comprising plant extracts of at least one of Tulbaghia violacea, Annona muricata, Dicoma capensis and Dodonaea viscosa and instructions for producing a conjugate as claimed in any one of claims 17 to 25.

27. The kit as claimed in claim 26, including PEG and / or folic acid.

28. A method of treating cancer in a subject, the method including the step of administering a therapeutically effective amount of a conjugate according to any one of claims 1 to 10 or a composition according to claim 1 1 or 12 to a subject in need thereof.

29. The method of claim 28, wherein the cancer is breast cancer or colorectal cancer.

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