WO2010085876A1 - Thylakoids and their functional derivatives, and their uses - Google Patents

Abstract

Thylakoid vesicles stably incorporating an heterogeneous agent. Methods of using thylakoids and their functional derivatives for applications such as photodynamic therapy, medical imaging, and delivery system.

Description

TITLE OF THE INVENTION

THYLAKOIDS AND THEIR FUNCTIONAL DERIVATIVES, AND THEIR USES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a PCT application no PCT/2010/^* filed on January 27, 2010 and published in English under PCT Article 21(2), which itself claims benefit of U.S. provisional application serial No. 61/147,604, filed on January 27, 2009. All documents above are incorporated herein in their entirety by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] N/A.

FIELD OF THE INVENTION

[0003] The present invention relates to thylakoids and their functional derivatives, and their uses. More specifically, the present invention is concerned with therapeutic, medical and environmental uses of thylakoids or of their functional derivatives.

BACKGROUND OF THE INVENTION

[0004] Thylakoids are membrane-bound compartments inside chloroplasts and cyanobacteria. They are the site of light-dependent reactions of photosynthesis.

[0005] They have been used in a number of applications that take advantage of their ability to generate a photosynthetic response to outside factors. For instance, stabilized thylakoids have been used for detecting toxic molecules in effluents, and thylakoids conditioned to express anti-oxidants have been used to scavenge reactive oxygen species.

[0006] There is a need to further exploit the properties of thylakoids in novel applications.

[0007] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

[0008] More specifically, in accordance with the present invention, there is provided thylakoid vesicles stably incorporating an heterogeneous agent. [0009] In a specific embodiment, the heterogeneous agent has a size of about 100 Da to about 400 Da. In another specific embodiment, the heterogeneous agent is a fluorescent agent. In another specific embodiment, the thylakoid vesicles further comprise a ligand for targeting tumor cells.

[0010] In accordance with another aspect of the present invention, there is provided a method of using the thylakoid vesicles of the present invention for medical imaging in a subject, comprising administering the thylakoid vesicles to a tissue of the subject, applying light on the tissue and visualizing fluorescence in the tissue.

[0011] In accordance with yet another aspect of the present invention, there is provided a method of sensitizing tumor cells to photodynamic therapy (PDT), comprising contacting the tumor cells with the thylakoid vesicles of the present invention, and applying light to the tumor cells, whereby tumor cells are sensitized to PDT.

[0012] In accordance with another aspect of the present invention, there is provided a method of sensitizing tumor cells to photodynamic therapy (PDT), comprising contacting the tumor cells with thylakoids or thylakoid functional derivatives and applying light to the tumor cells, whereby tumor cells are sensitized to PDT.

[0013] In a specific embodiment of the methods, the light comprises a wavelength of at least about 680 nm. In another specific embodiment, the tumor cells are melanoma cells, colon tumor cells or breast cancer cells. In another specific embodiment, the light is applied for between about 1 and 2 hours V-.

[0014] In accordance with another aspect of the present invention, there is provided a method of detecting a filtration membrane defect, comprising introducing the thylakoid vesicles of the present invention upstream of the filtration membrane and detecting the presence of fluorescence downstream of the filtration membrane, wherein the presence of fluorescence downstream of the filtration membrane is an indication that there is a defect in the filtration membrane .

[0015] In another specific embodiment, the heterogeneous agent is a cosmetic or a therapeutic agent.

[0016] In accordance with another aspect of the present invention, there is provided a pharmaceutical composition comprising the thylakoid vesicles of the present invention, and a pharmaceutically acceptable carrier.

[0017] In accordance with another aspect of the present invention, there is provided a kit comprising the thylakoid vesicles of the present invention. In accordance with another aspect of the present invention, there is provided a kit comprising the pharmaceutical composition of the present invention. [0018] In accordance with another aspect of the present invention, there are provided thylakoids or thylakoid functional derivatives for use in photodynamic therapy (PDT). In accordance with another aspect of the present invention, there is provided a use of thylakoids or thylakoid functional derivatives for photodynamic therapy (PDT).

[0019] Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In the appended drawings:

[0021] Figure 1 shows the principal components of thylakoids. PSII (photosystem II) and its antenna pigments, Cyt. bβ/f (cytochrome complex bβ/f). PSI (photosystem I) and its antenna pigments, NADP reductase and the ATP synthetase complex;

[0022] Figure 2 shows the emission spectrum for the fluorescence of the control thylakoid vesicles (without riboflavin) , excitation at 475 nm (dilution in water 1/1000) with a fluorimeter Varian Cary Eclipse™, an excitation slot of 10, an emission slot of 10 and a voltage of 800 applied to the photomultiplicator at 800;

[0023] Figure 3 shows the emission spectrum of riboflavin alone excited at 450 nm (dilution in water 1/1000) with a fluorimeter Varian Cary Eclipse™, an excitation slot of 10, an emission slot of 10 and a voltage of 800 applied to the photomultiplicator at 800;

[0024] Figure 4 shows the emission spectrum of the fluorescence of thylakoid vesicles with riboflavin, excited at 475 nm (dilution in water 1/1000) with a fluorimeter Varian Cary Eclipse™, an excitation slot at 10, an emission slot at 10 and a voltage of 800 applied to the photomultiplicator at 800;

[0025] Figure 5 shows the emission spectrum of rhodamine 6G alone excited at 480 nm (dilution in water 1/1000) with a fluorimeter Varian Cary Eclipse™, an excitation slot at 10, an emission slot at 10 and a voltage of 800 applied to the photomultiplicator at 800;

[0026] Figure 6 shows the emission spectrum of the fluorescence of thylakoid vesicles with rhodamine 6G, excited at 475 nm (dilution in water 1/1000) from a fluorimeter Varian Cary Eclipse™, an excitation slot at 10, an emission slot at 10 and a voltage of 800 applied to the photomultiplicator at 800;

[0027] Figure 7 shows the emission spectrum of Figure 6 enlarged so as to see chlorophyll bands at 680 nm; [0028] Figure 8 shows the survival rate obtained with the cell line HT-29 (human colon) after treatment under light for various duration with the thylakoids (A), under light with various chlorophyll concentrations (B) and in the dark (C);

[0029] Figure 9 shows the survival rate obtained with the cell line M21 (human melanoma) after treatment under light with the thylakoids (A), under light with various chlorophyll concentrations (B) and in the dark (C); and

[0030] Figure 10 shows the survival rate obtained with the cell line MCF-7 (human breast) after treatment under light with the thylakoids (A), under light with various chlorophyll concentrations (B) and in the dark (C).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0031] According to one aspect, the present invention takes advantage of the discovery that thylakoids can stably incorporate molecules. In another aspect the present invention takes advantage of the discovery that thylakoids and their functional derivatives can generate reactive oxygen species in an amount sufficient to display cytotoxicity to surrounding cells.

[0032] Thylakoids are made of protein complexes comprising antenna pigments, photosystems I and II, reaction centers of the photosystems I and Il (PSI and PSII), cytochrome bβ/f complex and the ATP synthetase complex (Murphy, 1986) (Figure 1). The specific organization of these thylakoids favors optimal activity of PSI and PSII, where photochemical reactions take place. The thylakoid membrane preparations of the present invention retain photosystems properties and form vesicles that can incorporate substances of interest. Riboflavin and rhodamine, both molecules of about 300 kDa, were shown to be stably incorporated. Many pharmaceutical agents have similar molecular weights.

[0033] The terminology "thylakoid vesicles" refers herein to thylakoid membranes having a closed inner space. Advantageously, this inner space can incorporate hydrosoluble molecules. The membrane itself can also incorporate lipophilic molecules. These vesicles have an average size varying between 0.45 μm and 1.2 μm. Typical methods of preparing thylakoids will generate thylakoids vesicles.

[0034] The terminology "thylakoids" without further specification refers herein to thylakoid membranes which may be in the form of vesicles or not. It thus encompasses thylakoid vesicles.

Stable incorporation of molecules

[0035] Thylakoid vesicles may for instance incorporate fluorescent agents and take advantage of this fluorescence in a number of applications. They may also incorporate therapeutic agents and act as delivery systems for these agents.

[0036] The present invention encompasses various methods of using the inherent or enriched fluorescence of thylakoids or of their functional derivatives. The fluorescence of thylakoids can be enriched by inserting in thylakoid vesicles one or more agents exhibiting fluorescence. It can be advantageous to enrich the fluorescence of thylakoids since their inherent fluorescence generated by chlorophyll fades over time and/or is not very intense. These characteristics may make the detection of thylakoids' inherent fluorescence more difficult in certain applications (e.g., detection of filtration membrane defect). For many applications of the present invention, stable fluorescence is thus advantageous. Without being so limited, fluorescent agents appropriate for methods of the present invention include fluorescent dyes such as riboflavin, rhodamine, Texas Red, Fluorescein, etc. For certain applications, it may be useful to incorporate two or more fluorescent agents in thylakoids to detect or monitor at least two physiological events or characteristics.

[0037] Methods taking advantage of the inherent or enriched fluorescence of thylakoids include, without being so limited, medical imaging, determination of membrane integrity in medical system, cell recognition in cytology.

[0038] Usual techniques for measuring lluorescence can be used in the methods in accordance with the present invention. However, because certain non photochemical parameters cannot be obtained in isolated thylakoids, pulsed light may not be necessary. Furthermore, pulsed light fluorometers are relatively complex, costly and bulky apparatuses. Fluorometers using non-modulated light do not have these drawbacks and are nevertheless able to adequately measure the photosynthetic efficiency of thylakoids. Spectrofluorimeter can also be used such as those by Varian, Pekin Elmer, SPEX, Shimadsu, etc.

Medical imaging

[0039] Due to their inherent fluorescence properties, and to their capacity to incorporate various fluorescent agents, thylakoids and their functional derivatives can be used as tracer agents for medical imaging. Medical imaging refers to techniques and processes used to create images of the human body (or parts thereof) for clinical purposes (medical procedures seeking to reveal, diagnose or examine disease) or medical science (including the study of normal anatomy and physiology). Thylakoids and their functional derivatives can be used for instance as in vivo tracer or in organs during a biopsy. In accordance with an aspect of the present invention, fluorescent agents can be incorporated into thylakoids which can then be administered to the targeted tissue (e.g., by direct contact) and produce an image of the targeted tissue. In specific applications such as photodynamic therapy (PDT), the ability of thylakoids and their functional derivatives to be used in medical imaging can be combined to their ability to produce reactive oxygen species and, in more specific embodiments, be targeted to tumoral tissues via specific ligands such as antibodies (see below).

Determination of filtration membrane integrity

[0040] The terminology "filtration system" refers herein to a filtration apparatus comprising one or more membranes. Membrane filtration systems in many configurations are increasingly being used to treat drinking water. In these systems water is passed through a thin membrane barrier in the form of hollow fiber or spiral-wound composite sheets. Organic and other contaminants are filtered and retained on the high- pressure side and frequently must be removed by reversing the flow and flushing the waste.

[0041] The terminology "filtration system defect" refers herein to the inability of the membrane(s) to block particles that it is/they are intended to block. Without being so limited, such particles include microorganisms and any particle larger than about 0.02 μm, and in more specific embodiments, particles larger than 0.45 μm. Without being so limited, this inability can be due to holes or tears in the membrane, an increased porosity of the system, and cracks in the joints connecting the membrane to the liquid enclosure (e.g., water reservoir or pipe).

[0042] The terminology "water sample" refers herein to any fluid containing microorganisms.

Without being so limited, it includes surface water, ground water, storm water, underground water, drinking water, agricultural effluents, industrial effluents (pulp and paper, municipal waste water, waste water landfills leachates, textile, petrochemical, chemical, mining), water extracted from food, sludge, sediments, scories, etc.

Delivery system

[0043] In accordance with an aspect of the present invention, thylakoids and their functional derivatives can be used as delivery system when they incorporate cosmetic agents, or therapeutic agents for instance. In a specific embodiment, vesicles of the present invention can incorporate molecules of about 100 to 400 Da, and more specifically of about 100 to 300 Da. Without being so limited, such cosmetic agents include vitamins and Botox™ for instance. Vesicles of thylakoids and of their functional derivatives are advantageously of low preparation costs, are stable at least 7 to 10 days at 4 degrees and could generate fewer side effects such as allergies due to their natural origin. Without being so limited, therapeutic agents that could advantageously be delivered by vesicles of thylakoids or of their functional derivatives include antitumoral agents and anti-aπgiogenic agents. They can advantageously be coupled to ligands targeting the tissues to treat using well-known techniques such as bio-conjugates on amines moieties of proteins at the surface of thylakoids. For instance, they may be coupled to antibodies specific to tumor antigens (e.g., PCA3 targeting prostate cancer, CA19-9 targeting gastrointestinal cancer, Ca125 targeting ovarian cancer, etc.).

Generation of reactive oxygen species

[0044] With an increasingly aging population, cancer is still one of the most common causes of death. Standard treatment includes surgery, chemotherapy and radiation therapy, with variable success rates. In search for new therapeutic options, photodynamic therapy (PDT) was found to be a promising alternative, given its mechanism of action and known low toxicity levels. PDT is used for the local treatment of benign and malignant conditions based on the administration of a photosensitizer followed by application of light to the area to be treated.

[0045] In accordance with yet another aspect, the present invention takes advantage of the ability of thylakoids and of their functional derivatives to generate reactive oxygen species (ROS) by activation of photosystems following light absorption. In a specific embodiment of the present invention, thylakoids and their functional derivatives (vesicles or not) are used in photodynamic therapy as photosensitizers. Although thylakoids and their functional derivatives can be used alone, they may also advantageously incorporate agents that can be one or more of other photosensitizers (e.g., porphyrin (water insoluble), rhodamine, chlorophyll (water insoluble), antitumoral or anti-angiogenic agents and fluorescent agents). The thylakoids for use as photosensitizers can be administered by direct contact (esophagus, stomach, uterus, bladder, eyes, mouth (e.g., for treatment of cancer or chronic bad breath)) for instance.

[0046] The terminology "thylakoid functional derivative" in its plural or singular form refers herein to thylakoid fragments that can generate reactive oxygen species through various mechanisms including charge separation (e.g., the reaction center of the P680 of PSII separates the electron donor and electron acceptor) but do not contain all components of a thylakoid per se. Minimally, these fragments contain the reaction center of the PSI or of the PSII which can induce charge separation contrarily to isolated chlorophyll perse. The reaction centers of PSI and PSII are the major generation site of ROS in thylakoids. In PSI, the primary reduced ROS is superoxide anion (Or), and its disproportionation produces H2O2 and O2. In PSII, oxygen of the ground triplet state (³θ2) is excited to singlet state (¹θ2) via charge recombination of the light-induced charge pair of the reaction center.

[0047] Thylakoids and their functional derivatives can be subjected to different treatments (e.g., treatment with urea and/or NaCI) that promote the generation ROS by affecting either the acceptor side or the donor side of the PSII (or of the PSI after activation of its ROS production with methyl viologen for example). As used herein thylakoids or their functional derivatives so treated are also encompassed within the meaning of thylakoid functional derivatives. Stabilized thylakoids as defined herein are also encompassed by the name functional derivatives.

[0048] Larger fragments of thylakoids may include for instance the complete PSI and/or the PSI, and other thylakoid components. Thylakoid functional derivatives can be in the form of vesicles. Without being so limited, "thylakoid functional derivatives" can be prepared by methods described below.

[0049] Any known methods of preparing thylakoids can be used to prepare the thylakoids or functional derivatives of the present invention. In addition to the method used in Examples below, thylakoids lyophilisation can also be used (Zimmermanπ etal., 1996). Advantageously, thylakoids used for the present invention can be isolated from any natural source of thylakoids. Hence, for instance any dark green leaf that is desirably not too fibrous or waxy can be used as a thylakoid source for the present invention (e.g., spinach, barley leaves, pea leaves, oregano, coriander, grapefruits leaves and orange tree leaves). Furthermore, thylakoids of the present invention and their functional derivatives do not need preconditioning of the plant prior to extraction. For instance, it is contrary to purposes of the present invention to promote the production of antioxidants (e.g., carotenoids) in plants prior to extraction of thylakoids. On the contrary, for their application in PDT, thylakoids must generate as much ROS as possible.

[0050] Thylakoid membranes spontaneously form microstructures (vesicles) when chloroplasts are lysed. These microstructures originate from the spontaneous reorganization of the components of the system (i.e., stacked membrane network, the grana, non stacked network and lamellar structures). Because these microstructures retain photosystem photochemical properties including photolysis and electron transport, they are likely made of the proteolipidic complexes of thylakoids (Boardman ef a/., 1964; Berthold ef a/., 1981).

[0051] As used herein the term "heterogeneous agent" refers to a molecule that is not naturally found in thylakoid vesicles and that is desirably incorporated into thylakoid vesicles in accordance with methods of the present invention. Without being so limited, such molecule may be a small molecule, a peptide or a protein. The present invention encompasses the use of one or more heterogeneous agents in thylakoids and/or functional derivatives of the present invention.

[0052] As used herein the terms "stably incorporated" when used in the context of heterogeneous agents stably incorporated into thylakoids vesicles of the present invention refers to agents that stay within the thylakoid vesicles for more than 10 days when refrigerated at 4 ° C and at least 10 days at room temperature.

[0053] As used herein the term "subject" is meant to refer to any animal, such as a mammal including human, mice, rat, dog, cat, pig, cow, monkey, horse, etc. In a particular embodiment, it refers to a human.

[0054] A "subject in need thereof or a "patient" in the context of the present invention is intended to include any subject that will benefit or that is likely to benefit from PDT or more generally from the thylakoids of the invention stably incorporating heterogeneous agents. In an embodiment, a subject in need thereof is a subject diagnosed with a cancer.

[0055] The invention also provides a pharmaceutical composition (medicament) comprising at least the thylakoid vesicles of the invention {e.g., incorporating or not at least one heterogeneous agent), and a pharmaceutically acceptable diluent, carrier, salt or adjuvant. Such carriers include, for example, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical composition may be adapted for the desired route of administration (e.g., oral, parental, intravenous, intramuscular, intraperitoneal, aerosol).

[0056] The invention also provides pharmaceutical compositions which comprise one or more agent(s).

[0057] The present invention also provides a kit or package comprising the above-mentioned thylakoids and/or their functional derivatives or pharmaceutical compositions. Such kit may further comprise, for example, instructions for the use of thylakoids and/or their functional derivatives in the treatment of cancer or in the treatment of aging skin conditions such as wrinkles and fine lines, in the detection of filtration system defects, containers, devices for administering the agent/composition, etc.

Dosage

[0058] The amount of the agent or cosmetic or pharmaceutical composition which is effective in the prevention and/or treatment of a particular disease {e.g., cancer), disorder or condition (e.g., skin condition such as wrinkle, fine lines) will depend on the nature and severity of the disease, the chosen prophylactic/therapeutic regimen, the target site of action, the patient's weight, special diets being followed by the patient, concurrent medications being used, the administration route and other factors that will be recognized by those skilled in the art. The dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient. Typically, 0.001 to 1000 mg/kg of body weight/day will be administered to the subject. In other embodiments, a daily dose range of about 0.01 mg/kg to about 500 mg/kg, of about 0.1 mg/kg to about 200 mg/kg, of about 1 mg/kg to about 100 mg/kg, or of about 10 mg/kg to about 50 mg/kg, may be used. The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial prophylactic and/or therapeutic response in the patient over time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration. Effective doses may be extrapolated from dose response curves derived from in vitro or animal model test systems. For example, in order to obtain an effective mg/kg dose for humans based on data generated from rat studies, the effective mg/kg dosage in rat may be divided by six.

[0059] The terms "treat/treating/treatment" and "prevent/preventing/prevention" as used herein, refer to eliciting the desired biological response, i.e., a therapeutic and prophylactic effect, respectively. In accordance with the subject invention, the therapeutic effect comprises one or more of a decrease/reduction in tumor, a decrease/reduction in the severity of the cancer (e.g., a reduction or inhibition of metastasis development), a decrease/reduction in symptoms and cancer-related effects, an amelioration of symptoms and cancer-related effects, and an increased survival time of the affected host animal, following administration of the agent/composition of the invention. In accordance with the invention, a prophylactic effect may comprise a complete or partial avoidance/inhibition or a delay of cancer development/progression (including a complete or partial avoidance/inhibition or a delay of metastasis development), and an increased survival time of the affected host animal, following administration of the thylakoid vesicles (or of a composition comprising the thylakoid vesicles).

[0060] As such, a "therapeutically effective" or "prophylactically effective" amount of an agent capable of reducing the tumor, or a combination of such agents, may be administered to an animal, in the context of the methods of treatment and prevention, respectively, described herein.

[0061] As used herein, the term "a" or "the" means "at least one".

[0062] Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims

[0063] The present invention is illustrated in further details by the following non-limiting examples.

EXAMPLE 1

Preparation of thylakoids [0064] I: Homogenisation : All steps were conducted in the dark or under green light and at cold temperature (samples on ice or procedure in cold room). A hundred gram of deveined spinach leaves were crushed in a mixer with 350 mL of homogenising buffer comprising TES-NaOH 2OmM pH 7.5, sorbitol 33OmM and MgCL.2 5mM. Other conventional buffers could be used including TES, Hepes, Tris, phosphate, tricine and MOPs. The homogenate was filtered on a cheese cloth and the filtrate was centrifuged 2 min at 2500X g at 4°C on a Eppendorf™5810-R, rotor # A-4-44.

[0065] II: Lysing chloroplasts: The pellet was then resuspended in 200 mL of hypotonic solution consisting of the homogenizing buffer diluted in water 1: 20 v/v (i.e. TES-NaOH 2OmM pH 7.5, sorbitol 33OmM and MgCL₂ 5mM diluted 1:20 v/v with water). This step was used to lyse the chloroplast membranes. The resulting solution was then crushed in a Wheaton™ mixer and centrifuged 3 min. at 3500Xg at 4⁰C on a Eppendorf™5810-R, rotor* A-4-44.

[0066] III: Resuspension: The resulting pellet contained the purified thylakoids. They were then resuspended in a buffer consisting of TES-NaOH 20 mM pH 7.5, sorbitol 330 mM, MgCL₂ 5 mM and NH₄CI 1mM so as to obtain a final concentration of chlorophyll between 2 and 3 mg/mL. The chlorophyll concentration was assessed according to the method taught in Porra (1989) described herein. The solution was then either stored at -80⁰C for later use or was stabilized as described in Example 2 below for storing at room temperature. The thylakoids obtained at the end of this process were in the form of vesicles.

EXAMPLE 2 Preparation of stabilized thylakoids

[0067] Thylakoids of the present invention may be stabilized using the method described in co- pending application US 2006-0147342 as described below or by other stabilization methods (e.g., lyophilisation). A preparation of 2 to 3 mg/mL of thylakoids were mixed with a pH 6.5 phosphate buffer containing: 0.02 M phosphate, sucrose 300 mM, NH₄CI 10 mM, MgCL₂ 20 mM, EDTA 10 mM, polyvinylpyrrolidine (PVP) (125 μL of solution 20% for 1 mL) and liposomes made of phosphatidylcholine and phosphatidylglycerol in a ratio of 10 mg mL¹:1 mg mL¹ to obtain a solution of 0.0125 mg of liposome/mL of chlorophyll/thylakoid membranes buffered solution. In this stabilized thylakoid membrane formulation, the final concentrations of PVP is of 2% v/v, the final concentration of liposomes is of 0.0125 mg/mL and that of chlorophyll is of 0.125mg/mL

[0068] The PVP increased the stability of the thylakoid preparations as compared to the same preparation without PVP but a useful stability was nevertheless obtained even without PVP. The amount of PVP may be varied without affecting the stability of the thylakoid membranes between about 0 and about 4% PVP v/v (data not shown). Other conventional buffers at pH ranging between about 6.2 and about 7.8 - a variation of about 10% is generated within this pH range (data not shown) - can also be used without affecting the usefulness of the thylakoid preparation of the present invention.

[0069] The quantity of liposomes used was determined via the ratio chlorophyll/liposome. This ratio may vary and is preferably at least equal to 10 mg mL¹:1 mg mL¹ and better results with regards to stability and sensitivity were obtained with a ratio 100 mg ml_-¹:1 mg mL¹. Any ratio higher than 100:1 is expected to work. The liposomes are believed to help to the formation of thylakoid membranes vesicles thereby increasing their stability and increasing the consistency of the readings. The quantity used can be varied as long as the final ratio of chlorophyll/liposomes is at least about 10 mg mL-¹:1 mg mL¹.

[0070] A hundred μL of this solution was poured into a 5 mL or 7 mL tube previously refrigerated.

The tubes were then frozen and then lyophilised with a speed-vac™ from Savant (#SS-22). Lyophilisation was conducted 4 hours in order to remove water completely from the 100 μL amount. The tubes were then closed and kept at the desired temperature. Table I presents the stability of various thylakoid preparations obtained as a function of the temperature of storage.

Table I Stability of various thylakoid vesicles functional derivatives

T° Conservation time

Natives PVP PVP (under vacuum) PVP (under vacuum + liposomes)

- 80

> 5 month > 5 month > 5 month N.D.

⁰C

- 20

> 3 month > 3 month > 3 month N.D. 0C

4⁰C 18 hours 12 days > 1 month N.D.

2O ⁰C 4 hours 7 days 11 days 12 days

37⁰C N.D. 2 days 4 days 4 days

N.D. = not determined

EXAMPLE 3

Preparation of a concentrated stabilized thylakoid formulation

[0071] A concentrated batch of 50 tests was prepared in a total volume of 20 mL of solution constituted of thylakoids at a chlorophyll/thylakoid concentration of 0,625 mg/mL, Tes-NaOH 25 mM pH 7.5, HEPES-NaOH 25 mM pH 7.5, sorbitol 330 mM, MgCI₂ 2 mM, NH₄C1 1 mM, PVP 2 % (p/v) and liposomes (phosphatidylcholine : phosphatidyglycerol 10 mg mL"¹:1 mg mL-¹). One mL of this solution was poured in an amber bottle. This preparation can obviously be adapted for the preparation of a smaller or larger number of tests. In this concentrated stabilized thylakoids formulation, the final concentrations of PVP was of 2% v/v, the final concentration of liposomes was of 0.0625 mg/mL v/v and that of chlorophyll was of 0.625mg/mL v/v. The quantity used can be varied as long as the final ratio of chlorophyll/liposomes is of at least about 10 mg mL-¹:1 rηg ml/¹. This concentrated formulation was used in Examples 5-10.

EXAMPLE 4

Resuspension of stabilized thylakoids [0072] The individual test tubes (100 μL stabilized thylakoid membranes) prepared in Example 2 were resuspended in 2 mL of water or tested samples. The concentrated batch of test tubes was resuspended in water, in Buffer 1 ( HEPES-NaOH 50 mM pH 7,5 buffer containing sucrose 330 mM, MgCb 20 mM, NH₄CI 10 mM, PEG 4000 4% (p/v)); or in Buffer 2 (phosphate buffer 0,1 M pH 6,5 containing sucrose 330 mM , NH₄CI 10 mM, MgCI₂ 20 mM, EDTA 10 mM, PEG 4000 4% (p/v)). A combination of buffered solutions 1 and 2 may also be used.

EXAMPLE S Integration of riboflavin into thylakoid vesicles

[0073] Step I of the preparation of thylakoids was as described in Example 1.

[0074] Step Il was identical to that in Example 1 except that the hypotonic solution was prepared in a solution saturated in riboflavin (i.e., about 1 mM).

[0075] Step III: The resulting pellet contained the purified thylakoid vesicles with incorporated riboflavin. They were then resuspended in 35 mL of buffer consisting of TES-NaOH 20 mM pH 7.5, sorbitol 330 mM, MgCL₂ 5 mM and NH₄C1 1 mM, crushed in a Wheaton™ mixer and centrifuged 3 minutes at 3500 X g to remove the non-incorporated riboflavin, The last centrifugation step was repeated 2-3 times to wash the vesicles. The final pellet was then resuspended in the same buffer to obtain a final concentration of chlorophyll between 2 and 3 mg/ml. The chlorophyll concentration was assessed according to the method described in Porra (1989) described herein. The solution was then either stored at -80⁰C for later use or stabilised for storing at room temperature.

[0076] In Figure 2 is presented the emission spectrum of the control thylakoid vesicle (i.e., without riboflavin) diluted 1:1000 v/v in water at an excitation of 475 nm. Emission bands of the thylakoid light harvesting complex are at 517 nm and chlorophyll peaks of PSII and PSI reaction centers are at 680 and 709 nm, respectively. The emission spectrum of riboflavin in solution is presented at Figure 3.

[0077] The maximal emission peak of riboflavin is observed at about 520 nm. In Figure 4 is presented the fluorescence spectrum of riboflavin incorporated in thylakoid vesicles. A peak is observed at 520 nm which corresponds to that of riboflavin. This peak masks the 517 nm thylakoid light harvesting complex peak. However the peaks at 680 and 709 nm are attributed mostly to thylakoid chlorophyll organization in PSII and PSI reaction center, respectively, as shown in the control Figure 2.

EXAMPLE 6

Integration of riboflavin into thylakoid vesicles with alternative method

[0078] Step I was identical to that in Example 1 except that the homogenizing buffer was prepared in a solution saturated in riboflavin 1 mM as in Example 1, the homogenate was then filtered on a cheese cloth and the filtrate was centrifuged 2 min at 2500X g at 4⁰C on a Eppendorf™5810-R, rotor # A-4-44.

[0079] Step Il was identical to that in Example 1 except that the hypotonic solution was prepared in a solution saturated in riboflavin 1 mM.

[0080] Step III: The pellet resulting from Step Il contained the purified thylakoid vesicles with incorporated riboflavin. They were then resuspended in 35 mL of buffer consisting of TES-NaOH 20 mM pH 7.5, sorbitol 330 mM, MgCL.25 mM and NH4C1 1 mM, crushed in a Wheaton™ mixer and centrifuged 3 minutes at 3500 X g to remove the non-incorporated riboflavin. The last centrifugation step was repeated 2-3 times to wash the vesicles. The final pellet was then resuspended in the same buffer to obtain a final concentration of chlorophyll between 2 and 3 mg/ml. The chlorophyll concentration was assessed according to the method described in Porra (1989). The solution was then either stored at -8O⁰C for later use or stabilised for storing at room temperature. A control was prepared with a hypotonic solution in a riboflavin saturated solution.

[0081] The fluorescence (at 515 nm and 680 nm) of the thylakoids of this Example, that of Example 5 and that of the control were compared to determine the integration efficacy of the two methods of preparation. Results are presented in Table II.

Table II: Comparison of integration efficacy of riboflavin in thylakoid preparations

[0082] A higher ratio of riboflavin fluorescence / chlorophyll fluorescence denotes a higher riboflavin integration.

EXAMPLE 7

Integration of rhodamine into thylakoid vesicles [0083] Step I of the preparation of thylakoids was as described in Example 1.

[0084] Step Il was identical to that in Example 1 except that the hypotonic solution was prepared in a solution saturated in rhodamine (i.e., about 5 rnM).

[0085] Step III: The resulting pellet contained the purified thylakoid vesicles with incorporated rhodamine. They were then resuspended in 35 mL buffered solution made of TES-NaOH 20 mM pH 7.5, sorbitol 330 mM, MgCL.2 5 mM and NH4CI ImM₁ crushed in a Wheaton™ mixer and centrifuged 3 min. at 3500 X g to remove the non-incorporated rhodamine. The last centrifugation step was repeated 2-3 times to wash the thylakoid vesicles, The final pellet was resuspended in the same buffer to obtain a final concentration of chlorophyll between 2 and 3 mg/ml. The chlorophyll concentration was assessed according to the method described in Porra (1989) described herein. The solution was then either stored at -8O⁰C for later use or stabilised for storing at room temperature.

[0086] As indicated above, in Figure 2 is presented the emission spectrum of the control thylakoid vesicle (i.e., without rhodamine) diluted 1:1000 v/v in water at an excitation of 475 NM. Emission bands of the thylakoid light harvesting complex are at 517 rim and chlorophyll peaks of reaction centers are at 680 and 709 nm. The emission spectrum of rhodamine in solution is presented at Figure 5 . The maximal emission peak of rhodamine is observed at about 550 nm. In Figure 6 is presented the fluorescence spectrum of rhodamine incorporated in thylakoid vesicles. A peak is observed at 550 nm that corresponds to that of rhodamine. This peak masked the 517 nm thylakoid peak. The peaks at 680 and 709 nm are attributed mostly to thylakoid chlorophyll organization in PSII and PSI, respectively, as shown in control Figure 7 (magnification of Figure 6).

EXAMPLE 8

Treatment of thylakoids to increase their ability to produce ROS

[0087] Chlorophyll as the main light-absorbing pigment in the light harvesting complex, the inner antenna, and also in the reaction centres, is very efficient in absorbing light and has the additional advantage that the excited states are long-lived enough (up to a few nanoseconds) to allow the conversion of the excitation energy into an electrochemical potential via charge separation. If the energy is not efficiently used, the spins of the electrons in the excited state can rephase and give rise to a lower energy excited state: the chlorophyll triplet state which has an even longer lifetime and can react with ³Ck to produce the very reactive ¹O₂ if no efficient quenchers are around. ¹O₂ can react with proteins, pigments, and lipids and is thought to be the most important species responsible for light-induced loss of PSII activity, the degradation of the D1 protein (protein of the reaction centre of PSII) and for pigment bleaching. When more light is absorbed than is used in photosynthesis, the reaction centers are subjected to photo-oxidative stress and photo inhibition takes place. Two distinct mechanisms will induce D1 degradation during photoinhibition of the electron transport in PSII: acceptor-side and donor-side of PSII. The reaction center of the P680 of PSII separates the electron donor and electron acceptor sides.

[0088] The acceptor side photoinhibition is related to the formation of ³P6βo by charge recombination in PSII, followed by ¹O₂ production. In isolated PSII reaction centres which lack QA and a functional donor side, the primary charge pair

recombines and a high yield of Pεso triplet is formed.

[0089] To produce PSII reaction centers without QA, PSII membranes have to be first isolated from thylakoids. For example this can be performed by incubating thylakoids 2 mg Chl/rnL with Triton X-100 25 mg/mg ChI or dodecyl maltoside 1.2 % (v/v). The incubation time, buffer used and the separation mode of PSII from the remaining thylakoid membranes depends on the preparation methods used (van Roon, 1993). Centrifugation or gel filtration or density gradient centrifugation are the most used method to isolate PSII. The subsequent steps of purification of PSII complexes (preparation of PSII core complexes and PSII reaction centres) will produce the PSII reaction centres without QA.

[0090] The donor side photoinhibition occurs in PSII with a non-functional or absent water-splitting complex. In this state, photoinhibition is caused by the accumulation of highly oxidizing species like Tyr_z ⁺/Pe8o⁺ generating different (ROS). The water splitting complex can be perturbed by an incubation of 30 minutes with 1 M NaCI or 2-2.5M urea, followed by a centrifugation to remove NaCI or urea.

[0091] When light is applied on thylakoids so treated, ROS production is increased.

EXAMPLE 9

Use of thylakoid vesicles with integrated fluorescence agent to determine integrity of water purification filtration membranes

[0092] Filtration membranes which physically extract micro-pollutants are increasingly used to purify water reserves and satisfy stricter public health requirements. To the Applicant's knowledge, there is no simple, fast and sensitive method able to determine a filtration system's integrity. Anomalies such as tears or holes, disruptions at the joints, could allow bacteria to flow through the filter into the drinking water reserves and cause public health hazards.

[0093] Membrane integrity is tested in accordance with a method of the present invention by filtrating fluorescent particles of a size corresponding to that of bacteria and measuring fluorescence on both sides (e.g., upstream and downstream of the membrane) knowing that with an intact membrane, no fluorescence is measured in the filtrate. It is desirable to choose a fluorescence that is stable for at least 2 hours so that the assay can safely be performed within about one hour.

[0094] The Applicants have determined that filtrations membranes having pores of 0.45 and 1.2 μm block about 92% and 58%, respectively of the thylakoid vesicles while a filter membrane having a pore size of about 0.22 μm stops about 99% of the thylakoid vesicles. It is thus assumed that thylakoid vesicles in accordance with a specific embodiment of the present invention have an average size of about 0.45 μm and 1.2 μm. This mean size corresponds to that of particles (e.g., microorganisms) that filtration membranes are meant to separate.

[0095] Assays were performed on two different filtration membranes: a nanofiltration membrane of 0.001 μm (NF270), and an ultrafiltration membrane of membrane de 0.01 μm (UE50). Each assay was performed as follows :

[0096] 1) Permeability to demineralized water (characterization of membrane per se) was determined by measuring the flow of the membrane permeate at three different pressures: 100, 150 et 200 psi, but not limited to these pressures.

[0097] 2) Filtration of thylakoid vesicles was performed in a closed circuit for 2 hours (i.e., concentrate and permeate are recirculated in the feed tank). Permeate flux measures were performed at filtration times of 5, 30, 60, 90 and 120 minutes. At each specified time, samples of feed and permeate were obtained for fluorescent measurement.

[0098] 3) The membrane was rinsed with demineralized water.

[0099] 4) The permeability of the clogged membrane to demineralized water was then performed (characterization of clogged membranes) to determine whether vesicles accumulate on the membrane or whether they clog pores.

[00100] The thylakoid vesicles was pumped into the tank that feeds the filtration reactor containing the membrane. The pump flow can be adjusted with a variable speed motor. A pressure gauge allows reading of the pressure at the input of the filtration and at the output of the filtration cell. The operating pressure was adjusted with a valve. Under pressure, the water is separated into a concentrate and a permeate. In a closed circuit operation, concentrate and a permeate are returned into the feed tank.

[00101] The assays were performed in the following conditions:

[00102] Chlorophyll concentration of the thylakoid vesicles: 6 mg/L, namely a concentration ensuring a detection threshold in fluorescence of 3 log so as to allow the detection of a concentration of 0.006 mg/L of the thylakoid vesicles.

[00103] Flow of 1 liter of thylakoid vesicles in a closed circuit (i.e., permeate and concentrate were returned to the feed tank, 2 mL of the solution in the feed tank and in the permeate are obtained to measure fluorescence).

[00104] The cross flow filtration was of 0.3 m/s for both tested membranes; the permeation flow was of: 94 UhIm² for the NF270 membrane and of 110 UhIm² for the UE50 membrane; Room temperature (i.e. about 15 to 25 ⁰C); Fluorescence measurements were performed with a Luminos (Lab_Bell inc) fluorescence Analyzer™ (Excitation LED 475 nm; reading time, 0,8 sec; Excitation LED current 10 mA).

[00105] Results are presented in Table III below.

Table III: Determination of filtration membranes integrity using a nanofiltration (NF270, 0.001 μm) and an ultrafiltration (UE50, 0.01 μm) membrane by a thylakoid vesicles of the invention

¹ The initial value (time zero) of the solution of thylakoid vesicles was performed for the blank fluorescence measurement; AU: arbitrary unit.

[00106] Table III shows that no significant fluorescence was measured in permeate samples for either membrane. Had thylakoid vesicles crossed the membrane, the fluorescence would have increased in the permeate. These results show that the thylakoid vesicles do not cross the tested membranes or the sealants between the permeate and feed tank compartments. Also, it confirms that the thylakoid fluorescence remains stable even after two hours. The decreased fluorescence observed with assays using the ultracentrifugation is due to a non specific adsorption of the thylakoid vesicles. It can be avoided by increasing tangential speed and decreasing pressure during assays which assisted in maintaining the thylakoid vesicles in solution. No adsorption or membrane clogging was observed.

EXAMPLE 10 Use of thylakoid vesicles as photosensitizers

[00107] It is demonstrated herein that thylakoids used in accordance with the methods of the present invention can produce reactive oxygen species (ROS) when illuminated and display toxicity against cells. To their knowledge, Applicants are the first to show that the capacity of thylakoids to produce ROS can be used in PDT applications.

[00108] Light can be applied directly on the skin for skin tumors or by laparoscopy and other techniques for tumors located elsewhere in the body (e.g., lungs, mouth, oesophagus, stomach, intestine, uterus, bladder, etc.). The use of an light wavelength of about 710 ± 10 nm (for excitation of the PSI) or about 680 nm ± 10 nm (for excitation of the PSII) will activate the PSI and/or PSII, which will transfer their energy of excitation to molecular oxygen, generating reactive oxygen species (ROS), mostly singlet oxygen. The photogenerated ROS will induce local oxidative degradation reactions, causing damage to vital cell functions. In addition to cytotoxic events, important local effects occur as a result of the destruction of the vascular system, with consequent occlusion and/or perforation of blood vessels and induction of local ischemia.

[00109] The chlorophyll used as photosensitizer in the present invention is comprised within the thylakoids components. It is mostly found in PSI and PSII. In the P680 of the PSII, two chlorophyll molecules are associated and designated chlorophyll pair. This chlorophyll pair is responsible for charge separation which provokes electron transport for the light phase of photosynthesis. Without this charge separation, no photosynthetic activity is possible and, by this, no ROS is generated. This reaction center is called P680 because it absorbs at a maximum wavelength of 680 nm.

[00110] The thylakoids photosensitizers of the present invention possess the following properties: A triplet state with high quantic yield or a high level of reactive oxygen species; an absorption peak of wavelength at about 680 or 710 (PSI and PSII have other lower absorption peaks. Longer wavelength advantageously penetrate tissues more deeply); and an absence of toxicity in the dark as shown in Figures 8C, 9C and 10C where cell growth in the dark is not influenced by the presence of thylakoids.

EXAMPLE 11 Cytotoxicity of the thylakoid vesicles

[00111] Assays were performed on tumor cells showing the cytoxicity of the thylakoids in accordance with the present invention. The assay was performed on colon cancer cells (HT-29), human melanoma (M21) and breast cancer (MCF-7). The cells were contacted with thylakoids vesicles in the dark, white light (ambient light (400-800 nm)) was then applied. Illuminations of one hour and then of two hours and a half were then performed. The lamp was then turned off. After light treatment, the cells were incubated at 37 C for 48 hours in the darkness. Growth rate was then measured. If the thylakoids generate a sufficient amounts of ROS, the cells survival rate will decrease in accordance with the vector effectiveness.

Growing cells

[00112] In 96-wells plates, were added 75 μl of culture medium (DMEM +5%(v/v) calf serum) containing the cells as follows: 1) HT-29 : 4000 cells/well; 2) M21: 3000 cells/well; and 3) MCF-7: 5500 cells/well. The plates were incubated (moist atmosphere, 37C, 5% CO2) for 24 hours so as to allow sufficient cell growth.

[00113] Thylakoids were then suspended in the same culture medium in the plates comprising the cells at a concentration 2X the initial tested concentration. This suspension was thereafter sequentially diluted 1/3 v/v in the cells culture medium. 75 μl of the thylakoid suspension was then added to the 75 μl of the tumor cell cultures. Light was applied to the plates for up to 2 Vz hours. Plates were then incubated 48 hours after light treatment to determine whether thylakoids influenced their growth.

Determination of cell survival

[00114] Two methods were used to determine cell survival and are described below:

[00115] Method 1 : Sulforhodamine B (SRB) coloration. This method is based on the following principle : cell proliferation is measured by determining total protein synthesis. SRB penetrates into cells and binds to proteins in an acidic medium while it detaches from proteins in a basic medium and solubilizes in the solution. Washes in acidic solutions (e.g., acetic acid) will remove unbound/solubilized SRB only. A basic solution is then used to unbind SRB and measure its concentration. Plates were thus emptied and washed delicately twice with 200 μl of PBS. This step is useful because at higher concentrations of chlorophyll, a thylakoid deposit was formed which hampered the measure. Fifty (50) μl of TCA 10% (w/v)/H2θ was then added. After one hour in cold temperature (about 4⁰C), plates were colored. They can also be maintained in the refrigerator a few days. [00116] Plates were emptied by inversion and rinsed under running water. Fifty (50) μl of SRB (sulforhodamine B) at 0.1% (w/v) / acetic acid 1% (v/v) were added. After one hour plates were washed by plunging them successively in each of the three tanks containing: 1 ) tap water; 2) acetic acid 1%; and 3) acetic acid 1%.

[00117] Plates were dried in a centrifuge having plate supports. Alternatively, they may be dried in an oven at about 5O⁰C. Dry plates were ready for use and stable for 1-2 days when refrigerated. Finally, SRB was solubilized by adding 200 μl of a Tris 0.02M solution and plates were read. No pH adjustment was necessary because the medium is already basic. Typically, a wavelength of about 530 nm is used (with a range of about 520 to about 580 nm). For a higher sensitivity, the wavelength can be increased to 565nm which is the maximum absorbance of SRB.

[00118] Method 2: metabolization of resazurine This method is based on the following principle : living cells metabolize resazurine (RZ) (blue and non fluorescent) into resorufin (RSF) (pink and fluorescent). 1) 50 μl of RZ at a concentration of 100 μg/ml are added to the 150 μl contained in the wells; and 2) plates are emptied and 200 μl of RZ at a concentration of 25 μg/ml are added.

[00119] When a slight coloration appears in the control wells, fluorescence is read. Ec (excitation) = 485nm, Em. (emission) = 590nm

[00120] This method does not kill cell. A higher sensitivity can thus be obtained by extending incubation. Significant differences of metabolism between cell lines and the significant effect of small variations temperature make the technique less interesting when there are more than one plate to evaluate.

[00121] Oxidative properties of the thylakoids were demonstrated by the decrease of the survival rate of the three cell lines tested. After one hour of exposition to ambient light in the presence of the 3 % v/v solution of thylakoid vesicles, only 62% of HT-29 cells grew while the survival rate fell at 36% after 2h30 light exposition (Figure 8A-B), only 51% of M21 cells grew while the survival rate fell at 31% after 2h30 light exposition (Figure 9A-B); and only 42% of MCF-7 cells grew while the survival rate fell at 25% after 2h30 light exposition (Figure 10A-B). After one hour of exposition to ambient light in the presence of the 1 % v/v solution of thylakoid vesicles only 60 % of MCF-7 cells grew after 1 hour while 50 % grew after 2,5 hours. As was observed in Figures 8C, 9C and 10C, when cells of the three cancer cell lines were incubated in the dark in the presence of the thylakoid vector, no toxicity was occurred since the survival rate of HT-29 and MCF-7 cells was over 95 %, while that of M21 cells was higher than 85%. EXAMPLE 12

Determination of toxicity of thylakoids and thylakoid functional derivatives at 680 nm with different light intensity

[00122] Assays are performed on colon cancer cells (HT-29), human melanoma (M21) and breast cancer (MCF-7). The cells are contacted with thylakoids vesicles in the dark, white light (680 nm) at varying intensity {e.g., a maximum of 500 to 2000 μmoles of photons per square meter per sec) is then applied. Illuminations of one hour and then of two hours and a half are then performed. The lamp is then turned off. After light treatment, the cells are incubated at 37 C for 48 hours in the darkness. Growth rate is then measured. Thylakoid functional derivatives are also similarly tested.

[00123] Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

REFERENCES

Aratό A, Bondarava N, Krieger-Liszkay A. 2004. Production of reactive oxygen species in chloride- and calcium-depleted photosystem Il and their involvement in photoinhibition. Biochim. Biophys. Acta. 1608(2- 3): 171 -80.

Asada K. 2006. Production and Scavenging of Reactive Oxygen Species in Chloroplasts and Their Functions. Plant Physiol. 141:391-96.

Baumgarten M, Philo JS, Dismukes GC. 1990, Mechanism of photoinhibition of photosynthetic water oxidation by Cl- depletion and F- substitution: oxidation of a protein residue. Biochem. 29(48):10814-22.

Bellemare F., Boucher N. and Lorrain L., 2001. Biosensors, and method and kits for using same BIOSENSORS, WO 2004/046717.

Berthold, Babcock, Yocum. 1981 A highly resolved oxygen-evolving photosystem Il preparation from spinach thylakoid membranes. FEBS Lett. 134, 231-234.

Boardman, N.D. and Anderson. 1964. Isolation from spinach chloroplastes of particules containing different proportions of chlorophyll b and their possible role in the light reaction of photosynthesis. Nature 203, 166- 167;

Durrant JR, Giorgi LB, Barber J, Klug DR, Porter G. 1990. caracterization of triplet-states in isolated photosystem Il reactions centres - oxygen quenching as a mechanism for photodamage. Biochim. Biophys. Acta 1017: 167-75.

Fufezan C, Gross CM, Sjόdin M, Rutherford AW, Krieger-Liszkay A, Kirilovsky D. 2007. Influence of the redox potential of the primary quinone electron acceptor on photoinhibition in photosystem ILJ. Biol. Chem. 282(17):12492-502.

Henmi T, Miyao M, Yamamoto Y. 2004. Release and reactive-oxygen-mediated damage of the oxygen- evolving complex subunits of PSII during photoinhibition. Plant Cell Physiol.45(2):243-50.

Krieger-Liszkay A. 2004. Singlet oxygen production in photosynthesis. J. Exp. Bot. 56(411): 337-46.

Krieger A, Rutherford AW, Vass I, Hideg E. 1998. Relationship between activity, D1 loss, and Mn binding in photoinhibition of photosystem II. Biochem. 37(46):16262-9.

Porra R.J., Thompson W.A., and Kriedemann P. E., 1989. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochimica Biophysics Acta, 975, 384-394.

Pospisil P, Aratό A, Krieger-Liszkay A₁ Rutherford AW. 2004. Hydroxyl radical generation by photosystem II. Biochem.43(21 ):6783-92.

Rinalducci S, Pedersen JZ, ZoIIa L. 2004. Formation of radicals from singlet oxygen produced during photoinhibition of isolated light-harvesting proteins of photosystem II. Biochim. Biophys. Acta 1608(1 ):63-73.

Song YG, Liu B, Wang LF, Li MH, Liu Y. 2006. Damage to the oxygen-evolving complex by superoxide anion, hydrogen peroxide, and hydroxyl radical in photoinhibition of photosystem II. Photosynth. Res. 90(1):67-78.

Stnzh I.G., Lysenko G.G., Neverov K.V. 2005. Photoreduction of Molecular Oxygen in Preparations of Photosystem Il under Photoinhibitory Conditions. Rus. J. Plant Physiol. 52(6):717-23.

Takahashi S, Murata N. 2008. How do environmental stresses accelerate photoinhibition? Trends Plant Sci. 13(4):178-82. van Roon et al., 2000; van Leeuwen et al., 1991; Eshaghi et al., 1999; Berthold et al., 1981. Vass I₁ Styring S. 1993. Characterization of chlorophyll triplet promoting states in photosystem Il sequentially induced during photoinhibition. Biochem. 32(13):3334-41.

Zimmermann G. M, Kramer G. N., Schnabl H. 1996. Lyophilization of thylakoids for improved handling in a bioassay. Environ. Toxicol. Chem. 15(9) : 1461-1463.

Claims

CLAIMS:

1. Thylakoid vesicles stably incorporating an heterogeneous agent.

2. The thylakoid vesicles of claim 1, wherein the heterogeneous agent has a size of about 100 Da to about 400 Da.

3. The thylakoid vesicles of claim 1 , wherein the heterogeneous agent is a fluorescent agent.

4. The thylakoid vesicles of claim 3, further comprising a ligand for targeting tumor cells.

5. A method of using the thylakoid vesicles of any one of claims 1 to 4, for medical imaging in a subject, comprising administering the thylakoid vesicles to a tissue of the subject, applying light on the tissue and visualizing fluorescence in the tissue.

6. A method of sensitizing tumor cells to photodynamic therapy (PDT) comprising contacting the tumor cells with the thylakoid vesicles of any one of claims 1 to 4, and applying light to the tumor cells, whereby tumor cells are sensitized to PDT.

7. A method of sensitizing tumor cells to photodynamic therapy (PDT) comprising contacting the tumor cells with thylakoids or thylakoid functional derivatives and applying light to the tumor cells, whereby tumor cells are sensitized to PDT.

8. The method of any one of claims 5 to 7, wherein the light comprises a wavelength of at least about 680 nm.

9. The method of any one of claims 5 to 7, wherein the tumor cells are melanoma cells, colon tumor cells or breast cancer cells.

10. The method of any one of claims any one of claims 5 to 7, wherein the light is applied for between about 1 and 2 hours V..

11. A method of detecting a filtration membrane defect, comprising introducing the thylakoid vesicles of claim 3, upstream of the filtration membrane and detecting the presence of fluorescence downstream of the filtration membrane, wherein the presence of fluorescence downstream of the filtration membrane is an indication that there is a defect in the filtration membrane.

12. The thylakoid vesicles of claim 1 or 2, wherein the heterogeneous agent is a cosmetic or a therapeutic agent.

13. A pharmaceutical composition comprising the thylakoid vesicles of any one of claims 1-4 and 12, and a pharmaceutically acceptable carrier.

14. A kit comprising the thylakoid vesicles of any one of claims 1-4 and 12.

15. A kit comprising the pharmaceutical composition of claim 13.

16. Thylakoids or thylakoid functional derivatives for use in photodynamic therapy (PDT).

17. Use of thylakoids or thylakoid functional derivatives for photodynamic therapy (PDT).