ALGA nanoparticles for treating cancer

Abstract

Provided herein are compositions including biocompatible, biodegradable, and low immunogenic alga(e) nanoparticles (aNPs) and methods of use thereof for treating, attenuating, and / or preventing progression of cancer and / or metastases, by parenteral route of administration.

Description

[0001] ALGA NANOPARTICLES FOR TREATING CANCER TECHNICAL FIELD

[0002] The present disclosure relates to compositions and methods for treating cancer. More specifically, the present disclosure pertains to alga(e) nanoparticles (aNPs) for cancer therapy.

[0003] BACKGROUND OF THE INVENTION

[0004] Cancer remains one of the most prevalent and life-threatening diseases worldwide, affecting millions of patients each year. Conventional cancer treatments, such as chemotherapy and radiotherapy, are widely used to kill cancer cells. Still, they are often associated with severe side effects, including damage to healthy cells, immune suppression, fatigue, nausea, and hair loss.

[0005] These adverse effects not only impact the quality of life of patients but can also limit the effectiveness of treatments due to the potential for drug resistance and the inability to target cancer cells. As a result, there is a growing need for more targeted, less toxic therapeutic options that can effectively combat cancer cells while minimizing damage to healthy tissues.

[0006] It has been proposed that natural compounds derived from marine organisms, such as algae, may possess a potential to perform as alternative cancer treatments since algae are rich sources of various bioactive compounds such as polysaccharides, proteins, lipids, vitamins, and antioxidants, which have demonstrated anti-cancer properties.

[0007] However, algae whole biomass and water extracts suffer from limited bioavailability and efficacy in achieving a desired anti-cancerous effect. Poor permeability through biological membranes reduces their effectiveness in reaching cancerous tissues.

[0008] Nanotechnology, liposomes, or other drug delivery systems are commonly utilized to overcome stability and absorption issues.

[0009] Nanoparticles (NPs) are transformative technology in cancer therapy, offering precision targeting and enhanced drug delivery. Their unique properties - small size, large surface area, and the ability to penetrate biological tissues - make them ideal for addressing bioavailability challenges in cancer treatments. In addition, tumors have leaky vasculature and poor lymphatic drainage, allowing NPs to accumulate preferentially in cancerous tissues.As an alternative cancer therapy NPs offer a platform for non-toxic and natural -derived therapies. For example, NPs can encapsulate bioactive natural compounds with anti-cancer properties, improving their solubility and stability in blood.

[0010] Nevertheless, despite a promising potential of NPs in cancer therapy, biocompatibility issues hinder their widespread clinical application, for example, non-biodegradable NPs, such as metal-based or carbon-based NPs, can accumulate and cause long-term toxicity in healthy tissues, and some NPs can trigger immune responses.

[0011] There is therefore an unmet need for expanding the repertoire of bioavailable biocompatible, non-immunogenic, biodegradable NPs for selective and less toxic cancer therapy.

[0012] SUMMARY OF THE INVENTION

[0013] According to one aspect, disclosed herein are compositions including Alga(e) derived nanoparticles (aNPs), which include non-soluble and amphiphilic alga(e) components, for use in treating, attenuating, and / or preventing progression of cancer in a subject.

[0014] Advantageously, in some embodiments, the compositions comprising the aNPs are formulated for parenteral administration at a therapeutic effective amount.

[0015] According to some embodiments, it was advantageously and surprisingly found that exposure of various cancer cell lines to NPs made of algae (such as, algae Arthospira platensis (Spirulina NPs (sNPs)) exerts a selective toxic effect towards cancerous cells, in comparison to non-cancerous cells. In some embodiments, the induced toxicity includes increased mortality rate and / or reduced viability of cancer cells.

[0016] In some embodiments, advantageously, the sensitivity of cancer cells to the cytotoxic effect increases when the aNPs dosage is elevated and the exposure is prolonged (i.e., dosedependent and / or time-dependent effect).

[0017] In some embodiments, the induced mortality and / or reduced viability includes increased cellular uptake by cancerous cells, relative to non-cancerous cells. In some embodiments, the increased cellular uptake by cancerous cells includes enhanced endocytosis by cancer cells relative / compared to non-cancerous cells.

[0018] According to some embodiments, disclosed herein is a method for treating, attenuating, and / or preventing progression of cancer in a subject in need thereof, the method includesparenterally administering to the subject a therapeutically effective amount of a composition including Alga(e) derived nanoparticles (aNPs).

[0019] According to some embodiments, there is provided a composition including alga-derived nanoparticles (aNPs) including non-soluble and amphiphilic alga(e) components, for use in treating, attenuating, and / or preventing progression of cancer in a subject in need thereof, wherein the aNPs have an average particle diameter in the range of between about 50 and about 650 nm, and wherein the composition is formulated for parenteral administration.

[0020] According to some embodiments, the non-soluble and amphiphilic alga(e) components include membranes and membrane-associated proteins of the alga(e).

[0021] According to some embodiments, the aNPs may have a spherical shape, and include a shell and inner core.

[0022] According to some embodiments, the alga(e) may belong to a genus selected from a group consisting of one or more of: Spirulina Arthrospira, Chondracanthus, Gracilaria, Chondrus, Undaria, Sargassum, Laminaria, Ecklonia, Macrocystis, Palmaria, Gigartina, Chlorella, and Haematococcus, or any combination thereof.

[0023] According to some embodiments, the alga(e) is Spirulina Arthrospira. According to some embodiments, the Spirulina Arthrospira includes one or more species selected from Arthrospira Platensis, Arthospira Maxima, Arthrospira ardissonei, Arthrospira erdosensis, Arthrospira fusiformis, Arthrospira indica, Arthrospira innermongoliensis, Arthospira jenneri, Arthrospira massartii, or any combination thereof. According to some embodiments, the Spirulina Arthospira is Spirulina Arthrospira Platensis.

[0024] According to some embodiments, the average particle diameter may be in the range between about 50 nm and 200 nm and / or a surface charge in the range between about -30 mV and about -45 mV.

[0025] According to some embodiments, the composition may include at least one pharmaceutically acceptable excipient.

[0026] According to some embodiments, the composition may further include an active ingredient.

[0027] According to some embodiments, the active ingredient may be associated with the aNPs. In some embodiments, the association includes the active ingredient attached to the aNPs shell or enclosed within the aNPs core.According to some embodiments, the composition may be administered with at least one additional active ingredient.

[0028] According to some embodiments, the composition may be administrated concomitantly with and / or sequentially to the at least one additional active ingredient.

[0029] According to some embodiments, the active ingredient may include an anti-cancer medication.

[0030] According to some embodiments, the cancer may be a primary solid, liquid tumor and / or metastases thereof.

[0031] According to some embodiments, the cancer may be buccal carcinoma, colorectal adenocarcinoma, breast cancer, brain, ovarian cancer, glioblastoma, kidney cancer, pancreas, liver, and leukemia, or any combination thereof.

[0032] According to some embodiments, the cancer may be selected from one or more of: buccal carcinoma, colorectal adenocarcinoma, and breast cancer, or any combination thereof.

[0033] According to some embodiments, the parenteral administration may include intradermal (ID) injection, subcutaneous (SQ) injection, intramuscular (IM) injection, intravenous (IV) injection, and / or intraperitoneal (IP) injection.

[0034] According to some embodiments, the administering of the composition may induce selective toxicity towards cancerous cells, in comparison to non-cancerous cells.

[0035] According to some embodiments, there is provided a method for treating, attenuating, and / or preventing progression of cancer in a subject in need thereof, the method includes parenterally administering to the subject a therapeutically effective amount of a composition including alga(e) derived nanoparticles (aNPs) including non-soluble and amphiphilic alga(e) components, and wherein the aNPs have an average particle diameter in the range of between 50 nm and about 650 nm.

[0036] According to some embodiments, the method further includes administering the composition concomitantly with and / or sequentially to at least one additional active ingredient.

[0037] According to some embodiments, the administering includes intradermal (ID) injection, subcutaneous (S.C.) injection, intramuscular (I.M.) injection, intravenous (I V.) injection, intraperitoneal (I.P.) injection, or any combinations thereof.Certain embodiments of the present disclosure may include some, all, or none of the above advantages. One or more technical advantages may be readily apparent to those skilled in the art from the figures, descriptions, and claims included herein. Moreover, while specific advantages have been enumerated above, various embodiments may include all, some, or none of the enumerated advantages.

[0038] BRIEF DESCRIPTION OF THE FIGURES

[0039] The invention will now be described in relation to certain examples and embodiments with reference to the following illustrative figures.

[0040] FIGs. 1A-1E show bar graphs presenting percentage of mortality (% Mortality) of TR-146 cells (human buccal carcinoma cell line) exposed to nanoparticles (NPs) derived from the algae Spirulina Arthospira platensis (sNPs) at concentrations of 25, 50, 125, 250, 375, and 500 mg / mL for (FIG. 1A) 3 hours, (FIG. IB) 6 hours, (FIG. 1C) 12 hours, (FIG. ID) 24 hours, and (FIG. IE) 48 hours before their viability was assessed using XTT assay. Statistical significance was assessed using one-way ANOVA with Dunnett' s post hoc test, comparing each concentration against the concentration showing the highest mortality at each time point. Values represent the mean ± SD from at least three independent experiments. Statistical significances are denoted as *p < 0.05, ** p < 0.01, **** p < 0.0001, and ns for nonsignificant.

[0041] FIGs. 2A-2E show bar graphs presenting percentage of mortality (% Mortality) of Caco-2 cells (human colorectal adenocarcinoma cell line) exposed to nanoparticles (NPs) derived from the algae Spirulina Arthospira platensis (sNPs) at concentrations of 25, 50, 125, 250, 375, and 500 mg / mL for (FIG. 2 A) 3 hours, (FIG. 2B) 6 hours, (FIG. 2C) 12 hours, (FIG. 2D) 24 hours, and (FIG. 2E) 48 hours before their viability was assessed using XTT assay. Statistical significance was assessed using one-way ANOVA with Dunnett' s post hoc test, comparing each concentration against the concentration showing the highest mortality at each time point. Values represent the mean ± SD from at least three independent experiments. Statistical significances are denoted as *p < 0.05, **** p < 0.0001, and ns for non-significant.

[0042] FIGs. 3A-3E show bar graphs presenting percentage of mortality (% Mortality) of HT-29 cells (human colorectal adenocarcinoma cell line) exposed to nanoparticles (NPs) derived from the algae Spirulina Arthospira platensis (sNPs) at concentrations of 25, 50, 125, 250, 375, and 500 mg / mL for (FIG. 3A) 3 hours, (FIG. 3B) 6 hours, (FIG. 3C) 12 hours, (FIG. 3D) 24 hours, and (FIG. 3E) 48 hours before their viability was assessed using XTT assay. Statisticalsignificance was assessed using one-way ANOVA with Dunnett' s post hoc test, comparing each concentration against the concentration showing the highest mortality at each time point. Values represent the mean ± SD from at least three independent experiments. Statistical significances are denoted as *p < 0.05, **** p < 0.0001, and ns for non-significant.

[0043] FIGs. 4A-4E show bar graphs presenting percentage of mortality (% Mortality) of MCF-7 cells (human breast cancer cell line) exposed to nanoparticles (NPs) derived from the algae Spirulina Arthospira platensis (sNPs) at concentrations of 25, 50, 125, 250, 375, and 500 mg / mL for (FIG. 4A) 3 hours, (FIG. 4B) 6 hours, (FIG. 4C) 12 hours, (FIG. 4D) 24 hours, and (FIG.

[0044] 4E) 48 hours before their viability was assessed using XTT assay. Statistical significance was assessed using one-way ANOVA with Dunnett' s post hoc test, comparing each concentration against the concentration showing the highest mortality at each time point. Values represent the mean ± SD from at least three independent experiments. Statistical significances are denoted as * p < 0.05, **** p < 0.0001, and ns for non-significant.

[0045] FIGs. 5A-5E show bar graphs presenting percentage of mortality (% Mortality) of MCF-10A cells (non-tumorigenic human breast epithelial cells; control) exposed to nanoparticles (NPs) derived from the algae Spirulina Arthospira platensis (sNPs) at concentrations of 25, 50, 125, 250, 375, and 500 mg / mL for (FIG. 5A) 3 hours, (FIG. 5B) 6 hours, (FIG. 5C) 12 hours, (FIG. 5D) 24 hours, and (FIG. 5E) 48 hours before their viability was assessed using XTT assay. Statistical significance was assessed using one-way ANOVA with Dunnett' s post hoc test, comparing each concentration against the concentration showing the highest mortality at each time point. Values represent the mean ± SD from at least three independent experiments.

[0046] FIGs. 6A-6E show bar graphs presenting percentage of mortality (% Mortality) in HDFn cells exposed to sNPs concentrations of 25, 50, 125, 250, 375, and 500 mg / mL for (FIG. 6A) 3 hours, (FIG. 6B) 6 hours, (FIG. 6C) 12 hours, (FIG. 6D) 24 hours, and (FIG. 6E) 48 hours before their viability was assessed using XTT assay. Statistical analysis was performed using one-way ANOVA with Bonferroni correction for multiple comparisons. Values represent the mean ± SD from at least three independent experiments. Statistical significance is denoted as ns for non-significant.

[0047] FIG. 7 shows a line graph presenting the IC50 values of the different cancer cell lines (MCF-7, Caco-2, TR-146, and HT-29) at varying time points (3, 6, 12, 24, and 48 hours), illustrating the time-dependent cytotoxicity and effectiveness of the Spirulina NPs (sNPs) on the different cancerous cell lines. Related to FIGs. 1 A-1E, FIGs. 2A-2E, FIGs. 3A-3E, FIGs. 4A-4E.FIG. 8 shows fluorescence microscopy images presenting the uptake of sNPs labeled with Dil (red) by TR-146 buccal cancer cells. The top left image shows the Dil-labeled sNPs (red), the top right image displays the DAPI-stained nuclei of TR-146 cells (blue), and the bottom image is a merged overlay showing the internalization of sNPs within the cells, as evidenced by the overlap of red and blue signals. Scale bars represent 100 pm.

[0048] FIGs. 9A-9E show bar graphs presenting the effect of endocytosis inhibitors - methyl-P-cyclodextrin (MpCD) which disrupts lipid rafts and 5-N-Ethyl-N-isopropyl amiloride (EIP A) which is an inhibitor of macropinocytosis - on the uptake of sNPs into the cell lines (FIG. 9A) TR-146, (FIG. 9B) Caco-2, (FIG. 9C) HT-29, (FIG. 9D) MCF-7, and (FIG. 9E) MCF-10A. These inhibitors were used to assess the aNPs mechanism of action. Values represent the mean ± SD from at least three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Fisher’s LSD post hoc test, with significance indicated as follows: *p < 0.05, **p < 0.01, and ****p < 0.0001.

[0049] DETAILED DESCRIPTION

[0050] In the following description, various aspects of the disclosure will be described. For the purpose of explanation, specific configurations, and details are set forth in order to provide a thorough understanding of the different aspects of the disclosure. However, it will also be apparent to one skilled in the art that the disclosure may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the disclosure.

[0051] Definitions

[0052] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well unless the context clearly indicates otherwise, “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the stated object, unless the context clearly dictates otherwise. By way of example, “a composition” means one or more compositions.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In case of conflict, the patent specification, including definitions, governs.

[0054] As used herein, the terms “prevent”, “reduce”, “attenuate”, “ameliorate”, and “inhibit” are used interchangeably. As used herein, the terms “Enhanced”, “increased”, and “elevated” are used interchangeably.As used herein, the term "about" or “approximately” are synonymous and when referring to a measurable value such as an amount, a temporal duration, and the like, the terms are meant to encompass variations of ± 20% or, in some instances ± 10%, or in some instances ± 5%, or in some instances ± 1%, or in some instances, ± 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. The terms about and approximately.

[0055] As used herein, the terms “essentially” and “substantially” are synonymous and when referring to a stated material such as a composition, a substance, and the like, is meant to encompass variations of in some embodiments, ±0.1%, or in some embodiments, ±1%, or in some embodiments, ±2%, or in some embodiments, ±5% from a stated amount, as such variations / deviations are appropriate to perform the disclosed methods.

[0056] According to some embodiments, the term “essentially devoid of’ may refer to a stated material as either entirely absent, or present in a residual amount, such as less than 5%, or less than 2%, or less than 1%, or less than 0.1% are present with respect to the initial amount of the stated material or with respect to the total % of all other components. Each possibility is a separate embodiment. According to some embodiments, the term “substantially made of’ may refer to a stated material as either entirely present, or absent in a neglectable amount, such as more than 95%, or more than 98%, or more than 99%, or more than 99.9% are present with respect to the initial amount of the stated material or with respect to the total % of all other components. Each possibility is a separate embodiment.

[0057] As used herein, the term “comprising” is synonymous with the terms "including," "containing," or "characterized by" and is inclusive or open-ended, i.e., does not exclude additional, unrecited elements. According to some embodiments, the term comprising may be replaced with the term with the term “consisting of’ which excludes any element, step, or ingredient not specified in the claim. According to some embodiments, the term comprising may be replaced with the term “consisting essentially of’ which limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristics" of the claimed invention.

[0058] As used herein, the terms "subject", "patient" or "individual" may be used interchangeably and generally refer to a human, although the methods of the invention are not necessarily limited to humans and should be useful in other mammals or non-mammal animal s / vertebrates, including, for example, but not limited to: farm animals, pets and the like.In some embodiments, the subject includes mammals and / or humans. Each possibility is a separate embodiment. In some embodiments, the subject is a human.

[0059] The term “treatment” as used herein, refers to anti-cancer therapeutic treatment, prophylactic and / or preventative measures. In some embodiments, those in need of treatment include those already having a disorder as well as those in which the disorder is to be prevented. The "treatment" refers to an approach for obtaining beneficial or desired results in treating cancer or metastases thereof, including clinical results. Beneficial or desired clinical results can include but are not limited to alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of disease, stabilization of the state of disease, prevention of deterioration of the disease or condition, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total). As used herein, the terms “prevent”, “reduce”, “attenuate”, “ameliorate”, and “inhibit” may be used interchangeably. In some embodiments, the disease or condition refers to cancer. In some embodiments, the cancer includes solid and / or liquid tumors and / or metastases thereof.

[0060] As used herein, the terms “composition”, “therapeutic compositions”, or “pharmaceutical composition” are used interchangeably to refer to a composition including the aNPs of the present invention, and in some embodiments, including one or more physiologically acceptable excipient / s. The compositions of the present invention facilitate processing of the aNPs into preparations that can be used pharmaceutically, whether the compositions further comprise an active ingredient such as an anti-cancer drugs, or not.

[0061] In some embodiments, the compositions of the present invention may further include an active ingredient. In some embodiments, the active ingredient is an anti-cancer medication / agent.

[0062] In some specific embodiments, the pharmaceutical compositions of the present disclosure are suitable for injection. The pharmaceutical forms suitable for injection may include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.According to some embodiments, preparations of the composition of the invention for injection include sterile aqueous or non-aqueous solutions, suspensions, or emulsions, each representing a separate embodiment of the present invention.

[0063] In some embodiments, the injection refers to parenteral route of administration.

[0064] In some specific embodiments, the pharmaceutical compositions of the present disclosure generally comprise a buffering agent, an agent who adjusts the osmolarity thereof, and, in some embodiments, one or more pharmaceutically acceptable excipients, carriers, additive / s, stabilizer / s, and / or diluent / s as known in the art.

[0065] The term “pharmaceutically acceptable excipient / s” refers to any of carrier / s, auxiliary / s, stabilizer / s, and / or diluent / s conventionally used in the production of pharmaceutical compositions as so it is physiologically acceptable to the subject and is also compatible with the activity of aNPs, namely the anti-cancer activity, and any further active ingredient included in the composition, and include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents and the like. In some embodiments, the carrier can be solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol (PEG), and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactant. In some embodiment, polyethylene glycol (PEG) is used to increase blood circulation. The use of such media and agents for pharmaceutical substances is well known in the art. Except as any conventional media or agent is incompatible with the aNPs, its use in the therapeutic composition is contemplated.

[0066] In some embodiments, the composition includes at least one pharmaceutically acceptable excipient / s, carrier / s, auxiliary / s, stabilizer / s, and / or diluent / s. Each possibility is a separate embodiment. The terms “excipient / s”, “carrier / s”, “auxiliary / s”, “stabilizer / s”, and / or “diluent / s” are broadly interpreted according to the meaning normally ascribed to the term in the art.

[0067] A non-limiting example of a pharmaceutically acceptable carrier is buffered or unbuffered normal saline (approximately 0.9% which is about 0.15MNaCl). Pharmaceutically acceptable carriers, excipients or stabilizers are well known in the art, for example Remington's Pharmaceutical Sciences, 16th edition, Osol, A Ed. (1980).In some embodiments, the pharmaceutical compositions of the present disclosure, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the aNPs, and any other active ingredient further included in the composition, with the pharmaceutical carrier / s, excipient / s, auxiliary / s, stabilizer / s, and / or diluent / s. In general formulations are prepared by uniformly and intimately bringing into association the aNPs with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0068] Cancer treatment often involves significant side effects, necessitating the exploration of more selective therapeutic options.

[0069] In some embodiment, the present disclosure is based on the surprising finding that nanoparticles (NPs) made of alga(e) (aNPs) induced at least 20% (e.g. between 30% to 80%) mortality across different cancer cell lines. Thus, it is herein disclosed that aNPs can be utilized for a targeted anti-cancer treatment.

[0070] Algae, particularly in the form of whole biomass and water extracts, contains high levels of phycocyanin, polysaccharides, vitamins, and essential fatty acids, which have immunomodulatory, anti-inflammatory, and antioxidant properties.

[0071] Notwithstanding the above beneficial properties, but differently from algae whole cell biomass, or algae water extracts, or algae combined with metallic NPs, the present disclosure pertains to aNPs containing non-soluble and amphiphilic algae components, such as, but not necessarily limited to membranes and membrane proteins derived from one or more alga(e) species by fractionation.

[0072] The NPs made of the alga(e), or, in some examples, specifically made of Spirulina, may be prepared according to the herein disclosed method of preparation wherein a non-soluble and amphiphilic fraction including membranes and membrane proteins derived from alga(e) cells / biomass (i.e., the aNPs) is isolated from top of a density gradient after homogenization (e.g., by sonication) of the alga(e) cells / biomass starting material, its fractionation using centrifugation and ultracentrifugation, and its subsequent separation on the density gradient, and without using any potentially cytotoxic materials such as ethanol or methanol.

[0073] In some embodiments, the alga(e) belongs to a genus selected from a group of consisting of one or more of: Spirulina Arthrospira, Chondracanthus, Gracilaria, Chondrus, Undaria, Sargassum, Laminaria, Kombu, Ecklonia, Macrocystis, Palmaria, Gigartina,Chlorella, and Haematococcus, or any combination thereof. Each possibility is a separate embodiment.

[0074] The terms “algal-based NPs”, “NPs made of alga(e)”, “aNPs” are interchangeably used to refer to “nanoparticles (NPs) derived from alga(e)” (“aNPs”), namely, from any one of the 14 types of edible algae specified in Table 1 below.

[0075] Among the different types of algae herein used to generate aNPs, Spirulina Platensis, a blue-green microalga is herein considered as having a great potential in selective cancer therapy.

[0076] As exemplified herein such aNPs, in some instances Spirulina Platensis NPs (sNPs), can promote cancer cell death and / or reduced viability. Each possibility is a separate embodiment.

[0077] The terms “Spirulina” and “Spirulina Arthospira” may be used interchangeably to refer to any species belonging to Spirulina Arthospira.

[0078] In some embodiments, the alga(e) is Spirulina Arthrospira.

[0079] In some embodiments, the Spirulina Arthrospira comprises one or more species selected from Arthrospira Platensis, Arthospira Maxima, Arthrospira ardissonei, Arthrospira erdosensis, Arthrospira fusiformis, Arthrospira indica, Arthrospira innermongoliensis, Arthospira jenneri, Arthrospira massartii, or any combination thereof. Each possibility is a separate embodiment.

[0080] In some embodiments, the Spirulina Arthospira comprises Spirulina Arthrospira Platensis.

[0081] In some embodiments, the aNPs refer to Spirulina (Arthospira) nanoparticles (sNPs). In some embodiments, aNPs consist of sNPs. In some embodiments, aNPs are sNPs.

[0082] The terms “spirulina aNPs”, “spirulina NPs”, “sNPs” are interchangeably used to refer to “nanoparticles (NPs) derived from Spirulina Arthospira” (“sNPs”)

[0083] According to some embodiments, as exemplified herein, the aNPs were evaluated for their cytotoxic effects on various cells, including TR-146 buccal cancer, Caco-2 and HT-29 colorectal cancer, MCF-7 breast cancer cells, and the non-cancerous MCF-10A cells and HDFn cells. The cytotoxic effects of various concentrations and exposure times were tested. It is herein exemplified that aNPs induced at least 20% (e.g., about 30% to 80%) mortality acrossdifferent cancer cell lines depending on concentration and exposure time, compared to negligible mortality of less than about 5% in non-cancerous cells.

[0084] Thus, in some embodiments, exposure of cancer cells to aNPs promotes selective toxicity in comparison to non-cancerous cells exposed to aNPs under the same conditions.

[0085] In some embodiments, the administering of compositions including the aNPs induces selective toxicity towards cancerous cells, in comparison to non-cancerous cells.

[0086] In some embodiments, the induced toxicity towards cancerous cells includes induced mortality and / or reduced viability of cancer cells. Each possibility is a separate embodiment. In some embodiments, said enhanced mortality and / or reduced viability includes, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, (e.g., in the range of about 30%-80%) change.

[0087] In some embodiments, the induced mortality and / or reduced viability includes a dose dependent effect of the aNPs or the composition comprising the aNPs. Each possibility is a separate embodiment.

[0088] In some embodiments, the induced mortality and / or reduced viability includes a time dependent (temporal) effect of the aNPs or the composition comprising the aNPs. Each possibility is a separate embodiment.

[0089] In some embodiments, mortality includes decrease in IC50 values over time, indicating increased sensitivity to aNPs with prolonged exposure. In some embodiments, mortality includes apoptosis.

[0090] In some embodiments, the induced mortality or reduced viability includes increased cellular uptake by cancerous cells, relative to / compared to non-cancerous cells. Each possibility is a separate embodiment.

[0091] In some embodiments, non-cancerous cells show no significant cytotoxic effects, across various concentrations and / or exposure times.

[0092] In some embodiments, aNPs may be selected from Spirulina NPs. In some embodiments, aNPs includes Spirulina NPs.

[0093] Reference is made to FIGs. 1A-1E, FIGs. 2A-2E, FIGs. 3A-3E, FIGs. 4A-4E, FIGs.

[0094] 5A-5E, FIGs. 6A-6E, and FIG. 7, showing the cytotoxic effects of the aNPs, (specifically sNPs), as percentage mortality (indicative of cytotoxicity) across different cancer cell lines, as demonstrated in Example 1.Cellular uptake was quantified through a fluorescence-activated cell sorter (FACS) and confirmed via fluorescence microscopy, while endocytic inhibitors were used to identify the pathways involved in aNPs uptake. It was found that MpCD, a blocker of caveolae-mediated endocytosis, had the highest impact on the internalization of aNPs, and subsequently on their cytotoxic effect.

[0095] In some embodiments, the induced mortality and / or reduced viability includes increased cellular uptake by cancerous cells, relative to non-cancerous cells. In some embodiments, the increased cellular uptake by cancerous cells includes enhanced internalization or endocytosis by cancer cells, in comparison to non-cancerous cells.

[0096] In some embodiments, aNPs are capable of penetrating / internalized cancer cells with enhanced efficacy in comparison to non-cancerous cells. In some embodiments, the enhanced efficacy includes increase of up to about 20 % (e.g., 30%, 40% or more) in cellular uptake.

[0097] In some embodiments, caveolae-mediated endocytosis is a crucial pathway for aNPs, in particular for sNPs, internalization and / or cytotoxicity in cancer cells. In some embodiments, cytotoxicity in cancer cells includes caveolae-mediated endocytosis and / or internalization.

[0098] Reference is made to FIG. 8, Table 2 and FIGs.9A-E showing internalization of aNPs by cancer cells, specifically sNPs, and the effect of inhibiting endocytosis on cellular uptake and subsequent mortality of cancer cells, as demonstrated in Example 2 and Example 3.

[0099] Therefore, as exemplified herein, aNPs, in particular sNPs, have a surprising effect as a selective and efficient anti -cancer agent.

[0100] According to some embodiments, the aNPs (e.g., the sNPs) are drug free. In some embodiments, the aNPs (e.g., the sNPs) do not encapsulate a drug. In some embodiments, the aNPs (e.g., the sNPs) are free of anti-cancer agent / drug / medication. In some embodiments, the aNPs (e.g., the sNPs) do not encapsulate an anti-cancer agent / drug / medication.

[0101] According to some embodiments, exemplified herein are difference in sensitivity that were observed between colorectal cancer cell lines, with male-derived Caco-2 cells being more sensitive to aNPs than female-derived HT-29 cells, suggesting sex-based differences. In accordance, in some embodiments, the composition of the present invention may promote enhanced selective toxicity towards cancer cells in male subjects, in comparison to female subjects.In accordance with the abovementioned findings, the present disclosure provides according to one aspect, a composition(s) including alga(e) derived nanoparticles (aNPs) including non-soluble and amphiphilic alga(e) components, for use in treating, attenuating, and / or preventing progression of cancer in a subject in need thereof, wherein the composition(s) are formulated for parenteral administration. In some embodiments, the aNPs may have an average particle diameter in the range of between about 100 nm and about 650 nm.

[0102] As used herein, the term “administration” or “administering” refers to parenteral routes of administering to the subject the compositions of the present invention, as carried out using known procedures, at dosages, and for periods of time effective to provide the desired effect. The effective amount of therapeutic compositions including aNPs according to the present invention (whether the compositions further include an active ingredient, or not) necessary to achieve a desired therapeutic effect may vary according to factors such as the age, sex, and weight of the subject and the ability of the aNPs to treat the malignant disease / condition in the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response.

[0103] In some embodiments, the administration or administering includes parenteral routes of administration that allow the compositions of the invention to perform their intended function. In some embodiments, the administration or administering includes parenteral routes of administration that allow the compositions of the invention to perform their intended function.

[0104] In some embodiments, the composition including the aNPs for use in the treatment of cancer is administered parenterally. In some embodiments, parenteral routes of administration include injection.

[0105] In some embodiments, parenteral administration includes injection to thereby achieve a systemic and / or local anti -cancerous therapeutic effect. Each possibility is a separate embodiment. In some embodiments, parenteral administration includes injection to thereby achieve a systemic anti-cancerous therapeutic effect

[0106] In some embodiments, the parenteral administration includes intradermal (ID) injection, subcutaneous (SQ) injection, intramuscular (IM) injection, intravenous (IV) injection, and / or intraperitoneal (IP) injection. Each possibility is a separate embodiment.

[0107] According to another embodiment, administration systemically is through a parenteral route. According to some embodiments, parenteral administration is administrationintravenously, intra-arterially, intramuscularly, intraperitoneally, intradermally, intravitreally, or subcutaneously. Each of the abovementioned administration routes represents a separate embodiment of the present invention. According to another embodiment, parenteral administration is performed by bolus injection. According to another embodiment, parenteral administration is performed by continuous infusion.

[0108] In some embodiments, the administration does not include oral administration or topical application. Each possibility is a separate embodiment.

[0109] In some embodiments, the administration excludes (does not include) oral administration to the gastrointestinal (Gl)-tract and / or topical application to the nasal epithelium / cavity, buccal epithelium / cavity, and / or vaginal epithelium. Each possibility is a separate embodiment. In some embodiments, the administration does not include oral administration to mucosal epithelium or topical application on mucosal epithelium. Each possibility is a separate embodiment.

[0110] According to some embodiments, the administration may include any suitable administration regime, depending, inter alia, on the medical condition, patient characteristics, administration route, and the like. Each possibility is a separate embodiment. In some embodiments, administration may include administration twice daily, every day, every other day, every third day, every fourth day, every fifth day, once a week, once every second week, once every third week, once every month, and the like. Each possibility is a separate embodiment.

[0111] Notwithstanding the beneficial properties mentioned hereinabove somewhere, (i.e., immunomodulatory, anti-inflammatory, and antioxidant properties) and without wishing to be bound by any theory or mechanism of action, aNPs protein content of membrane proteins and / or non-membrane proteins may contribute to the beneficial properties of the aNPs disclosed herein.

[0112] In some embodiments, the aNPs include non-soluble and amphiphilic alga(e) components. In some embodiments, the aNPs consist essentially of non-soluble and amphiphilic alga(e) components. In some embodiment, the aNPs comprise or consist essentially of non-soluble and amphiphilic alga(e) components comprising membranes and membranal proteins. Each possibility is a separate embodiment.

[0113] In some embodiments, non-soluble and amphiphilic alga(e) components include membranes and membrane proteins of the alga(e). In some embodiments, non-soluble andamphiphilic alga(e) components include membranes and membrane proteins of the alga(e), and may further include non-membrane proteins and polysaccharides of the alga(e).

[0114] According to some embodiments, the term “non-soluble and amphiphilic components” refer to alga(e) derived cellular structures and / or molecules, in water or water-based solution / buffer. Such water insoluble structures and / or molecules may refer to phospholipid bilayer (biological membranes) and membranal proteins, and may also refer in some embodiments to non-membrane proteins and / or polysaccharides. Such structures and / or molecules may reside, for example, in a water insoluble fraction of an extract of alga(e) biomass homogenized and fractionated in a water or water-based solution.

[0115] In some embodiments, the term “membrane proteins” has the meanings normally ascribed to it in the art, referring to proteins that are part of, or interact with membranes, permanently or transiently, and herein it refers to proteins that are part of or interact with aNPs, including, for example, integral proteins or non-integral proteins. The terms “membrane proteins,” “membranal proteins,” and “membrane-associated proteins” are interchangeably used. As used herein, the term “non-membrane proteins” refers to proteins that are not membrane proteins.

[0116] In some embodiments, the aNPs include a plurality of different membrane proteins of the alga(e). In some embodiments, the aNPs comprise a plurality of different membrane proteins of the alga(e) and further comprise a plurality of non-membrane proteins of the alga(e).

[0117] As used herein, the term “plurality” refers to at least two (two or more), and may relate to membrane proteins or non-membrane proteins. According to some embodiments, the plurality includes at least two, or at least ten, or at least twenty, or at least about thirty, or at least about forty, or at least about fifty, or at least about sixty, or at least about seventy, or at least about eighty, or at least about ninety, or at least about one hundred, or at least about two hundreds, or at least about three hundreds, or more membrane-associated proteins. Each possibility is a separate embodiment.

[0118] According to some embodiments, the aNPs include at least 2, or at least 5, or at least 10, or at least 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 80, or at least about 90, or at least about 100, or at least about 200, or about 230, or about 250 or more, membrane-associated proteins. Each possibility is a separate embodiment.In some embodiments, the aNPs include between 2 and about 400 membrane proteins, or between 2 and 10 membrane proteins, or between 2 and 20 membrane proteins, or between 2 and 50 membrane proteins, or between 2 and 100 membrane proteins, or between 2 and 250 membrane proteins, or between 180 and 300 membrane proteins. Each possibility is a separate embodiment.

[0119] In some embodiments, the aNPs include about 200-250 (e.g., 229) membrane associated proteins. In some embodiments, the plurality of membrane-associated proteins is selected from those demonstrated in Example 4.

[0120] Reference is made to Example 4 presenting the protein content of the aNPs as identified by LC-MS.

[0121] According to some embodiments, as exemplified herein, the selective toxicity of algal NPs (aNPs) towards cancer cells, and its impact on the mortality rate, apoptosis or proliferation rate of cancer cells and non-cancer cells is herein evaluated in cell lines and primary cells in vitro with different aNPs concentrations and exposure times, as well as in vivo in tumor models. In addition, the biodistribution of aNPs in vivo is considered herein, in order to assess the capability of aNPs to specifically target tumor cells (cancerous tissues) in vivo and to assess the distribution of the aNPs in the major organs in vivo, such as heart, liver, spleen, lung, and kidney.

[0122] In some embodiments, as exemplified herein, also evaluated, using different aNPs concentrations and exposure times, in the presence or absence of endocytosis inhibitors, is the presumed connect! on / relati on between cellular uptake / internalization of aNPs and the selective toxicity of aNPs.

[0123] According to some embodiments, non-limiting examples of cell lines that may be utilized to assess the abovementioned effects (e.g., cytotoxicity, mortality, proliferation, apoptosis, cellular uptake, endocytosis) in vitro or in vivo in tumor models, may include, but not limited to: TR-146 (human buccal carcinoma), Caco-2 (human colorectal adenocarcinoma), HT-29 (human colorectal adenocarcinoma), and MCF-7 (human breast cancer), MDA-MB-231 (triple-negative breast cancer), A549 (non-small-cell lung cancer), HeLa (cervical cancer), U87 / LN-18 (glioblastoma), and HEK293 (embryonic kidney), MRC-5 (human lung fibroblasts), BJ (human foreskin fibroblasts), as well as non-cancerous control cell lines MCF-10A (epithelial cell line, control), HDFn cells (Human Dermal Fibroblasts, neonatal fibroblast cells), and HaCaT (human keratinocytes). Human primary keratinocytes and hepatocytes arealso utilized as primary cells that mimic the behavior and responses of cells in vivo more closely than immortalized cell lines.

[0124] According to some embodiments, the foregoing effects may be evaluated using aNPs derived alga(e) belonging to any one of: Spirulina Arthrospira, Chondracanthus, Gracilaria, Chondrus, Undaria, Sargassum, Laminaria, Kombu, Ecklonia, Macrocystis, Palmaria, Gigartina, Chlorella, and Haematococcus, or any combination thereof. Each possibility is a separate embodiment.

[0125] According to some embodiments, the cancer includes primary solid and / or liquid tumor and / or metastases thereof. Each possibility is a separate embodiment. In some embodiments, the cancer includes primary malignancy. In some embodiments, the cancer includes primary malignancy and metastases thereof.

[0126] According to some embodiments, the cancer is selected from one or more of: buccal carcinoma, colorectal adenocarcinoma, breast cancer, brain, kidneys, pancreas, liver, lung, ovarian cancer, glioblastoma, leukemia, or any combination thereof. Each possibility is a separate embodiment.

[0127] According to some embodiments, the cancer is selected from one or more of: buccal carcinoma, colorectal adenocarcinoma, and breast cancer, or any combination thereof. Each possibility is a separate embodiment.

[0128] According to some embodiments, the herein disclosed compositions including aNPs may further include an active ingredient. The active ingredient may be, or may not be, associated with the aNPs. For active ingredients that are associated with the aNPs, said association includes the active ingredient attached to the aNPs shell or enclosed within the aNPs core as described herein below.

[0129] The provided aNPs are suitable for targeting cancer cells and facilitating uptake into cancer cells of one or more active ingredients associated (i.e., encapsulated) with the aNPs. Each possibility is a separate embodiment.

[0130] According to some embodiments, the aNPs includes an active ingredient associated therewith, said association includes the active ingredient attached to the aNPs shell or enclosed within the aNPs hydrophilic core. In some embodiments, the active ingredient is a medication known to be beneficial for anti-cancer treatment.The term “active ingredient” as used herein refers to a biologically active pharmaceutical, drug or therapeutic agent that is further included in the herein disclosed compositions and may or may not be associated with the alga(e) nanoparticles (aNPs), and is capable of inducing a sought-after effect upon administration of the composition, the effect is related to an anti-cancer therapy. Non-limiting examples of active ingredients include anticancer drugs such as small molecules, nucleic acid-based biologies, or amino acid-based biologies.

[0131] Non-limiting examples of anti-cancer drugs include such drugs as, but not limited to: rituximab, trastuzumab, cetuximab, bevacizumab, 5-fluorouracil, pembrolizumab, nivolumab, and the like, or any combination thereof. Each possibility is a separate embodiment.

[0132] According to some embodiments, as used herein the terms “associated with aNPs” “associated therewith” or “encapsulated with aNPs” or “comprised with aNPs” are used interchangeably and should be broadly interpreted to refer to the formation of any kind of a complex / physical combination between one or more active ingredient(s) and the aNPs. The complex can be initiated / formed by mixing the alga(e) nanoparticles (aNPs) with one or more active ingredient(s). The terms “comprised with”, “associated”, and “encapsulated” are interchangeably used.

[0133] According to some embodiments, the aNPs include one or more active ingredient(s). According to some embodiments, the aNPs are associated with one or more active ingredient(s). According to some embodiments, the aNPs are encapsulated with one or more active ingredient(s).

[0134] According to some embodiments, the “association” or “encapsulation” includes the active ingredient attached to the aNPs shell or enclosed within the aNPs hydrophilic core. Each possibility is a separate embodiment.

[0135] In some embodiments, as used herein the term “enclosed” refers more specifically to the one or more active ingredient(s) being at least partially solubilized or bounded inside the aNPs hydrophilic core. Each possibility is a separate embodiment.

[0136] In some embodiments, as used herein the term “attached” refers more specifically to the active ingredient(s) being at least partially attached to the aNPs membrane, either from the inner side or the outer side of the bilayer. Each possibility is a separate embodiment.In some embodiments, the association or encapsulation of one or more active ingredients with the aNPs includes attachment of the active ingredient(s) to the aNPs shell (i.e., membrane).

[0137] In some embodiments, the association or encapsulation of one or more active ingredients with the aNPs includes enclosure of the active ingredient(s) within the aNPs hydrophilic core.

[0138] In some embodiments, the association with aNPs may facilitate selectivity of the active ingredient towards cancer cells, or improve its bioavailability and stability by protecting the active ingredient from being degraded, for example by blood protease, providing sustained release, enhancing retention time, and overall prolonging the therapeutic effect. Each possibility is a separate embodiment.

[0139] The encapsulation or association may be affected by the hydrophilic, hydrophobic, or amphipathic properties of the active ingredient. For example, an active ingredient(s) having hydrophilic properties would be more prone to be encapsulated / associated inside the aNPs hydrophilic core (at least partially enclosed inside), while an active ingredient(s) having hydrophobic properties would be more prone to be encapsulated / associated by attachment to the aNPs membrane (at least partially attached).

[0140] According to some embodiments associating / encapsulating one or more active ingredients includes mixing or incubating aNPs with one or more active ingredients.

[0141] According to some embodiments, the association of one or more active ingredients with the aNPs comprises mixing or incubating the aNPs with the one or more active ingredients and subjecting the mixture to sonication. According to some embodiments, the association comprises mixing the aNPs with one or more active ingredients, subjecting the mixture to sonication and further to ultra-centrifugation.

[0142] In some embodiments, the aNPs are associated or encapsulated with drugs for cancer therapy prior to parenteral administration of compositions including the aNPs.

[0143] In some embodiments, as exemplified herein, association of aNPs with Fluorescein Isothiocyanate dextran (FITC-Dextran) is herein described in order to depict the ability of aNPs to encapsulate anti-cancer related substances or agents, including small molecules or larger entities such as miRNA, siRNA, peptides, enzymes, and antibodies having higher size and MW(up to about 250-300 kDa), ultimately carry them to cancerous tissue and facilitate their uptake by cancer cells.

[0144] In some embodiments, the one or more active ingredients is released from the aNPs into cancer cells.

[0145] In addition, regardless of the compositions including aNPs further including the active ingredient associated, or not associated with the active ingredient, the herein disclosed compositions for use in treating cancer, or method of treating cancer, may also include a combinational therapy with at least one additional active ingredient such as anti-cancer medication.

[0146] According to some embodiments, the composition including the aNPs is administrated with at least one additional active ingredient, said additional active ingredient comprises an anti-cancer medication.

[0147] In some embodiments, the composition including the aNPs is administrated concomitantly with and / or sequentially to the at least one additional active ingredient. Each possibility is a separate embodiment.

[0148] In some embodiments, to further facilitate selective targeting of the aNPs towards cancer cells, the aNPs may be modified with targeting moieties, such as antibodies and antibody fragments, nucleic acids aptamers, peptides, carbohydrates, and small molecules, which can selectively bind to tumor-specific antigens or receptors.

[0149] According to another aspect, the present disclosure provides a method for treating, attenuating, and / or preventing progression of cancer in a subject in need thereof, the method includes parenterally administering to the subject a therapeutically effective amount of a composition comprising alga(e) derived nanoparticles (aNPs) including non-soluble and amphiphilic alga(e) components. In some embodiments the aNPs have an average particle diameter in the range of between 50 nm and about 650 nm.

[0150] As used herein, the term “alga(e)” refers to a large and diverse group of photosynthetic organisms and should be broadly interpreted as referring to any alga(e) species / genus belonging to red algae, brown algae, green algae, and including blue-green algae (cyanobacteria), whether multicellular or unicellular, and whether edible or non-edible.

[0151] In some embodiments, the algae may include species or genus belonging, for example, but not limited to red algae (division Rhodophyta including class Florideophyceae), brownalgae (including class Phaeophyceae), green algae (including division Chlorophyta), and bluegreen algae (cyanobacteria including the family Spirulinaceae).

[0152] In some embodiments, the alga(e) may be selected from any one of the 14 types of the edible alga(e) species / genus disclosed hereinbelow in Table 1.

[0153] Table 1: Algae-based NPs (aNPs) may be prepared from edible alga belonging to different genera and species.

[0154] Type of aNPs Type of alga

[0155] Species Genus (or Class) Chondracanthus Chondracanthus Chondracanthus

[0156] Chamissoi aNPs Chamissoi (red algae of class Florideophyceae)

[0157] (C. chamissoi)

[0158] (C.Agardh) Kiitzing

[0159] Gracilaria aNPs Gracilaria

[0160] (red algae of class Florideophyceae) Irish Moss aNPs Chondrus Crispus Chondrus

[0161] (red algae of class Florideophyceae) (C. Crispos)

[0162] Wakame aNPs Undaria Pinnatifida Undaria

[0163] (brown algae of the class Phaeophyceae) (U. Pinnatifida)

[0164] Sargassum Sargassum

[0165] (brown algae of the class Phaeophyceae) Seaweed aNPs

[0166] Kelp Laminaria Laminaria Digitata Laminaria

[0167] Digitata aNPs (L. Digitata) (brown algae of the class Phaeophyceae) Kombu / Laminaria (brown algae of the class (Konbu) aNPs Phaeophyceae)

[0168] Ecklonia Cava Ecklonia Cava Ecklonia

[0169] (E. Cava) (brown algae of the class Phaeophyceae) Giant Kelp aNPs Macrocystis Pyrifera Macrocystis

[0170] (M. Pyrifera) (brown algae of the class Phaeophyceae) Dulse aNPs Palmaria Palmata Palmaria

[0171] (P. Palmata) (red algae of class Florideophyceae) Gigartina Red Gigartina Skottsbergii Gigartina

[0172] Marine aNPs (G. Skottsbergii) (red algae of class Florideophyceae) Spirulina aNPs Arthrospira Platensis Arthrospira

[0173] (A. Platensis) (blue-green algae; cyanobacteria) Chlorella aNPs Chlorella Vulgaris Chlorella

[0174] (C. Vulgaris) (green algae of the division Chlorophyta) H. pluvialis Haematococcus pluvialis Haematococcus

[0175]

[0176] aNPs (H. pluvialis) (green algae of the division Chlorophyta)In some embodiments, the alga(e) includes edible alga(e).

[0177] In some embodiments, the algae includes one or more red alga(e) species or genus belonging to the class Florideophyceae. Each possibility is a separate embodiment.

[0178] In some embodiments, the algae include one or more brown alga(e) species or genus belonging to the class Phaeophyceae. In some embodiments, the algae comprise brown one or more alga(e) species or genus belonging to the order Phaeophyceae. Each possibility is a separate embodiment.

[0179] In some embodiments, the algae include green alga(e) one or more species or genus belonging to the division Chlorophyta. Each possibility is a separate embodiment. In some specific embodiments, the algae include one or more species or genus of cyanobacteria.

[0180] In some other specific embodiments, the alga(e) belongs to a genus or class selected from a group of consisting of one or more of: Spirulina Arthrospira, Chondr acanthus, Gracilaria, Chondrus, Undaria, Sargassum, Laminaria, Ecklonia, Macrocystis, Palmaria, Gigartina, Chlorella, and Haematococcus, or any combination thereof. Each possibility is a separate embodiment.

[0181] In further specific embodiments, the algae include one or more species belonging to the genus Arthrospira (herein referred to as spirulina), including Arthrospira platensis species.

[0182] In some embodiments, the aNPs have an average particle diameter of less than about 1000 nm and / or surface charge more negative than about -10 mV.

[0183] In addition, in some embodiments, the term used herein “made of alga(e)” may refer to the nanoparticles (NPs) being produced from alga(e) cells / biomass as starting material (including whole / complete cells or any processed form thereof, such as, but not limited to algal dry powder, dissolved alga cell / biomass (i.e., algal cell suspension), or algal cell lysate), which is subjected to lysis and homogenization and fractionation, while a certain fraction, namely a fraction including non-soluble and amphiphilic components (i.e., structures and molecules), mainly membranes and proteins, are continuously being collected / preserved, as this isolated non-soluble and amphiphilic fraction contain purified aNPs (reference is made to the method of preparation of aNPs).

[0184] Further, the aNPs may be prepared / extracted solely or essentially from alga(e) cells / biomass as starting material. In some embodiments, the nanoparticles (NPs) are prepared / made substantially or solely from alga(e) cells / biomass as starting material.In some embodiments, nanoparticles (NPs) made of alga(e) (aNPs) consist essentially of non-soluble and amphiphilic alga(e) components of the alga(e) / alga(e) cells / biomass, including membranes and membrane proteins.

[0185] Also, the term made of alga(e) may refer to the nanoparticles (NPs) and the method of preparing them, being produced / extracted solely / substantially from alga(e) cells / biomass as starting material (including any processed form of the alga(e) cells / biomass such as algal dry powder, dissolved algal cells / biomass, or algal cell lysate), and in some embodiment excluding any addition (i.e., external addition to the alga(e) cells / biomass at any stage of their growth or after harvesting the cells) of natural or synthetic polymers, including biopolymers.

[0186] In some embodiments, the alga(e) comprises an edible alga(e). In some embodiments, the alga(e) consists essentially of edible alga(e). In some embodiments, the alga(e) consists of edible alga(e). In some embodiments, the alga(e) / alga(e) cell / biomass is substantially made of an edible alga(e). Each possibility is a separate embodiment.

[0187] As used herein, the term “edible” may refer to algal species known in the art as algae that can be eaten or used for culinary purposes, including for example, but not limited to those listed in Table 1 above.

[0188] In some embodiments, the alga(e) / alga(e) cell / biomass comprises, or consists essentially of one or more types of alga(e) selected from a group consisting of Chondracanthus Chamissoi, Gracilaria, Irish Moss, Wakame, Sargassum Seaweed, Kelp Laminaria Digitata, Kombu, Ecklonia Cava, H. pluvialis, Giant Kelp, Dulse, Gigartina Red Marine, Spirulina Arthospira, and Chlorella, or any combination thereof. Each possibility is a separate embodiment.

[0189] In some embodiments, the alga(e) / alga(e) cell / biomass comprises, or consists essentially of, one or more types of alga(e) selected from a group consisting of Chondracanthus Chamissoi, Gracilaria, Irish Moss, Wakame, Sargassum Seaweed, Kelp Laminaria Digitata, Kombu, Ecklonia Cava, Giant Kelp, Dulse, Gigartina Red Marine, Spirulina Arthospira, and Chlorella, or any combination thereof. Each possibility is a separate embodiment.

[0190] In some embodiments, the alga(e) comprises one or more of a unicellular alga(e). In some embodiments, the alga(e) comprises one or more species of cyanobacteria.

[0191] In some embodiments, the cyanobacteria comprise (or consists essentially of) one or more species belonging to the genus Arthrospira, including but not limited to Arthrospiraplatensis (also known as spirulina Arthrospira platensis). Each possibility is a separate embodiment.

[0192] In some embodiments, the alga(e) comprises (or consists essentially of) one or more species belonging to the genus Arthrospira, including but not limited to Arthrospira platensis (also known as spirulina Arthrospira platensis).

[0193] In some embodiments, the alga(e) comprises Spirulina Arthospira. In some embodiments, the alga(e) comprises one or more species belonging to Spirulina Arthospira In some embodiments, the alga(e) consists essentially of Spirulina Arthrospira. In some embodiments, the alga(e) consists essentially of one or more species belonging to Spirulina Arthrospira. In some embodiments, the one or more species belonging to Spirulina Arthospira comprises Spirulina Arthospira Platensis. In some embodiments, the one or more species belonging to Spirulina Arthospira consists essentially of Spirulina Arthospira Platensis.

[0194] In some embodiments, the alga(e) comprises (or consists essentially of) Spirulina Arthospira Platensis. In some embodiments, the alga(e) consists of Spirulina Arthospira Platensis.

[0195] According to some embodiments, the structural characteristics of the aNPs are advantageous. For example, a small particle nano-size can be advantageous for solubility, drug loading, and contact-mediated interactions such as cellular uptake via endocytosis. Thus, the nano-sized characteristic of the aNPs implies that any of the tested aNPs may be an effective carrier of active ingredients to cancer cells, (especially, for example, Spirulina NPs that had the smallest particle size of 126 ± 2 nm among the tested algae).

[0196] According to some embodiments, the aNPs structural properties are herein characterized, including its size, shape, zeta potentials (surface charge), protein content, and PDI.

[0197] According to some embodiments, the aNPs may have characteristic shape and size. In some embodiments, the aNPs have a spherical shape. In some embodiments, said spherical shape comprises a shell and an inner core. In some embodiments, said spherical shape comprises a shell having water-insoluble and amphiphilic components, and an inner hydrophilic core.According to some embodiments, the aNPs have an average particle diameter of less than about 650 nm and / or surface charge more negative than about -10 mV. each possibility is a separate embodiment.

[0198] In some embodiments, the average particle diameter is less than about 1000 nm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 550 nm, less than about 500 nm, less than about 450 nm, less than about 400 nm, less than about 350 nm, preferably less than about 300 nm, less than about 275 nm, less than about 250 nm, less than about 225 nm, less than about 200 nm, less than about 190 nm, less than about 180 nm, less than about 170 nm, less than about 160 nm, less than about 150 nm, less than about 140 nm, less than about 130 nm, or less than about 100 nm. Each possibility is a separate embodiment.

[0199] According to some embodiments, the average particle diameter is less than about 650 nm. In some embodiments, the average particle diameter is less than 146 nm. In some embodiments, the average particle diameter is about 126 nm. In some embodiments, the average particle diameter is between about 100 nm and 146 nm.

[0200] In some embodiments, the average particle diameter is between about 60 nm and about 1000 nm, between about 100 nm and about 1000 nm, between about 100 nm and about 700 nm, or between 100 nm and about 650 nm, or between 100 nm and 157 nm, or between 100 nm and 146 nm, or between about 100 nm and 135 nm, or between 80 nm and 146 nm, or between about 126 nm and about 650 nm, or in the range between 80 nm and about 130 nm, or in the range between 105 nm and 145 nm or in the range between 120 nm and 130 nm. Each possibility is a separate embodiment.

[0201] In some embodiments, the aNPs have an average particle diameter in the range between about 100 nm and about 650 nm and / or a surface charge in the range between about -10 mV and about -45 mV. Each possibility is a separate embodiment.

[0202] In some embodiments, the aNPs have an average particle diameter in the range between about 100 nm and about 200 nm and / or a surface charge in the range between about -30 mV and about -45 mV. Each possibility is a separate embodiment.

[0203] In some embodiments, the aNPs have an average particle diameter in the range between about 100 nm and about 145 nm and / or a surface charge in the range between about -30 mV and about -45 mV. Each possibility is a separate embodiment. In some embodiments, the alga(e) comprises Spirulina Arthospira Platensis.In some embodiments, the surface charge of the aNPs is more negative than about -10 mV, more negative than about -12 mV, more negative than about -15 mV, more negative than about -17 mV, more negative than about -20 mV, more negative than about -22m V, more negative than about -25 mV, more negative than about -27 mV, more negative than about -30 mV, more negative than about -35 mV, more negative than about -40 mV. Each possibility is a separate embodiment.

[0204] In some embodiments, the aNPs comprise a surface charge in the range of between about -5 mV and about -55 mV, or between about -10 mV and about -38 ± 3 mV. Each possibility is a separate embodiment.

[0205] In some embodiments, the aNPs comprise a surface charge in the range of between about -10 mV and about -45 mV, or between about -15 mV and about -45 mV, or between about -30 mV and about -45 mV, or between -30 mV and -45 mV. Each possibility is a separate embodiment.

[0206] In some embodiments, the aNPs comprise a surface charge of about -38 ± 3 mV. In some embodiments, the Spirulina Arthospira aNPs comprise a surface charge of about -38 ± 3 mV. In some embodiments, the alga(e) comprises Spirulina Arthospira Platensis.

[0207] According to some embodiments, the aNPs are characterized by a poly dispersity index (PDI) of less than about 0.7, less than about 0.6, less than about 0.5, less than about 0.45, less than about 0.4, less than about 0.35, less than about 0.3, less than about 0.28, less than about 0.26, less than about 0.24, less than about 0.22, less than about 0.2, less than about 0.19, less than about 0.18, less than about 0.17, less than about 0.16, less than about 0.15, or less than about 0.14. Each possibility is a separate embodiment.

[0208] According to some embodiments, the aNPs are characterized by a poly dispersity index (PDI) of between about 0.05 and about 0.7, between about 0.05 and about 0.5, or between about 0.12 and about 0.5, or between about 0.10 and about 0.18.

[0209] According to some embodiments, the aNPs comprise a poly dispersity index (PDI) of about 0.14. In some embodiments, the alga(e) comprises Spirulina Arthospira Platensis. According to some embodiments, the aNPs comprise a protein content (mg / mL) of more than about 0.01 mg / mL.

[0210] According to some embodiments, the aNPs comprise a protein content (mg / mL) of more than about 0.01 mg / mL, than about 0.1 mg / mL, more than about 0.4 mg / mL, more thanabout 0.6 mg / mL, more than about 1.0 mg / mL, 2.4 mg / mL, more than about 2.6 mg / mL. Each possibility is a separate embodiment.

[0211] According to some embodiments, the aNPs comprise a protein content (mg / mL) of between about 0.01 mg / mL and about 3.0 mg / mL. According to some embodiments, the aNPs comprise a protein content of about 2.6 mg / mL.

[0212] In some embodiments, there is provided a method preparing alga(e) nanoparticles (aNPs), including the steps of: (i) obtaining alga(e) cells / biomass; (ii) homogenizing the alga(e) cells / biomass in water to receive a suspension of cell lysate; (iii) centrifuging the suspension and collecting supernatant, said supernatant comprises a fraction of non-soluble and amphiphilic components; (iv) applying / loading the supernatant onto a density gradient and subjecting it to ultra-centrifugation; (v) collecting a fraction comprising non-soluble and amphiphilic components on top of the density gradient; thereby obtaining Alga(e) nanoparticles (aNPs) comprising non-soluble and amphiphilic membranes and membrane proteins of the alga(e); wherein the aNPs have an average particle diameter of in a range of between about 100 nm and about 650 nm.

[0213] In some embodiments, homogenizing the alga(e) cells / biomass in water comprises homogenizing the alga(e) cells / biomass in water-based buffer such as but not necessarily limited to PBS or HEPES. Each possibility is a separate embodiment.

[0214] In some embodiments, the nanoparticles comprise membranes and membrane proteins, including for example, glycosylated or other modified forms thereof, derived from the obtained alga(e) cells / biomass.

[0215] As used herein, the terms “isolated" and “purified” may be used interchangeably to refer to the aNPs to mean either that they are: 1) separated from at least some of the components with which it is usually associated in nature; and / or 2) prepared or purified by a process that involves the hand of man; and / or 3) not occurring in nature.

[0216] In some embodiments, the method includes collecting a fraction comprising non-soluble and amphiphilic components of alga(e). In some embodiments, non-soluble and amphiphilic components or molecules including membranes lipids, phospholipids, and membrane proteins.

[0217] According to some embodiments, the alga(e) cells / biomass belongs to a genus selected from a group of consisting of one or more of: Spirulina Arthrospira, Chondr acanthus,Gracilaria, Chondrus, Undaria, Sargassum, Laminaria, Ecklonia, Macrocystis, Palmaria, Gigartina, Chlorella, and Haematococcus, or any combination thereof.

[0218] According to some embodiments, the alga(e) cells / biomass belongs to Spirulina Arthrospira.

[0219] In some embodiments, the Spirulina Arthrospira comprises one or more species selected from Arthrospira Platensis, Arthospira Maxima, Arthrospira ardissonei, Arthrospira erdosensis, Arthrospira fusiformis, Arthrospira indica, Arthrospira innermongoliensis, Arthospira jenneri, Arthrospira massartii, or any combination thereof

[0220] In some embodiments, the Spirulina Arthospira comprises Spirulina Arthrospira Platensis.

[0221] In some embodiments, the obtained aNPs have an average particle diameter in the range between about 100 nm and 200 nm and / or a surface charge in the range between about -30 mV and about -45 mV.

[0222] In some embodiments, the homogenization of the alga(e) cells / biomass includes sonication.

[0223] In some embodiments, the sonication comprises an ultrasonic transducer at an amplitude range between about 40% - 100% and / or at a range between about 40% - 60% duty cycle.

[0224] In some embodiments, the sonication comprises an ultrasonic transducer at an amplitude range between about 60% - 100% and / or at a range between about 40% - 60% duty cycle.

[0225] In some embodiments, the density gradient comprises one or more of sucrose cushion, CsCl cushion, D2O density gradient, Ficoll cushion, glycerol cushion, sorbitol cushion, and percoll cushion, or any combination thereof.

[0226] In some embodiments, the density gradient comprises between about 50% and about 70% sucrose solution, and wherein the collecting of the fraction comprising the non-soluble and amphiphilic components comprises collecting the fraction on top of the sucrose gradient.

[0227] In some embodiments, the ultracentrifugation comprises a centrifugal force of at least about 50,000 g.In some embodiments, the obtained aNPs comprise a plurality of different membrane proteins of the alga(e).

[0228] In some embodiments, there are provided alga(e) nanoparticles (aNPs), obtained or obtainable by the method of preparation.

[0229] In some embodiments, there are provided alga(e) nanoparticles (aNPs), comprising non-soluble and amphiphilic alga(e) components isolated from one or more species belonging to Spirulina Arthrospira, for use in treating, attenuating, and / or preventing progression of cancer in a subject in need thereof; wherein the aNPs have an average particle diameter in the range between 100 nm and 200 nm and / or surface charge ranging between -10 mV and -45 mV, preferably the one or more species belonging to Spirulina Arthrospira comprises Spirulina Arthospira Platensis.

[0230] In some embodiments, alga(e) cells / biomass is substantially devoid by depletion of one or more substances or polymers, and wherein the alga(e) cells / biomass comprise cultures, harvested, dry, dissolved, and lysed forms of the cells / biomass, or any combination thereof. Each possibility is a separate embodiment.

[0231] In some embodiments, the alga(e) cells / biomass is substantially devoid of polymer addition, wherein the addition comprises externally providing the polymer to the alga(e) cells / biomass, and wherein the alga(e) cells / biomass comprise cultured or harvested cells, dry, dissolved, and lysed forms of the cells / biomass. Each possibility is a separate embodiment.

[0232] As a non-limiting example, in some embodiments, H. pluvialis alga(e) cells / biomass may be devoid of the lipid-soluble keto-carotenoid pigment Astaxanthin by depletion.

[0233] According to related embodiments, the alga(e) / alga(e) cells / biomass are not being added (i.e., external addition to the alga(e) cells / biomass at any stage of their growth or after harvesting the cells) with any polymer, natural or synthetic, that contribute / facilitate / improves the structure of the produced aNPs.

[0234] In some embodiments, the alga(e) cells / biomass include whole / complete cells or any processed form thereof, such as, but not limited to algal dry powder, dissolved alga(e) cell / biomass, or algal cell lysate. Each possibility is a separate embodiment.

[0235] In accordance with some embodiments, the alga(e) cells / biomass consists of alga(e) including dry, dissolved, and lysed forms of the alga(e). In some embodiments, the alga(e)consists essentially of alga(e) cells / biomass, including dry, dissolved, and lysed forms of the cells / biomass.

[0236] In some embodiments, the density gradient (step (iv) of the method)) comprises about 60% sucrose solution, and wherein the collection of the fraction comprising the non-soluble and amphiphilic components or molecules comprising membranes and membrane proteins (step (v) of the method) comprises collecting the fraction on top of the about 60% sucrose solution.

[0237] In some embodiments, the density gradient (step (iv) of the method)) comprises at least about 40% sucrose solution. In some embodiments, the density gradient (step (iv) of the method) comprises between about 40% sucrose solution and about 80% sucrose solution, or between about 50% sucrose solution and about 70% sucrose solution, or between about 55% sucrose solution and about 65% sucrose solution. Each possibility is a separate embodiment.

[0238] In some embodiments, the density gradient is based on one or more of sucrose cushion, CsCl cushion, D2O density gradient, Ficoll cushion, glycerol cushion, sorbitol cushion, and percoll cushion, or any combination thereof. Each possibility is a separate embodiment. In some embodiments, the collecting of the fraction comprising the non-soluble and amphiphilic components comprises collecting the fraction on top of the gradient.

[0239] In some embodiments, homogenization of alga(e) cells / biomass comprises sonication / ultrasound insonation. In some embodiments, homogenization of alga(e) cells / biomass consists of sonication.

[0240] In some embodiments, homogenizing of alga(e) cells / biomass comprises sonication in water or water-based buffer. Each possibility is a separate embodiment.

[0241] In some embodiments, sonication comprises transducer at about 60% amplitude. In some embodiments, sonication comprises transducer at a range between 40% - 80% amplitude. In some embodiments, sonication comprises transducer at a range between 50% - 70% amplitude. In some embodiments, sonication comprises transducer at a range between 55% -65% amplitude.

[0242] In some embodiments, sonication comprises transducer at about 50% duty cycle. In some embodiments, sonication comprises transducer at a range between 30% - 70% duty cycle. In some embodiments, sonication comprises transducer at a range between 40% - 60% dutycycle. In some embodiments, sonication comprises transducer at a range between 45% - 55% duty cycle.

[0243] In some embodiments, the homogenization of the alga(e) cells / biomass comprises sonication, wherein insonation comprises a transducer at a range between 55% - 65% amplitude and / or at a range between 45% - 55% duty cycle. Each possibility is a separate embodiment.

[0244] In some embodiments, sonication comprises transducer at a range between 55% - 65% amplitude and / or transducer at a range between 45% - 55% duty cycle. Each possibility is a separate embodiment.

[0245] In some embodiments, centrifugation is performed at a temperature of about 4°C. In some embodiments, centrifugation is performed at a temperature in the range between 4°C and 20°C. In some embodiments, centrifugation is performed at a temperature in the range between 4°C and 18°C. In some embodiments, centrifugation is performed at a temperature in the range between 4°C and 16°C.

[0246] In some embodiments, centrifugation comprises centrifugal force in the range of between about 3000 g and about 15,000 g. In some embodiments, the method comprises one or more centrifugation steps, each comprises a centrifugal force in the range of between about 3000 g and about 15,000 g.

[0247] In some embodiments, the alga(e) comprises one or more alga(e) selected from Chondracanthus Chamissoi, Gracilaria, Irish Moss, Wakame, Sargassum Seaweed, Kelp Laminaria Digitata, Kombu, Ecklonia Cava, H. pluvialis, Giant Kelp, Dulse, Gigartina Red Marine, Spirulina Arthrospira and Chlorella, or any combination thereof. Each possibility is a separate embodiment.

[0248] According to some embodiments, the association / encapsulation comprises mixing the obtained aNPs with one or more active ingredients and subjecting the mixture to sonication.

[0249] According to some embodiments, the association / encapsulation comprises mixing the obtained aNPs with one or more active ingredients, subjecting the mixture to sonication, and ultra-centri fugati on .

[0250] In some embodiments, the aNPs are spherical. In some embodiments, the aNPs are spherical and have a shell and a hydrophilic core.

[0251] According to some embodiments, demonstrated herein is the use of aNPs, particularly sNPs, as selective and effective anticancer therapy. In some embodiments, the sNPs showed asignificant cytotoxic effect on a range of cancer cell lines, including TR-146 buccal cancer, Caco-2 and HT-29 colorectal cancer, and MCF-7 breast cancer cells, while having minimal impact on the non-cancerous MCF-10A and HDFn cells. The observed selectivity indicates that sNPs could target cancer cells without affecting normal cells, reducing the likelihood of adverse effects typically associated with conventional cancer treatments.

[0252] As exemplified herein, the flow cytometry analysis confirmed a higher uptake of sNPs by cancer cells than non-cancerous cells. Without wishing to be bound by any theory or mechanism of action, this supports the notion that aNPs, specifically sNPs, are internalized more efficiently by cancer cells due to their enhanced endocytic activity. The evaluation of endocytosis inhibitors revealed that caveolae-mediated endocytosis plays a predominant role in sNPs uptake, with macropinocytosis also contributing to cancer cells to a lesser extent.

[0253] The following examples are presented in order to illustrate some embodiments of the invention more fully. They should in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0254] EXAMPLES

[0255] Materials and Methods

[0256] Materials - The cell lines MCF-10A, MCF-7, Caco-2, HT-29, and TR-146 were obtained from ATCC®. For culturing the MCF-10A cells, the growth medium included StableCell™ DMEM / F12, Insulin solution human, Cholera Toxin from Vibrio cholerae, Hydrocortisone solution, Epidermal Growth Factor Protein (Human recombinant), and 0.05% Trypsin-0.02% EDTA. The remaining cell lines (MCF-7, Caco-2, HT-29, and TR-146) were cultured using Dulbecco’s Modified Eagle Medium (DMEM), L-Glutamine (L-Glu), and Penicillin-Streptomycin (P / S). Phosphate-buffered saline (PBS) and fetal bovine serum (FBS) for all cell lines were obtained from Biological Industries (Kibbutz Beit Haemek, Israel). The Spirulina (Arthospira platensis) powder used to produce the Spirulina NPs (sNPs) was purchased from NutriCargo, USA. A sucrose solution used to produce and purify sNPs was also sourced from Sigma-Aldrich Israel. Endocytosis inhibitors, methyl-P-cyclodextrin (MpCD) and 5-(N-ethyl-N-isopropyl) amiloride (EIPA), were purchased from Sigma-Aldrich Israel. The Cell Proliferation Kit II (XTT), the fluorescent dye Dil used for sNPs labeling, DAPI (300 nM) fornuclear staining, and 4% paraformaldehyde (PF A) for fixation were all obtained from Sigma-Aldrich Israel.

[0257] Preparation of Alga derived NPs (aNPs) made of Spirulina (sNPs) - To produce the sNPs, 0.6 g of Spirulina (Arthospira platensis) powder was accurately weighed and mixed with 240 mL of double-distilled water. The suspension was then sonicated (Q700, Qsonica) in an ice bath using a 1.3 cm diameter probe at full amplitude (100%) with a 50% duty cycle for 2 min and centrifuged at 3,200 * g for 5 minutes at 4°C. The resulting supernatant containing the disrupted cell components was subjected to a second sonication and centrifugation step under identical conditions to refine the particle size further. Afterward, the supernatant containing the sNPs was centrifuged at 10,000 x g for 60 minutes at 4°C. The supernatant was carefully transferred onto a 6 mL of 60% sucrose layer and underwent ultracentrifugation (Sorvall wX, Thermo Scientific, Israel) at 150,000 x g for 45 minutes at 4°C. Approximately 4 mL of the sNPs solution was gently extracted from the layer above the sucrose. The sNPs solution was then frozen at -80°C and subsequently placed in a lyophilizer (Labconco FreeZone 4.5 L) to convert into powder form, which was later stored in a refrigerator for further use.

[0258] Evaluation of Spirulina NPs (sNPs) impact on cell mortality - Five cell types (MCF-10 A, MCF-7, Caco-2, HT-29, and TR-146) were cultured in a 96-well plate (Greiner, Monroe, NC, USA), seeding approximately 8,000 cells per well to ensure 70-80% confluence after 24 hours of incubation. The cells were washed twice with PBS. Before incubation with the cells, the sNPs solutions were filtered to ensure sterility using a 0.22 pm PVDF -membrane syringe filter (Romical Israel). The filtered sNPs were then diluted in the growth medium to various concentrations (25, 50, 125, 250, 375, and 500 mg / mL), and 100 pL of this solution was added to the cells. The incubation times were set at 3, 6, 12, 24, and 48 h. Following the incubation, 50 pL of the XTT reagent was added to each well. This reagent was prepared by mixing 5 mL of XTT labeling reagent with 0.1 mL of electron coupling reagent, as required for one microplate assay. After an additional incubation period of 4.5 hours with the XTT reagent, absorbance was measured at a wavelength of 500 nm using a microplate reader (Infinite M200 Tecan). The mortality rate for each cell type was calculated relative to the control group (cells cultured with growth medium only).

[0259] Assessment of endocytosis - To assess the role of endocytosis in sNPs toxicity mechanism of action, two inhibitors, MpCD at a concentration of 40 pM and EIPA at 10 mM, were added to the cells and incubated at 37°C for 40 min. Following this incubation, the sNPs were added at two concentrations: 125 mg / mL and 500 mg / mL. The total volume per well wasadjusted to 100 pL with the growth medium. After treatment with these inhibitors, cell mortality was evaluated using the XTT assay under the same conditions as described above.

[0260] Preparation of Dil-Labeled sNPs - sNPs were labeled with a fluorescent lipophilic dye, Dil, to assess cellular uptake. A 0.1 mg / mL Dil solution (100 pL) was added to 10 mL of 500 mg / mL sNPs, followed by incubation for 30 minutes at 37°C. The Dil-labeled sNPs were then purified by ultrafiltration at 150,000 x g for 45 minutes at 4°C, and the pellet was collected for further experimentation.

[0261] Evaluation of Cellular Uptake by Flow Cytometry and Fluorescence Microscopy - The cell lines MCF-10A, MCF-7, Caco-2, HT-29, and TR-146 were seeded in a 6-well plate (Greiner, Monroe, NC, USA) at a density of 1 x io5cells per well and cultured overnight at 37°C until they reached 70-80% confluence. The culture medium was then replaced with fresh medium containing 500 pg / mL Dil-labeled sNPs, and the cells were incubated for an additional 3 hours to allow for uptake. After this incubation, the cells were washed twice with PBS, and 1 mL of trypsin containing 300 nM DAPI was added to each well for 10 min to detach the cells. The cells were centrifuged at 500 x g for 12 minutes, and the pellet was collected .The cells were fixed with 4% PFA and transferred to a 96-well plate on ice. FACS instrument (CytoFLEX Beckman Coulter, Indianapolis, IN, USA) was then performed to quantitatively assess the uptake and Mean fluorescence intensity (MFI) of Dil-labeled sNPs.

[0262] To further confirm the cellular uptake of sNPs, TR-146 buccal cancer cells were incubated with 500 pg / mL Dil-labeled sNPs for 3 hours. Following incubation, the cells were washed with PBS, fixed with 4% PFA, and stained with DAPI to visualize the nuclei. The cells were then transferred onto a glass slide for fluorescence microscopy (Olympus™ CKX53) analysis. Images were captured to observe the red fluorescence from the Dil-labeled sNPs and the blue fluorescence from the DAPI-stained nuclei. The captured fluorescence images were processed for qualitative analysis using Imaged version ij 154 software. The red (Dil-labeled sNPs) and blue (DAPI-stained nuclei) channels were merged to confirm the co-localization of sNPs within the TR-146 cells.

[0263] Encapsulation of Fluorescent Molecule in aNPs - each of six types of aNPs made of Spirulina Arthospira, Kombu, giant Kelp, Sargassum, Gracilaria, and Chondracanthus Chamissoi were encapsulated with 3 different FITC-dextran (FD) having molecular weights (MWs) of 4 kDa, 40 kDa, and 250 kDa were. First, 2 mL of the aNPs were mixed with 0.1 mL of 0.5 mg / mL FD40 and 7.4 mL of PBS. Then, the mixture was placed in an ice bath andsonicated using a microtip (0 0.3 cm) ultrasound transducer at 60% amplitude and 75% DC for two minutes. After sonication, the solution was refrigerated for 10 minutes. The sonication and refrigeration were repeated one more time. Then, the FD-loaded aNPs were harvested. For the measurement of encapsulation efficiency (EE), first, 200 pL of the (final) supernatant was transferred into a 96-well flat black plate (by Greiner) to determine the amount of free FD and analyzed using a fluorometer (Infinite M200 Tecan) at excitation and emission wavelengths of 490 nm and 525 nm, respectively.

[0264] Statistical Analysis - GraphPad Prism (version 10) was used to analyze the data. The Shapiro-Wilk test was applied to check data normality, while the Brown-Forsythe test assessed the homogeneity of variances. Group differences were evaluated using a one-way ANOVA analysis with Fisher’s Least Significant Difference (LSD) post hoc test at a = 0.05. A one-way ANOVA analysis with Dunnett was used to compare one variable. Presented values are the mean ± standard deviation (SD) of a minimum of n = 3 per condition. The IC50 values were calculated using non-linear regression analysis (log(inhibitor) vs. response - variable slope) with GraphPad Prism, fitting the concentration-response data to determine the concentration of sNPs required to inhibit 50% of cell viability.

[0265] Characterization of Spirulina aNP’s protein content - the non-soluble and amphiphilic fraction collected from the layer above the 60% sucrose solution of the Arthrospira platensis sample after ultracentrifugation (see method of preparation hereinabove somewhere) were digested by trypsin, analyzed by LC-MS / MS on Q-Exactive HF (Thermo) and identified by Discoverer 2.4 software with the search algorithms Sequest (Thermo) against the Arthrospira platensis section or the human proteome from the Uniprot database, and a decoy database (in order to determine the false discovery rate). All the identified peptides were filtered with high confidence. High-confidence peptides have passed the 1% FDR threshold. (*FDR =false discovery rate, is the estimated fraction of false positives in a list of peptides). A protein identified with a single peptide was not considered as a certain identification. Semi -quantitation was done by calculating the peak area of each peptide. The abundance of the protein is the sum of all associated peptide group abundances.

[0266] Example 1 - algal NPs (aNPs) promote cancer cell mortality

[0267] The cytotoxic effect of nanoparticles (NPs) derived from the algae Spirulina Arthospira platensis (sNPs) was evaluated for the cancer cell lines TR-146 (human buccal carcinoma), Caco-2 (human colorectal adenocarcinoma), HT-29 (human colorectal adenocarcinoma), andMCF-7 (human breast cancer), as well as for the non-cancerous control cell line MCF-10A (epithelial cell line, control).

[0268] The cytotoxic effect was evaluated by measuring cell mortality and the results for the TR-146, Caco-2, HT-29, MCF-7, MCF-10A and HDFn cell lines are presented in FIGs. 1A-1E, FIGs. 2A-2E, FIGs. 3 A-3E, FIGs. 4A-4E, FIGs. 5A-5E and FIGs. 6A-6E, respectively.

[0269] A statistical test was performed comparing all groups against the selected concentration at each time point, where this concentration represented the one that resulted in the highest observed mortality. Concentrations above this level showed no significant increase in mortality, while concentrations below it demonstrated a statistically significant difference.

[0270] As can be seen in FIGs. 1A-1E, TR-146 buccal cancer cells showed the highest cytotoxic effect among the tested cell lines. TR-146 cells exhibited approximately 60% to 80% mortality when exposed to sNPs concentrations of 250 mg / mL for 3 hours (FIG. 1A), and approximately 60% to 80% mortality at concentrations of 125 mg / mL sNPs and above, across all exposure times above 3 hours, with no further increase in mortality at the higher concentrations of 250, 375, and 500 mg / mL (FIG. 1B-1E).

[0271] As can be seen in FIGs. 2A-2E, Caco-2 cells required exposure to higher sNPs concentrations of 375 mg / mL to reach similar cytotoxic effect of approximately 60% to 80% mortality at 3 hours (FIG. 2A), while increasing the exposure time to sNPs correlated with reduced concentrations required to reach a similar cytotoxic effect at, so that at 6 hours of exposure, and on, a similar cytotoxic effect of approximately 60% to 80% mortality was reached at concentrations of 250 mg / mL (FIGs. 2B-2E) and at 24 hours of exposure, and on, concentrations of 125 mg / mL were sufficient to reach approximately 60% to 80% mortality (FIGs. 2D-2E)

[0272] As can be seen in FIGs. 3A-3E, HT-29 cells also demonstrated the highest mortality at sNPs concentrations of 375 mg / mL reaching a cytotoxic effect of approximately 30% to 60% mortality at 3 and 6 hours of exposure to the sNPs (FIGs. 3A-3B), whereas 12, 24, and 48 hours of exposure to sNPs concentrations of 250 mg / mL were sufficient for the cells to reach approximately 60% to 70% mortality (FIGs. 3C-3E).

[0273] As can be seen in FIGs. 4A-4E, MCF-7 breast cancer cells were slightly affected by 3 hours of exposure to sNPs (FIG.4A), whereas increasing the exposure time to sNPs correlated with reduced concentrations required to reach a similar cytotoxic effect at, so that at 6 and 12 hours of exposure mortality increased and reached approximately 60% at sNPs concentrationsof 375 mg / mL (FIGs. 4B-4C), at 24 hours of exposure similar mortality was achieved by 250 mg / mL (FIG. 4D), and at 48 hours of exposure 125 mg / mL were sufficient to reach the same 60% mortality (FIG4E).

[0274] In stark contrast, as can be seen in FIGs. 5A-5E, the non-cancerous MCF-10A cells showed no significant cytotoxic effects across all concentrations and exposure times1highlighting the selective cytotoxicity of sNPs towards cancerous cells.

[0275] Likewise, as can be seen in FIGs. 6A-6E, the non-cancerous HDFn cells showed no significant cytotoxic effects across all concentrations and exposure times^highlighting the selective cytotoxicity of sNPs towards cancerous cells.

[0276] These results strongly suggest that sNPs have selective toxicity towards cancerous cells, offering promising potential for cancer therapy.

[0277] Overall, exposing cancerous cells to sNPs resulted in a dose-and time-dependent increase in mortality, ranging from approximately 30% to 80%.

[0278] To further assess the effect of increased exposure time of cancerous and noncancerous cells to sNPs, IC50 was calculated based on the above data and plotted to show the dependency of IC50 values for the tested cell lines on exposure times.

[0279] As can be seen in FIG. 7, presenting the dependency between the different cell lines IC50 values and the exposure time to spirulina aNPs, all four tested cancer cell lines exhibited a decrease in IC50 values over time, indicating increased sensitivity to sNPs with prolonged exposure.

[0280] TR-146 and Caco-2 cancer cells yielded the lowest IC50 values (highest sensitivity), achieving values of approximately 200 ng / mL at 3 hours of exposure to sNPs and approximately 100 ng / mL at 6, 12, 24 and 48 hours of exposure to sNPs, with TR-146 cells consistently displaying the lowest IC50 values across all exposure times, indicating the highest sensitivity to sNPs, whereas MCF-7 and HT-29 cancer cells yielded higher IC50 values (lower sensitivity), achieving values of approximately 300-400 ng / mL as between 3-24 hours of exposure to sNPs, and approximately 100 ng / mL at 48 hours of exposure to sNPs.

[0281] Notably no IC50 values could be calculated for the non-cancerous MCF-10A cells since no significant mortality was observed. MCF-10A cells exhibited minimal cell death across all concentrations and time points, highlighting the selective cytotoxicity of sNPs towards cancerous cells.The results are indicative of the ability of aNPs, herein specifically demonstrated by sNPs, to exert a cytotoxic effect selectively on cancer cells (such as buccal carcinoma, colorectal adenocarcinoma, and breast cancer), but not on non-cancerous cells, thereby reducing viability of the cancer cells and / or inducing cancer cell mortality by at least 10% (e.g., between 30% and 80%), and promoting increased sensitivity to the aNPs, particularly sNPs, at higher concentrations and / or as exposure time is longer (i.e., dose dependent and / or time dependent).

[0282] Further, a difference was observed in sensitivity between the colorectal cancer cell lines, with Caco-2 cells (derived from a male) being more sensitive to sNPs than HT-29 cells (derived from a female). This increased sensitivity of Caco-2 cells may be attributed to sexbased differences in cancer, which can result from a combination of genetic and epigenetic factors, as well as differences in gene regulation and expression. Differences in the expression of / / RNAs, autophagy, apoptosis, and the activation of the X chromosome, may contribute to the observed disparities in response to sNPs treatment between male and female-derived colorectal cancer cells.

[0283] Example 2 - Enhanced cellular uptake of algal NPs (aNPs) by cancer cells in comparison to non-cancerous cells

[0284] Following the evaluation of the cytotoxic effects of the nanoparticles (NPs) derived from the algae Spirulina Arthospira platensis (sNPs) on different cancerous and non-cancerous cell lines, the cellular uptake of these sNPs by the cancerous and non-cancerous cells was assessed using flow cytometry and fluorescence microscopy, to better understand and determine whether the differences in mortality rates result from different penetration rates. The results are presented in Table 2 below.

[0285] Table 2: Mean fluorescence intensity (MFI) and percentage uptake of 500 mg / mL of Dil-labeled sNPs into TR-146, Caco-2, MCF-7, HT-29, and MCF-10A after 3 hours of exposure. The MFI values represent the added fluorescence intensity in cells exposed to sNPs over the autofluorescence intensity.

[0286] Cell line MFI Uptake

[0287] TR-146 12222 100%

[0288] Caco-2 2883 100%

[0289] MCF-7 7509 100%HT-29 2819 100%

[0290] MCF-10A 2749 60%

[0291] The most prominent result in Table 2 is that all cancerous cells showed 100% cellular uptake, while the non-cancerous cells (i.e., MCF-10A) showed only 60% cellular uptake after 3 hours of exposure. This observation may also partially account for the selective toxicity of sNPs towards cancerous cells. Moreover, it suggests that sNPs need to penetrate the cells to be effective. Even though all cancerous cells exhibited 100% cellular uptake, the extent of penetration, indicated by the MFI parameter, slightly varied between the different cancer cells.

[0292] TR-146 buccal cancer cells showed the highest MFI (12,222), followed by the MCF-7 breast cancer cells with an MFI of 7,509, approximately 40% less. The Caco-2 and HT-29 colorectal cancer cells displayed significantly lower MFIs approximately 77% less compared to TR-146) of 2,883 and 2,819, respectively (with 100% cellular uptake) (Table 2).

[0293] These results are indicative of a correlation between the extent of penetration and the sensitivity of the cell line to sNPs, as demonstrated previously in Example 1. The TR-146 (67%) cells, with the highest MFI, also demonstrated the highest mortality (FIGs. 1A-1E), while the Caco-2 (62%) and HT-29 (32%) cells, with the lowest MFIs, showed comparatively lower mortality rates (FIGs. 2A-2E and FIGs. 3 A-3E).

[0294] This suggests that the selective cytotoxicity of sNPs is closely related to their ability to penetrate the cells, with higher internalization leading to more significant toxicity.

[0295] In contrast, the non-cancerous MCF-10A cells showed lower uptake of 60% (Table 2), with an MFI of 2,749. However, even though the cellular uptake into MCF-10A was lower than the cellular uptake into cancer cells, they still showed no mortality post-incubation with sNPs. This observation suggests that sNPs are more potent and toxic toward cancer cells, further supporting their selective toxicity capability.

[0296] To visually confirm the internalization of the sNPs by cancer cells, fluorescence microscopy was performed on TR-146 buccal cancer cells rather than mere surface adhesion assay. The sNPs were labeled with the fluorescent dye Dil, enabling to track their penetration and localization within the cells. The DAPI stain was used to visualize the cell nuclei, confirming that the sNPs had been internalized and were not simply attached to the cell membrane.As can be seen in FIG. 8, the representative fluorescence microscopy images confirm the cellular uptake of sNPs by TR-146 buccal cancer cells. The images show sNPs labeled with the fluorescent dye Dil (marked sNPs-Dil, appearing in red), indicating the presence of sNPs in proximity to the cells. The images further show the nuclei of the TR-146 cells stained with DAPI (marked TR-146-DAPI, highlight in blue), showing the cellular structure. In the merged image, the overlap of the red signal from the Dil-labeled sNPs and the blue signal from the DAPI-stained nuclei demonstrates that the sNPs have been internalized or attached by the TR-146 cells. This overlap confirms that the sNPs are present within the cellular boundaries.

[0297] Collectively, the results indicate that the flow cytometry and fluorescence microscopy confirm that the aNPs, herein demonstrated by sNPs, undergo increased cellular uptake by cancerous relative to non-cancerous cells. Notwithstanding, forgoing the different penetration rates does not fully explain the stark difference in mortality rates between cancerous and non-cancerous cells, further supporting aNPs, particularly sNPs, selective toxic potency towards cancerous cells. As manifested by the observed significant uptake to the healthy cell line of MCF-10A of 60% (Table 2) resulting with negligent mortality (Fig 5A-E).

[0298] Example 3 - Algal NPs (aNPs) undergo endocytosis into cancer cells

[0299] The observed differences in uptake of nanoparticles (NPs) derived from the algae Spirulina Arthospira platensis (sNPs) demonstrated in Example 2, may be attributed to variations in endocytic activity. Therefore, the endocytic activity of sNPs uptake was herein assessed.

[0300] Endocytosis is a primary pathway through which cells internalize NPs, and inhibiting specific pathways can help identify how aNPs, and particularly sNPs, are internalized by cancerous and non-cancerous cells. To explore this, two inhibitors were used: 1) MpCD, which disrupts caveolae-mediated endocytosis by removing cholesterol from cell membranes, and 2) EIP A, a macropinocytosis inhibitor.

[0301] The impact of these inhibitors on aNPs cellular uptake was herein evaluated using sNPs for the cell lines of TR-146, Caco-2, HT-29, MCF-7, and MCF-10A.

[0302] As can be seen in FIGs. 9A-9E, caveolae-mediated endocytosis, which MpCD inhibited, plays a crucial role in the uptake of aNPs, particularly sNPs, in the tested cancer cells, with macropinocytosis (inhibited by EIP A) being less influential.Blocking caveolae-mediated endocytosis with MpCD resulted in a statistically significant decrease in mortality of MCF-7 breast cancer cells from 31% to 19% at the 500 mg / mL concentration (*p < 0.05), suggesting that caveolae-mediated endocytosis is crucial for sNPs uptake into these cells and induced toxicity (FIG. 9A). No significant effects were seen at 125 mg / mL, likely due to the lower quantity of NPs at this concentration, nor with EIPA treatment, indicating that macropinocytosis does not play a role in sNPs uptake to MCF-7 cells.

[0303] The non-cancerous MCF-10A cells showed no significant changes in mortality at both concentrations with MpCD or EIPA, indicating that these endocytosis pathways do not impact sNPs uptake or cytotoxicity, consistent with the lower uptake and mortality rates observed in these cells (FIG. 9B).

[0304] Different patterns emerged for the colorectal cancer cell lines. In the Caco-2 cells, a significant reduction in mortality from 39% to 25% was observed with MpCD treatment at 125 mg / mL (*p < 0.05) and 500 mg / mL (**** < 0.0001) from 63% to 34%, suggesting that caveolae-mediated endocytosis is the dominant pathway for sNPs uptake in these cells. In contrast, EIPA treatment had no significant effect, highlighting that macropinocytosis is not a major route for sNPs internalization into Caco-2 cells (FIG. 9C). This aligns with their increased mortality rates.

[0305] In HT-29 colorectal cancer cells, a statistically significant decrease in mortality from 32% to 14% was observed at 500 mg / mL with MpCD (**p < 0.01) and EIPA treatments from 32% to 13%, suggesting that both caveolae-mediated endocytosis and macropinocytosis contribute to sNPs uptake into HT-29 (FIG. 9D).

[0306] Finally, in TR-146 buccal cancer cells, MpCD treatment at 500 mg / mL resulted in a significant reduction in mortality from 66% to 31% (** / ? < 0.01), suggesting that caveolae-mediated endocytosis is a significant pathway for sNPs uptake into these cells. No significant effect was observed with EIPA treatment (FIG. 9E).

[0307] A comparison between Caco-2 and HT-29 cells (FIGs. 9C-9D) reveals again a plausible sex-based differences in endocytosis, as Caco-2 cells responded significantly only to MpCD, whereas HT-29 cells responded to MpCD and EIPA. This suggests that variations in cholesterol metabolism and endocytic pathways might contribute to differences in sNPs uptake between male-derived Caco-2 and female-derived HT-29 cells.

[0308] The inhibition patterns indicate that caveolae-mediated endocytosis is a crucial pathway for aNPs, in particular for sNPs, internalization and / or cytotoxicity in cancer cells.To conclude, the significant reduction in cytotoxicity observed when caveolae-mediated endocytosis was blocked, underscores the importance of this pathway for sNPs uptake. The lack of significant effects in non-cancerous MCF-10A cells is related to the fact that sNPs are internalized more by cancer cells, supporting their potential for targeted cancer therapy. Additionally, this finding supports the hypothesis that the internalization of the sNPs likely causes the killing of the cells.

[0309] Example 4 - Characterization of Spirulina aNP’s protein content

[0310] To characterize the protein content of Spirulina ArthrospiraNPs liquid chromatography mass-spectrometry (LC-MS / MS) analysis was performed.

[0311] The method identified, among other non-membrane proteins, 229 proteins that were annotated as proteins associated with Spirulina’ s membranes. In a non-limiting example, each of these 229 membranal proteins was classified into one or more classes of ‘plasmamembranes’ and / or ‘other-membranes’. According to this analysis, 129 proteins were classified as other-membranes proteins; 100 proteins were classified as both ‘plasmamembranes’ and ‘other-membranes’.

[0312] Example 5 - Assessment of endocytosis pathways of algal NPs (aNPs) at additional concentrations and exposure times and using additional endocytosis inhibitors Further assessment of cellular uptake of aNPs, particularly of sNPs, at additional concentrations and exposure times in the presence or absence of endocytosis inhibitors is performed to validate the presumed connection between cellular uptake and the selective toxicity of aNPs.

[0313] To this aim, cancer and normal cell lines are seeded in 6-well plates, 105cells per well and cultured until they reach >70% confluency. Then, the cells are incubated for 3, 6, or 24 h with fresh medium containing, 50, 125, or 500 mg / mL of DiL-labeled aNPs. Next, the cells are washed twice with phosphate-buff ered saline (PBS) and 1 mL of a trypsin solution, containing 300 nM DAPI, are added for 10 min. The cells are then be centrifuged at 500 g for 12 min, fixed with 4% PF A, and transferred to a 96-well plate on ice. Finally, the cellular uptake and the MFI within the cells is analyzed using FACS (CytoFLEX by Beckman Coulter).

[0314] To further investigate the mechanism of aNPs penetration into cancer cells, cellular uptake is measured in the presence of additional endocytosis inhibitors, including, for example, but not limited to: chlorpromazine, which targets receptor-mediated endocytosis; dynasore,which is related to clathrin- and caveolin-mediated endocytosis; and fllipin, which is involved in lipid rafts and caveolin-dependent uptake. The inhibitors are added to the cells after they reach more than 70% confluency and incubated at 37°C for 40 min. Then, the cells are exposed to aNPs, and their cellular uptake is assessed as exemplified above. In addition, the exposed cells are imaged using confocal microscopy, with the Z-stack function, to confirm and visualize cellular uptake.

[0315] Example 6 -Assessment of the effect of algal NPs (aNPs) on the mortality rate of additional cancer and non-cancerous cell lines in vitro., and in vivo in tumor models

[0316] The selective toxicity of the aNPs, particularly of sNPs, is assessed for obtaining a larger pool of potential candidates for in vivo tumor models, as some cell lines are limited in their ability to form tumors in vivo. Based on global cancer prevalence and / or the lack of efficient targeted therapies, the following non-limiting examples of cell lines are chosen: MDA-MB-231 (triple-negative breast cancer), A549 (non-small-cell lung cancer), HeLa (cervical cancer), U87 / LN-18 (glioblastoma), and HEK293 (embryonic kidney), MRC-5 (human lung fibroblasts), BJ (human foreskin fibroblasts), HDFn cells, and HaCaT (human keratinocytes) as additional non-cancer cell lines.

[0317] The effect of aNP, particularly sNPs, exposure on normal cells, namely, human primary keratinocytes and hepatocytes, as primary cells mimic the behavior and responses of cells in vivo more closely than immortalized cell lines.

[0318] For in vitro experiments, cell mortality is assessed using the XTT method according to standard protocol.

[0319] Example 7 - Assessment of algal NPs (aNPs) biodistribution in vivo

[0320] Assessment of the capability of aNPs, particularly sNPs, to specifically target cancerous tissues (tumors) in vivo is performed, as well as assessment of the biodistribution of the aNPs in the major organs in vivo, and confirmation that the in vitro cellular adhesion and / or uptake correlates with the tissue targeting capabilities in vivo. Briefly, a rat bearing two types of tumors, one per hind limb is established. Each hind limb is injected subcutaneously with, for example, 5* 106cells from the cancer cell lines that exhibited the highest cellular adhesiveness or uptake of aNPs. Then, 300 pL of DiL-stained aNPs are injected via the tail vein at three concentrations (n = 4 rats per group, including one control, untreated group). After 24 h, the rats are anesthetized and imaged via IVIS Spectrum CT to detect the location of the DiL-stainedaNPs. To test the biodistribution of the sNPs, the rats are sacrificed 24 h post-injection, and the tumors and major organs (heart, liver, spleen, lung, and kidney) are harvested, dissected, washed with cold saline, and their average fluorescence intensities recorded via IVIS Spectrum CT.

[0321] Example 8 - Assessment of anti-cancer efficacy of the algal NPs (aNPs) in vivo

[0322] Cancer-bearing rats are used to assess the anti -cancer efficacy of the aNPs, particularly sNPs, in vivo. To this end, three cancer cell lines, for which sNPs showed the highest cancerous tissue specific targeting are used. Briefly, rats (n = 4 per group, including an untreated control group) bearing tumors (as described above, but in one hind limb) are administered periodically with high, medium, or low dosages of sNPs, for example, on days 1, 4, 7, 14, and 28 after the tumor has reached -100 mm3(tumors are harvested on these days and their volume is calculated as V = (W2L) / 2, where W and L are the shortest and longest diameters, respectively). The sNPs are administered in PBS, which also serves as the medium administered to the control group. Then, the tumor volume, rat body weight, and rat survival are recorded daily for 12 weeks.

[0323] For in vivo toxicity assessment, healthy rats are treated with a high dose of sNPs, and the heart, liver, spleen, lung, and kidneys are collected after 28 days (or earlier if the rats show toxic symptoms) for histological examinations by a hematoxylin and eosin (H&E) staining (4% PFA fixation, paraffin embedding, sectioning to 5 pm thick slices, and staining with H&E on polylysine-coated slides). In parallel, the tumors are harvested for an immunofluorescence assessment of proliferation by Ki67 (green Ki67 is found only in dividing cells) and apoptosis via caspase-3 (red active caspase-3, a cysteine-aspartic acid protease that executes cell death). Five fields are selected randomly per section to calculate the proliferation or apoptotic rate in each tumor, and the total number of Ki67- or caspase-3 -positive cells are counted at 200* magnification.

[0324] For in vivo experiments, aNPs are administered by parenteral route. Administration may include intradermal (ID) injection, subcutaneous (SQ) injection, intramuscular (IM) injection, intravenous (IV) injection, and / or intraperitoneal (IP) injection, to achieve a systemic or local therapeutic effect.Example 9 - Assessment of capability of additional algal NPs (aNPs) to promote cancer cell mortality, cellular uptake, and engage in endocytosis

[0325] The cytotoxic effect, and the selectivity of the effect towards cancerous cells, is evaluated for nanoparticles (NPs) derived from alga(e) (aNPs) belonging to a genus of any one or more of: Spirulina Arthrospira, Chondracanthus, Gracilaria, Chondrus, Undaria, Sargassum, Laminaria, Kombu, Ecklonia, Macrocystis, Palmaria, Gigartina, Chlorella, and Haematococcus.

[0326] Evaluation is performed in a similar manner to the evaluation of Spirulina Arthrospira NPs (sNPs) as demonstrated, for example, in hereinabove example 1. The cytotoxic effect and its selectivity are evaluated by exposing cancerous cells, such as, but not limited to cancerous cell lines: TR-146, Caco-2, HT-29, MCF-7, MDA-MB-231, A549, HeLa, U87 / LN-18, HEK293, MRC-5, and BJ, as well as exposing non-cancerous cells, such as, but not limited to non-cancerous cell lines: MCF-10A, HDFn, or HaCaT, to different concentrations and incubation time of aNPs and measuring cell mortality.

[0327] In addition to cell mortality, cellular uptake and endocytosis are evaluated in a similar manner to the evaluation of Spirulina Arthrospira NPs (sNPs) demonstrated, for example, in hereinabove Examples 2-3.

[0328] Example 10 - aNPs can be associated / encapsulated with drugs for cancer therapy prior to parenteral administration

[0329] The herein disclosed aNPs are parenterally administered to selectively target cancer cells for promoting anti-cancerous therapeutic effect and cancer cell mortality. In addition, the aNPs, including Spirulina NPs, may be used as carriers of active ingredients for further enhancing the therapeutic effect of the selective cancer therapy, by associating with the aNPs drugs such as small molecules and / or higher molecular weight biologies including nucleic acids-based therapeutics, such as, but not limited to miRNAs and siRNA, or protein-based therapeutics, such as, but not limited to peptides, enzymes, and antibodies, prior to the parenteral administration, thereby promoting enhanced anti-cancerous effect and cancer cell mortality in comparison to the effect resulting from parenteral administration of aNPs alone.

[0330] The association with aNPs may facilitate selectivity of the active ingredient towards to cancer cells, or improve its bioavailability and stability by protecting the active ingredient fromdegradation by blood protease, providing sustained release, enhancing retention time, and overall prolonging the therapeutic effect.

[0331] The association of one or more active ingredients with the aNPs is performed by mixing or incubating the aNPs with the one or more active ingredients and subjecting the mixture to sonication, followed by ultra-centrifugation.

[0332] To evaluate the ability of algal-based NPs to encapsulate / associate active ingredients the encapsulation efficiency (EE) of the hydrophilic Fluorescein Isothiocyanate dextran (FITC-Dextran) having different MWs ranging from 4 kDa to 250 kDa with the aNPs is tested.

[0333] Example 11 - characterization of aNPs structure

[0334] Methods:

[0335] Preparation of Algal NPs (aNPs) - to prepare aNPs, each algae powder, such as powder of Spirulina Arthospira Platensis, was weighed and dispersed in 80 mL double distilled water. Subsequently, the algae solution was insonated in an ice bath using a 0 1.3 cm transducer at 60% amplitude, 50% duty cycle (DC), for two minutes. After sonication, the solution was transferred to a centrifuge tube and centrifuged at 3200 g for 5 min at 4°C. Then, the supernatant was subjected to another sonication cycle and centrifugation at the same conditions. The final supernatant containing aNPs was centrifuged at 10000 g for 60 min at 4°C. Later, the supernatant was gently poured over 2 mL of 60% sucrose followed by ultracentrifugation at 200000 g for 30 min at 4°C, and from that, 600 pL of solution containing non-soluble and amphiphilic components or molecules such as membranes and proteins was carefully removed from the layer above the 60% sucrose solution.

[0336] Characterization of aNPs - the size, zeta potential, and concentration of aNPs were measured by a dynamic light scattering (DLS) instrument in water. The aNPs yield was defined as the number of obtained aNPs divided by the initial algae mass. Additionally, protein in aNPs solution was assayed via the Bradford method. The sample was analyzed using an ELISA reader at 595 nm.

[0337] Results - 13 different types of edible algae were processed according to the method of preparation to produce 13 algal -based NPs (aNPs made of alga(e)). The 13 aNPs were comprehensively characterized for their structure.aNPs structural properties were evaluated. The evaluation of their structure included analyses of their size, poly dispersity index (PDI), protein content, surface charge (i.e., zeta potential), and shape.

[0338] The results of the structural evaluation are presented in Table 3 below.

[0339] Table 3 - The obtained size, PDI, and protein content of the NPs produced from the tested alga. Values represent the average ± SD of at least three repetitions. *x 109NPs / (mLxg).

[0340] Type of aNPs Size PDI Protein

[0341] (nm) Content (mg) Chondracanthus Chamissoi 605 ± 67 0.42 ± 0.01 0.04 ± 0.01 aNPs

[0342] Gracilaria aNPs 232 ± 03 0.24 ± 0.02 0.07 ± 0.001 Irish Moss aNPs 275 ± 24 0.33 ± 0.03 0.01 ± 0.001 Wakame aNPs 466 ± 08 0.29 ± 0.01 0.44 ± 0.01 Sargassum aNPs 170 ± 07 0.24 ± 0.01 0.68 ± 0.06 Kelp Laminaria Digitata aNPs 212 ± 20 0.27 ± 0.01 0.11 ± 0.00 Kombu aNPs 217 ± 10 0.19 ± 0.02 0.68 ± 0.001 Ecklonia Cava aNPs 235 ± 12 0.19 ± 0.01 0.81 ± 0.01 Giant Kelp aNPs 252 ± 20 0.29 ± 0.03 0.15 ± 0.02 Dulse aNPs 245 ± 33 0.44 ± 0.12 0.77 ± 0.09 Gigartina Red Marine aNPs 381 ± 13 0.48 ± 0.01 0.01 ± 0.01 Spirulina Arthospira Platensis 126 ± 02 0.14 ± 0.00 2.61 ± 0.81 aNPs (sNPs)

[0343] Chlorella aNPs 157 ± 11 0.25 ± 0.05 0.80 ± 0.04

[0344]

[0345] As can be seen in Table 3, the aNP size ranged from 126 nm to 605 nm, and their PDI from 0.14 to 0.48. The relative aNP concentration varied between 1.5 to 60.8xl09NPs / (mLxg), while the protein content / concentration ranged from 0.01 to 3.24 mg / mL.

[0346] Spirulina Arthospira Platensis NPs are characterized by having the smallest particle size of 126 ± 02 nm (average particle diameter) and the lowest PDI of 0.14. Furthermore, Spirulina NPs had the highest relative concentration with 60.8 ± 2.9 xlO9NPs / (mLxg).

[0347] Additionally, there were differences in protein content between the different algae, wherein Spirulina had 2.61 ± 0.81 mg / mL while for all other aNPs, the protein concentration was lower than 1 mg / mL.

[0348] As mentioned above, Spirulina NPs had the smallest particle size of 126 ± 2 nm among the tested algae. This nano-size can be advantageous for solubility, drug loading, and contact-mediated interactions such as cellular uptake via endocytosis. Thus, the nano-sized characteristic implies that the tested aNPs may be an effective carrier of active ingredients to cancer cells.

[0349] Also, the shape of SpirulinaNPs was visualized using transmission electron microscopy (TEM). SpirulinaNPs are spherical. SpirulinaNPs have a hydrophilic core and an amphiphilic shell (bi-layered biological membrane).

[0350] In addition, Spirulina NPs had the lowest PDI among the tested algae of PDI=0.14. PDI indicates the monodispersity of the NPs population. The narrower it is, the more likely the NPs will exert a similar effect. And vice versa, the more polydisperse it is (higher PDI), the more their effect would vary. For carry of active ingredients to cancer cells, PDI<0.3, and preferably PDI<0.2, is considered mono / homodisperse.

[0351] Furthermore, Spirulina NPs displayed the highest initial concentration of 60.8 ± 2.9 xlO9NPs / (mL*g), indicating a substantial yield and cost-effective production process.

[0352] Finally, the zeta potentials were measured in DDW for the harvested aNPs. The zeta potential values observed for the aNPs range from about -38 to about -9 mV, reflecting variety in the harvested components from the tested aNPs. Among these, Spirulina NPs displayed the most negative zeta potential of -38 ± 3 mV.

[0353] In summary, disclosed are advantageous structural characteristics of the edible aNPs, especially of Spirulina NPs, including their spherical shape, nano-size, and negative surface charge. While certain embodiments of the invention have been illustrated and described, it will be clear that the invention is not limited to the embodiments described herein. Numerous modifications, changes, variations, substitutions, and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the present invention as described by the claims which follow.

Claims

CLAIMSWhat we claim is:

1. A composition comprising alga-derived nanoparticles (aNPs) comprising non-soluble and amphiphilic alga(e) components, for use in treating, attenuating, and / or preventing progression of cancer in a subject in need thereof, wherein the aNPs have an average particle diameter in the range of between about 50 nm and about 650 nm, and wherein the composition is formulated for parenteral administration.

2. The composition for use according to claim 1, wherein the non-soluble and amphiphilic alga(e) components comprise membranes and membrane-associated proteins of the alga(e).

3. The composition for use according to claim 1 or 2, wherein the aNPs have a spherical shape, said spherical shape comprises a shell and inner core.

4. The composition for use according to any one of claims 1-3, wherein the alga(e) belongs to a genus selected from a group consisting of one or more of: Spirulina Arthrospira, Chondracanthus, Gracilaria, Chondrus, Undaria, Sargassum, Laminaria, Ecklonia, Macrocystis, Palmaria, Gigartina, Chlor ella, rAHaemalococciis, or any combination thereof.

5. The composition for use according to claim 4, wherein the alga(e) is Spirulina Arthrospira.

6. The composition for use according to claim 5, wherein the Spirulina Arthrospira comprises one or more species selected from Arthrospira Platensis, Arthospira Maxima, Arthrospira ardissonei, Arthrospira erdosensis, Arthrospira fusiformis, Arthrospira indica, Arthrospira innermongoliensis, Arthospira jenneri, Arthrospira massartii, or any combination thereof.

7. The composition for use according to any one of claims 4-6, wherein the Spirulina Arthospira comprises Spirulina Arthrospira Platensis.

8. The composition for use according to any one of claims 4-7, wherein the average particle diameter is in the range between about 50 nm and 200 nm and / or a surface charge in the range between about -30 mV and about -45 mV.

9. The composition for use according to any one of claims 1-8, further comprising at least one pharmaceutically acceptable excipient / s.

10. The composition for use according to any one of claims 1-9, further comprising an active ingredient.

11. The composition for use according to claim 10, wherein the active ingredient is associated with the aNPs, said association comprises the active ingredient attached to the aNPs shell or enclosed within the aNPs core.

12. The composition for use according to any one of claims 1-11, administered together with at least one additional active ingredient.

13. The composition for use according to claim 12, wherein the composition is administrated concomitantly with and / or sequentially to the at least one additional active ingredient.

14. The composition for use according to any one of claims 10-13, wherein the active ingredient comprises an anti -cancer medication.

15. The composition for use according to any one of claims 1-14, wherein the cancer comprises primary solid, liquid tumor and / or metastases thereof.

16. The composition for use according to any one of claims 1-15, wherein the cancer comprises buccal carcinoma, colorectal adenocarcinoma, breast cancer, brain, ovarian cancer, glioblastoma, kidney cancer, pancreas, liver, and leukemia, or any combination thereof.

17. The composition for use according to claim 16, wherein the cancer is selected from one or more of: buccal carcinoma, colorectal adenocarcinoma, and breast cancer, or any combination thereof.

18. The composition for use according to any one of claims 1-17, wherein the parenteral administration comprises intradermal (ID) injection, subcutaneous (SC) injection, intramuscular (IM) injection, intravenous (IV) injection, intraperitoneal (IP) injection, or any combinations thereof.

19. The composition for use according to any one of claims 1-18, wherein the administering of the composition induces selective toxicity to cancerous cells, in comparison to non-cancerous cells.

20. A method for treating, attenuating, and / or preventing progression of cancer in a subject in need thereof, the method comprises parenterally administering to the subject a therapeutically effective amount of a composition comprising alga(e) derived nanoparticles(aNPs) comprising non-soluble and amphiphilic alga(e) components, and wherein the aNPs have an average particle diameter in the range of between 50 nm and about 650 nm.

21. The method according to claim 20, wherein the non-soluble and amphiphilic alga(e) components comprise membranes and membrane-associated proteins of the alga(e).

22. The method according to claim 20 or 21, wherein the aNPs have a spherical shape, said spherical shape comprises a shell and inner core.

23. The method according to any one of claims 20-22, wherein the alga(e) belongs to a genus selected from a group of consisting of one or more of: Spirulina Arthrospira, Chondracanthus, Gracilaria, Chondrus, Undaria, Sargassum, Laminaria, Ecklonia, Macrocystis, Palmaria, Gigartina, Chlor ella, rAHaemalococciis, or any combination thereof.

24. The method according to any one of claims 20-23, wherein the alga(e) is Spirulina Arthrospira.

25. The method according to claim 24, wherein the Spirulina Arthrospira comprises one or more species selected from Arthrospira Platensis, Arthospira Maxima, Arthrospira ardissonei, Arthrospira erdosensis, Arthrospira fusiformis, Arthrospira indica, Arthrospira innermongoliensis, Arthospira jenneri, Arthrospira massartii, or any combination thereof 26. The method according to claim 24 or 25, wherein the Spirulina Arthospira comprises Spirulina Arthrospira Platensis.

27. The method according to any one of claims 20-26, wherein the aNPs have an average particle diameter in the range of between about 50 nm and 200 nm and / or a surface charge in the range between about -30 mV and about -45 mV.

28. The method according to any one of claims 20-27, wherein the composition further comprises at least one pharmaceutically acceptable excipient / s.

29. The method according to any one of claims 20-28, wherein the composition further comprises an active ingredient.

30. The method according to claim 29, wherein the active ingredient is associated with the aNPs, said association comprises the active ingredient attached to the aNPs shell or enclosed within the aNPs core.

31. The method according to any one of claims 20-30, further comprising administering at least one additional active ingredient.

32. The method according to claim 31, wherein the method comprises administering the composition concomitantly with and / or sequentially to the at least one additional active ingredient.

33. The method according to any one of claims 29-32, wherein the active ingredient comprises an anti-cancer medication.

34. The method according to any one of claims 20-33, wherein the cancer comprises primary solid tumor, liquid tumor and / or metastases thereof.

35. The method according to any one of claims 20-34, wherein the cancer is selected from one or more of: buccal carcinoma, colorectal adenocarcinoma, breast cancer, brain, kidneys, pancreas, liver, and leukemia, or any combination thereof.

36. The method according to claim 35, wherein the cancer is selected from one or more of: buccal carcinoma, colorectal adenocarcinoma, and breast cancer, or any combination thereof.

37. The method according to according to any one of claims 20-36, wherein parenterally administering comprises intradermal (ID) injection, subcutaneous (SC) injection, intramuscular (IM) injection, intravenous (IV) injection, intraperitoneal (IP) injection, or any combinations thereof.

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