WO2025088609A1

WO2025088609A1 - Alga(e) nanoparticles and uses thereof for drug delivery

Info

Publication number: WO2025088609A1
Application number: PCT/IL2024/051028
Authority: WO
Inventors: Aharon AZAGURY; Eliyahu DRORI
Original assignee: Ariel Scientific Innovations Ltd
Current assignee: Ariel Scientific Innovations Ltd
Priority date: 2023-10-25
Filing date: 2024-10-22
Publication date: 2025-05-01
Anticipated expiration: 2026-04-25

Abstract

Disclosed are biocompatible, biodegradable, and low immunogenic alga(e) Nanoparticles (aNPs) that can adhere and deliver active ingredient(s) to mucosal epithelium tissue, as well as compositions comprising the aNPs, methods of preparing the aNPs, and uses thereof in methods of treatment.

Description

ALGA(E) NANOPARTICLES AND USES THEREOF FOR DRUG DELIVERY

TECHNICAL FIELD

The present disclosure relates to alga(e) nanoparticles (NPs) and uses thereof, including for drug absorption into the mucosal epithelium.

BACKGROUND OF THE INVENTION

Drug administration by oral delivery is the preferred route, regardless of some remaining challenges, such as acidic degradation by the stomach or enzymatic degradation in the gastrointestinal (GI) tract, short intestinal resident time, ineffective mass transfer across or into intestinal tissue, and toxicity issues.

One strategy to overcome these barriers is utilizing a drug delivery system (DDS), for example, nanocarriers. Various nanocarriers have been evaluated as oral DDS, including polymeric NPs, liposomes, exosomes, membrane -based NPs, nano-sized hydrogels, and NPs derived from edible plants (e.g., corn, grapefruit, and ginger).

However, even when intestinal resident time is increased, the binding of nanocarriers to the intestinal mucus layer can be complex, as the mucus layer closer to the intestinal lumen is looser than the layer closer to the epithelial cells. The mucoadhesion of NPs to the loose mucus layer may cause a rapid release of the drug, thereby preventing its binding to the epithelium.

Mucoadhesive nanocarriers were recognized as a promising strategy for effective drug uptake within the intestine. Mucoadhesive carriers, including NPs, microparticles, hydrogels, and films, have been investigated as potential mucoadhesive platforms based on their ability to firmly attach to the intestinal epithelium's mucosal lining, swiftly transit through the GI tract, circumvent enzymatic degradation, and extend resident time, thereby to enhance drug bioavailability and augment drug absorption (e.g., systemic, or localized uptake).

Indeed, by employing mucoadhesive nanocarriers, the pharmacokinetics of drug absorption within the intestinal milieu undergo transformative shifts, effectively enabling systemic and localized drug delivery. Nevertheless, aside from effectively facilitating the bioavailability of drug cargo, mucoadhesive nanocarriers are also required to exhibit inherently advantageous qualities that make them biocompatible, non-immunogenic, biodegradable, and enable reasonable cost of production, preferably from sustainable and renewable sources of production.

There is, therefore, an unmet need to expand the repertoire of mucoadhesive nanocarriers that can effectively perform as a platform for delivering drugs to mucosal tissue.

SUMMARY OF THE INVENTION

According to one aspect, there are provided alga(e) Nanoparticles (aNPs) that can adhere and facilitate delivery of active ingredients to mucosal epithelium tissue.

In some embodiments, the disclosed algal-based NPs (aNPs) have advantageous structural and functional characteristics, including their spherical shape, desired nano-size, surface charge that ranges from negative to almost neutral zeta potential, capability of adhering to mucosal epithelium tissue, and potency in promoting cellular uptake of active ingredient(s), that make it suitable for use as a drug delivery system (DDS) to mucosal tissue.

Notwithstanding its nano-size (the majority are between 100-300 nm), the aNPs can surprisingly efficiently encapsulate active ingredient(s) having a molecular weight (MW) corresponding to that of an average antibody, in some embodiments.

Further advantages are the inherent sustainability, biocompatibility, biodegradability, and low immunogenicity of the edible algae it is made of, which increase its potential as oral DDS.

Finally, some types of alga(e) were found to be surprisingly potent with regard to mucoadhesion force and cellular uptake of their corresponding aNPs, particularly potent were those NPs made of Spirulina Arthospira, Kombo, Giant Kelp, Dulse, Gigartina Red Marine, and Gracilaria, but especially advantageous were NPs made of Spirulina Arthospira platensis which exemplified surprisingly superior functionality.

Advantageously, Spirulina Arthospira platensis NPs, exhibited a superior ability to deliver active ingredient(s), in comparison to other aNPs made of edible algae or inedible alga(e) species as disclosed herein, including their mucoadhesion, cellular uptake, and release of the active ingredient(s) into human epithelial cells. According to additional aspects, there are provided compositions comprising the aNPs, methods of preparing the aNPs, aNPs produced by those methods, and uses of aNPs or composition comprising the same in methods of treatment including drug delivery to mucosal tissue.

According to one aspect, there are provided alga(e) derived nanoparticles (aNPs), comprising non- soluble and amphiphilic alga(e) components, wherein the aNPs have an average particle diameter in the range of between 100 nm and about 650 nm and/or surface charge ranging between about -10 mV and -45 mV (or +10 mV and +45 mV), and wherein the aNPs are capable of adhering to mucosal epithelium tissue and wherein the aNPs are capable of facilitating delivery of one or more active ingredients associated with the aNPs to the mucosal epithelium tissue.

According to one aspect, there are provided alga(e) nanoparticles (aNPs), comprising non-soluble and amphiphilic components of the alga(e), wherein the aNPs have an average particle diameter of less than 650 nm and/or surface charge more negative than about -10 mV, wherein the aNPs are capable of adhering to mucosal epithelium tissue, and wherein the aNPs are capable of facilitating delivery of one or more active ingredients to the mucosal epithelium tissue. Each possibility is a separate embodiment.

According to some embodiments, the aNPs have a spherical shape. According to some embodiments, the aNPs have a spherical shape and/or a hydrophilic core. Each possibility is a separate embodiment.

In some embodiments, the aNPs consist essentially of non-soluble and amphiphilic alga(e) components. In some embodiments, non-soluble and amphiphilic alga(e) components comprise membranes and membrane-associated proteins of the alga(e).

According to some embodiments, the non-soluble and amphiphilic alga(e) components comprise membranes and membrane proteins of the alga(e). In some embodiments non-soluble and amphiphilic alga(e) components further comprise non-membrane proteins and/or polysaccharides of the alga(e). Each possibility is a separate embodiment.

In some embodiments, the aNPs comprise a plurality of different membrane proteins of the alga(e). In some embodiments, the alga(e) components or the aNPs are substantially devoid of polymer addition thereto. Each possibility is a separate embodiment.

In another embodiment, the aNPs are capable of adhering to mucosal epithelium tissue at a fracture strength of more than about 200 p N/mnr when exposed to forces between 20 mN and 200 mN. Each possibility is a separate embodiment.

According to some embodiments, the aNPs comprise one or more active ingredient(s) associated with the aNPs. In some embodiments, the one or more active ingredient(s) is encapsulated with the aNPs.

In some embodiments, the aNPs can facilitate delivery of one or more active ingredients to the mucosal epithelium tissue.

In some embodiments, the delivery of the one or more active ingredients to the mucosal epithelium tissue comprises adherence of the aNPs to the mucosal epithelium tissue and cellular uptake of the one or more active ingredients.

In some embodiments, the alga(e) comprises one or more edible alga(e).

According to some embodiments, the alga(e) comprises one or more alga(e) species belonging to a genus selected from Chondracanthus, Gracilaria, Chondrus, Undaria, Sargassum, Laminaria, Ecklonia, Macrocystis, Palmaria, Gigartina, Spirulina Arthrospira, Chlorella, and Haematococcus, or any combination thereof. Each possibility is a separate embodiment.

In some specific embodiments, alga(e) comprises one or more alga(e) species belonging to a genus selected from Spirulina Arthrospira, Laminaria, and Gracilaria, or any combination thereof. Each possibility is a separate embodiment.

In further specific embodiments, the alga(e) comprises one or more species belonging to Spirulina Arthrospira.

In some embodiments, the alga(e) comprises a combination of Spirulina Arthrospira with one or more additional alga(e) species. In some embodiments, a combination of Spirulina Arthrospira with one or more additional alga(e) species comprises at least about 20% (w/w) Spirulina Arthrospira species relative to other alga(e) species. In some embodiments, the alga(e) or the aNPs comprises at least about 50% (w/w) of the one or more species belonging to Spirulina Arthrospira relative to other species. In some embodiments, the alga(e) or the aNPs comprises at least about 75% (w/w) of one or more species belonging to Spirulina Arthrospira relative to other species.

In some embodiments, the one or more species belonging to Spirulina Arthospira comprises Spirulina Arthrospira Platensis.

According to some embodiments, the aNPs have a surface charge more negative than about -30 mV. According to some embodiments, the average particle diameter is less than 157 nm. According to some embodiments, the average particle diameter is less than 130 nm.

According to some embodiments, the aNPs have an average particle diameter in the range between about 100 nm and 160 nm and/or a surface charge in the range between about - 30 mV and about -45 mV.

According to some embodiments, the aNPs have an average particle diameter in the range between about 100 nm and about 130 nm and/or a surface charge in the range between about -30 mV and about -45 mV.

In some embodiments, the alga(e) comprises a combination of Spirulina Arthrospira with one or more additional alga(e). In some specific embodiments, the combination comprises a combination of Spirulina Arthrospira with Chlorella. In some specific embodiments, the combination comprises a combination of Spirulina Arthrospira Platensis with Chlorella. Each possibility is a separate embodiment.

According to some embodiments, the aNPs have a poly dispersity index (PDI) of less than 0.7. According to some embodiments, the aNPs have a polydispersity index (PDI) of less than 0.3.

In some embodiments, the aNPs are capable of adhering to mucosal epithelium tissue of a human.

In some embodiments, the mucosal epithelium tissue is selected from one or more of: gastrointestinal (GI) epithelium tissue, nasal epithelium tissue, buccal epithelium tissue, and vaginal epithelium tissue, or any combination thereof. Each possibility is a separate embodiment. In some specific embodiments, the mucosal epithelium tissue is gastrointestinal (GI) epithelium tissue.

In some other embodiments, the aNPs comprise enhanced capability of adhering to the mucosal epithelium tissue with respect to adhering to at least one of any one of the nanocarriers made of: 5% alginate, 5% gelatine, 5% chitosan, Nori algae, Chlorella, a combination of Spirulina with Chlorella, or an inedible alga; and wherein the enhanced capability of adhering comprises an increase of at least 3-fold. Each possibility is a separate embodiment.

According to some embodiments, the one or more active ingredient(s) is released from the aNPs for a period of at least about 12 hours.

In some embodiments, the one or more active ingredient(s) is characterized by having hydrophilic and/or amphipathic properties. Each possibility is a separate embodiment.

In some embodiments, the active ingredient comprises one or more of a pharmaceutical/drug, a tag, and a food supplement, or any combination thereof. Each possibility is a separate embodiment.

In some embodiments, the active ingredient comprises a pharmaceutical drug. In some embodiments, the pharmaceutical drug comprises a biological drug. In some embodiments, the biological drug comprises a protein-based drug. In some embodiments, the active ingredient comprises a protein-based drug.

According to some aspects, there is provided a composition comprising the aNPs and a pharmaceutically acceptable carrier.

According to some embodiments, the aNPs or the composition comprising the same for use in delivery of one or more active ingredients to mucosal epithelium tissue of a subject in need, wherein the aNPs or the composition comprise one or more active ingredient(s), and wherein the delivery of the one or more active ingredients to the mucosal epithelium tissue comprises adherence of the aNPs to the mucosal epithelium tissue and cellular uptake of the one or more active ingredients.

In some embodiments, the aNPs or the composition comprising the same for use, wherein the mucosal epithelium tissue is selected from one or more of: gastrointestinal (GI) epithelium tissue, nasal epithelium tissue, buccal epithelium tissue, and vaginal epithelium tissue, or any combination thereof.

In some embodiments, the aNPs or the composition comprising the same for use, wherein the delivery of the of one or more active ingredients to mucosal epithelium tissue comprises local and/or systemic effects.

In some related embodiments, the aNPs or the composition comprising the same for use in treating, attenuating, and/or preventing progression of a gastrointestinal (Gl)-disease in a subject in need thereof, wherein the aNPs or the composition comprise one or more active ingredient(s). In some embodiments, the treating, attenuating, and/or preventing progression of a gastrointestinal (Gl)-disease in a subject in need thereof comprises delivery of one or more active ingredients to the GI mucosal epithelium tissue of the subject, and wherein the delivery of the one or more active ingredients to the mucosal epithelium tissue comprises adherence of the aNPs to the mucosal epithelium tissue and cellular uptake of the one or more active ingredients.

In some specific embodiments, the aNPs or the composition comprising the same for use, wherein the GI disease is selected from Inflammatory Bowel Disease (IBD) and/or cancer.

In some additional embodiments, the aNPs or the composition comprising the same for use, administrated orally at a therapeutically effective amount.

In some additional embodiments, the aNPs or the composition comprising the same for use, wherein the subject comprises a human subject.

According to another aspect, there is provided a method for delivery of one or more active ingredients to mucosal epithelium tissue, in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of the aNPs or the composition comprising the same, wherein the aNPs or the composition comprising the same comprise one or more active ingredient(s), and wherein the delivery of the one or more active ingredients to the mucosal epithelium tissue comprises adherence of the aNPs to the mucosal epithelium tissue and cellular uptake of the one or more active ingredients.

According to related aspect, there is provided a method for treating, attenuating, and/or preventing progression of gastro- or intestinal- disease in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of the aNPs or the composition comprising the same, wherein the aNPs or the composition comprise one or more active ingredient(s).

According to yet another aspect, there is provided a method for preparing nanoparticles made of alga(e) (aNPs), comprising 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; (iv) applying/loading the supernatant onto a density gradient and subjecting it to ultracentrifugation; (v) collecting a fraction comprising non-soluble and amphiphilic components; thereby obtaining the nanoparticles made of alga(e) (aNPs) comprising nonsoluble and amphiphilic alga(e) components; and wherein the aNPs have an average particle diameter in a range of between about 100 nm and 650 nm and/or surface charge in a range of between about -10 mV and about -45 mV, and wherein the aNPs are capable of adhering to mucosal epithelium tissue. Each possibility is a separate embodiment.

In some embodiments, the method comprises collecting a fraction that comprise or consist essentially of non-soluble and amphiphilic components of the alga(e). Each possibility is a separate embodiment. In some embodiments, non-soluble and amphiphilic alga(e) components comprise membranes and membrane proteins of the alga(e). Each possibility is a separate embodiment.

In some embodiments, the non-soluble and amphiphilic alga(e) components comprise membranes and membrane proteins of the alga(e), and further comprise non-membrane proteins and polysaccharides of the alga(e).

According to some embodiments, the aNPs comprise membranes and membrane- associated proteins derived from the obtained alga(e) cells/biomass.

In some embodiments, the aNPs comprise a plurality of different membrane proteins of the alga(e).

In some embodiments, the obtained alga(e) cells/biomass or the obtained aNPs are devoid of polymer addition thereto. 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. Each possibility is a separate embodiment.

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.

In some embodiments, the density gradient comprises between about 55% and about 65% sucrose solution, and wherein the collecting of the fraction comprising non-soluble and amphiphilic components comprises collecting the fraction on top of the about sucrose gradient.

In some other embodiments, the alga(e) comprises one or more alga(e) species belonging to a genus selected from Chondracanthus, Gracilaria, Chondrus, Undaria, Sargassum, Laminaria, Ecklonia, Macrocystis, Palmaria, Gigartina, Spirulina Arthrospira, Chlorella, and Haematococcus, or any combination thereof. Each possibility is a separate embodiment.

In some specific embodiments, the alga(e) comprises one or more alga(e) species belonging to a genus selected from Spirulina Arthrospira, Laminaria including Kombo, Giant Kelp, and Gracilaria, or any combination thereof. Each possibility is a separate embodiment.

In some further specific embodiments, the alga(e) comprises one or more species belonging to Spirulina Arthrospira.

In some embodiments, alga(e) comprises a combination of Spirulina Arthospira with one or more additional alga(e) species.

In some embodiments, the alga(e) cells/biomass comprise at least about 20% (w/w) Spirulina Arthrospira species relative to other alga(e) species. In some related embodiments, the alga(e) comprises at least about 50% (w/w) Spirulina Arthospira species relative to other alga(e) species.

In some further specific embodiments, the one or more Spirulina Arthospira species comprises Spirulina Arthospira Platensis. In some other embodiments, the alga(e) comprises a combination of Spirulina Arthospira with one or more additional alga(e). In some other specific embodiments, the combination of Spirulina Arthospira with one or more additional alga(e) comprises a combination of Spirulina Arthospira with Chlorella.

In some embodiments, the obtained aNPs have a polydispersity index (PDI) of less than 0.5. In some embodiments, the obtained aNPs have a poly dispersity index (PDI) of less than 0.3.

In some embodiments, the method for preparation of aNPs further comprises a step of associating/encapsulating one or more active ingredients, wherein said associating/ encapsulating comprises mixing the obtained aNPs with one or more active ingredients.

In some related embodiments, the encapsulating comprises encapsulation efficiency (EE) of at least about 15%.

In some embodiments, the ultracentrifugation comprises a centrifugal force of at least 50,000 g.

In some embodiments, the aNPs comprise an average particle diameter of less than about 650 nm and/or surface charge more negative than about -10 mV, and wherein the aNPs are capable of adhering to mucosal epithelium tissue.

In some embodiments, the obtained aNPs have an average particle diameter of less than 169 nm and/or surface charge more negative than about -30 mV. In some embodiments, the one or more alga(e) species comprises .

According to an additional aspect, there are provided nanoparticles made of algae (aNPs), obtained or obtainable by the method of preparation.

According to an additional aspect, there are provided Alga(e) nanoparticles (aNPs), comprising non-soluble and amphiphilic alga(e) components, wherein the aNPs have an average particle diameter of less than 157 nm and/or surface charge more negative than about -30 mV, and wherein the aNPs are capable of adhering to mucosal epithelium tissue and wherein the aNPs are capable of facilitating delivery of one or more active ingredients to the mucosal epithelium tissue, and wherein the alga(e) comprises one or more species belonging to Spirulina Arthrospira. In some embodiments, the one or more species belonging to Spirulina Arthrospira comprises Spirulina Arthospira Platensis. In some embodiments, the aNPs have an average particle diameter of less than 130 nm and/or surface charge more negative than -30 mV.

In some embodiments, the aNPs have an average particle diameter in the range between 100 nm and 169 nm and/or surface charge ranging between -15 mV and about -45, and wherein the alga(e) comprises one or more species belonging to Spirulina Arthrospira in combination with Chlorella, preferably the one or more Spirulina Arthrospira species comprises Spirulina Arthospira Platensis.

In some embodiments, the aNPs have an average particle diameter in the range between 120 and 130 nm and/or surface charge ranging between -30 mV and -42 mV, and wherein the alga(e) comprises one or more species belonging to Spirulina Arthrospira, preferably the one or more species comprises Spirulina Arthospira Platensis.

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.

BRIEF DESCRIPTION OF THE FIGURES

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

FIGs. 1A-1C show graphs presenting structural characteristics of harvested NPs made of alga (aNPs).

FIG. 1A shows a line graph presenting the distribution of particle diameter of Spirulina Arthospira NPs in six preparation repeats. The size distribution of the harvested Spirulina NPs was determined using dynamic light scattering (DLS), and the average particle size was determined to be 126nm (referring to Table IB).

FIG. IB shows a pictogram of cryo transmission electron microscopy (cryo TEM) visualizing Spirulina Arthospira NPs. The shape of the harvested Spirulina NPs is spheric. FIG. 1C shows a bar graph presenting surface charge measurements in DDW of 13 different harvested NPs made of alga (aNPs) and Astaxanthin. The values of the measured zeta potentials of the 13 aNPs and Astaxanthin are also listed in Table IB, ranging from -38 to -9 mV. The zeta potential of the harvested Spirulina NPs is -38 ± 3 mV. Statistical significance was determined by ANOVA test; the values represent the mean ± SD of n=3, where statistical significances are denoted as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 compared to Spirulina Arthospira NPs.

FIGs. 2A-2C show bar graphs presenting mucoadhesion fracture strength of the 13 different harvested NPs made of alga (aNPs) and Astaxanthin against the small intestines of mice (FIG. 2A) pigs (FIG. 2B) and sheep (FIG. 2C) for an applied force of 20 mN (left, blue) and 200 mN (right, red). Statistical significance was determined by ANOVA test; the values represent the mean ± SD of n=4, where statistical significances are denoted as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 compared to Spirulina Arthospira NPs.

FIGs. 3A-3B show bar graphs presenting mucoadhesive fracture strengths of six different aNPs subjected to applied forces of 20 mN and 200 mN (blue and red bars, respectively) and grouped as the three aNPs with the highest mucoadhesion (FIG. 3A; I. Spirulina Arthospira; II. Kombu; III. giant Kelp) and the three with the lowest mucoadhesion (FIG. 3B; I. Sargassum; II. Gracilaria; III. Chondracanthus Chamissoi). Statistical significance was determined by ANOVA test; the values represent the mean ± SD of n=4, where statistical significances are denoted as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 multiple variable comparisons.

FIGs. 4A-4B show histograms of FACS presenting cellular uptake of aNPs made of the same six algae shown in FIG. 3A-3B - three with the most significant mucoadhesive forces (Spirulina Arthospira, Kombu, and giant Kelp) and three exhibiting the lowest mucoadhesive forces (Sargassum, Gracilaria, Chondracanthus Chamissoi). All six aNPs were associated/encapsulated with Fluorescein Isothiocyanate dextran - FITC-Dextran - (FD40) and incubated with Caco-2 cells in two different incubation ratios of aNPs: Caco-2 cells: 1:1 ratio (FIG. 4A) and 100:1 ratio (FIG. 4B), respectively.

FIG. 4C shows the release profile of (40kDa) FITC-Dextran (FD40) encapsulated by spirulina Arthospira aNPs. FIG. 5A schematically illustrates the encapsulation of FITC-Dextran by mixing spirulina Arthospira aNPs with FITC-Dextran, followed by sonication and ultra-centrifugation of the mixture, according to some embodiments.

FIG. 5B shows bar charts presenting Spirulina Arthospira NPs encapsulation efficiency (EE%) of hydrophilic FITC-Dextran having three different molecular weights (MWs) of 4 kDa, 40 kDa, and 250 kDa, representing a characteristic MW of a conventional protein-based drug such as a peptide, a protein, and an antibody, respectively.

FIG. 6 shows bar graphs presenting mucoadhesive fracture of NPs made of Spirulina Arthospira (SNPs), hybrid NPs made of a combination of Spirulina Arthospira and Chlorella at a ratio of 1:1 (50%:50% (w/w)), chlorella NPs (CNPs), and control (water) respectively.

FIG. 7 shows bar graphs presenting mucoadhesive fracture of NPs made of Spirulina Arthospira (SNPs), a combination of 1:1 (50%:50% (w/w)) Spirulina with Chlorella, Chlorella NPs (CNPs), 5% chitosan, an inedible alga, 5% alginate, 5% gelatine, Nori algae, and control (water).

FIG. 8 schematically illustrates the preparation of NPs made of alga(e) (aNPs). aNPs are being produced, isolated/extracted, 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 (i.e., algal cell/biomass in water) or algal cell lysate, which is then subjected to lysis, homogenization, and fractionation, while certain fractions, namely those containing the aNPs and including non-soluble and amphiphilic components such as membranes and membrane-associated proteins are continuously being collected/ purified (reference is made to the method of preparation of aNPs as well as to the protocol of preparing aNPs made of alga(e) in the material and method section).

DETAILED DESCRIPTION

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. Definitions

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 particle” means one or more particles.

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.

As used herein, "optional" or "optionally" means that the subsequently described event or circumstance does or does not occur and that the description includes instances where said event or circumstance occurs and instances where it does not.

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" when referring to a measurable value such as an amount, a temporal duration, and the like, is 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.

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.

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.

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.

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 animals/vertebrates, including , for example, but not limited to: farm animals, pets and the like.

In some embodiments, the subject comprises mammals and/or humans. Each possibility is a separate embodiment. In some embodiments, the subject comprises a human.

As used herein, the term "treating" refers to an approach for obtaining beneficial or desired results, 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).

The term “treatment” as used herein, refers to both therapeutic treatment and prophylactic 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. As used herein, the terms “prevent”, “reduce”, “attenuate”, “ameliorate”, and “inhibit” may be used interchangeably.

As used herein, the term “composition” is intended to be used herein in its broader sense to include the aNPs of the present invention, whether encapsulated with an active ingredient (for example a pharmaceutical drug) or not encapsulated with an active ingredient, and whether formulated in a conventional manner using one or more physiologically acceptable excipient/carriers/stabilizer, which facilitate processing of the active compounds into preparations that can be used pharmaceutically, or not.

The term “active ingredient” as used herein refers to an effective ingredient/agent associated/encapsulated with the alga(e) nanoparticles (aNPs), and capable of inducing a sought-after effect upon administration, the effect may be, for example, a therapeutic effect achieved by a pharmaceutical; or a diagnostic effect achieved by tagging and reporting, for example, of a mucosal epithelium tissue; or a nutritional-related effect achieved by a food supplement. Non-limiting examples of active ingredient include drugs/therapeutic agents, small molecules, biologies, or other substances such as reporter molecules and food additives or alternatives, but especially suitable are biologies, particularly protein-based drugs.

As used herein, the term “administration/administering” to a subject can be carried out using known procedures, at dosages, and for periods of time effective to provide the desired effect. An effective amount of the aNPs of the present invention encapsulated with an active ingredient and the therapeutic composition including the same, 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 encapsulated with an active ingredient or the therapeutic composition comprising the same to treat the condition/disease in the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. The administration/administering includes routes of administration that allow the compositions of the invention to perform their intended function. In some embodiments, a variety of routes of administration are possible, including, but not necessarily limited to oral, nasal, buccal, and/or vaginal. Formulations may include, for example, but are not limited to tablets, capsules, and the like, for oral administration, and topical agents such as spray, drops, creams, ointment, oil, and the like, for topical administration to nasal and buccal cavities, and/or vaginal tissue.

The term “pharmaceutically acceptable carrier” refers to any carrier conventional used in the production of pharmaceutical compositions as so it is physiologically acceptable to the subject and is also compatible with the activity of the active ingredient. Also, such a carrier must not interfere with the ability of the aNPs of the present invention to perform their intended activity, namely, the binding to a mucosal epithelium tissue. A non-limiting example of a pharmaceutically acceptable carrier is buffered or unbuffered normal saline (approximately 0.9% which is about 0.15M NaCl). Pharmaceutically acceptable carriers, excipients or stabilizers are well known in the art, for example Remington's Pharmaceutical Sciences, 16th edition, Osol, A Ed. (1980).

According to an aspect of the disclosure, there are provided nanoparticles (NPs) made of alga(e) (aNPs), including an average particle diameter of less than about 157 nm and/or surface charge more negative than about -30 mV and wherein the aNPs are capable of adhering to mucosal epithelium tissue; and wherein the alga(e) comprises one or more species belonging to Spirulina Arthrospira . In specific embodiments, the alga(e) comprises Spirulina Arthrospira Platensis.

According to an aspect of the disclosure, there are provided nanoparticles (NPs) made of alga(e) (aNPs), including an average particle diameter of less than about 650 nm and/or surface charge more negative than about -10 mV and wherein the aNPs are capable of adhering to mucosal epithelium tissue; and wherein the alga(e) comprises one or more species belonging to a genus selected from Chondracanthus, Gracilaria, Chondrus, Undaria, Sargassum, Laminaria, Ecklonia, Macrocystis, Palmaria, Gigartina, Spirulina Arthrospira, and Chlorella, or any combination thereof. In specific embodiments, the alga(e) comprises species belonging to a genus selected from Spirulina Arthrospira, Laminaria, and Gracilaria, or any combination thereof.

According to an aspect of the disclosure, there are provided nanoparticles (NPs) made of alga(e) (aNPs), including an average particle diameter of less than about 1000 nm and/or surface charge more negative than about -10 mV and wherein the aNPs are capable of adhering to mucosal epithelium tissue.

As used herein, the term Alga(e) nanoparticles (aNPs) or “made of alga(e)” refers to nanoparticles (NPs) containing (comprise or consist essentially of) non- soluble and amphiphilic components, such as, but not necessarily limited to membranes and membrane proteins derived from one or more alga(e) species. Such aNPs can adhere to mucosal epithelium tissue. The NPs made of the alga(e) may or may not be prepared according to the herein disclosed method of preparation wherein a non-soluble and amphiphilic fraction comprising membranes and membrane proteins is isolated from alga(e) cells/biomass or from a starting material derived from the alga(e) cells/biomass. The terms “Nanoparticles made of alga(e)” (aNPs), “Alga(e) derived nanoparticles” (aNPs), “Algal-based NPs” (aNPs), and “Alga(e) nanoparticles (aNPs)” are interchangeably used.

The herein disclosed aNPs comprise or consist essentially of non-soluble and amphiphilic alga(e) components comprising membranes and membranal proteins. Each possibility is a separate embodiment.

In some embodiments, the aNPs comprise non-soluble and amphiphilic alga(e) components. In some embodiments, the aNPs consist essentially of non-soluble and amphiphilic alga(e) components.

In some embodiments, non-soluble and amphiphilic alga(e) components comprise membranes and membrane proteins of the alga(e).

In some embodiments, non-soluble and amphiphilic alga(e) components comprise membranes and membrane proteins of the alga(e), and further comprise non-membrane proteins and polysaccharides of the alga(e).

According to some embodiments, the term “non-soluble and amphiphilic components” may 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.

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. Reference is made to Example 8 presenting the protein content of the aNPs as identified by LC-MS. As used herein, the term “non-membrane proteins” refers to proteins that are not membrane proteins. In some embodiments, the aNPs comprise a plurality of different membrane proteins of the alga(e). In some embodiments, the aNPs further comprise non-membrane proteins of the alga(e).

As used herein, the term “plurality” refers to at least two (two or more), and may relate to 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.

In some embodiments, the aNPs have an average particle diameter of less than about 1000 nm or less than 650 nm, and/or have surface charge more negative than about -9 mV (or more than about +9 mV), and are capable of adhering to mucosal epithelium tissue with a fracture strength of more than about 200 pN/mm² when exposed to condition that replicate human GI forces (e.g., ex vivo exposure of an intestine tissue of a vertebrate, for example, mice, to an applied force of between about 20 mN and about 200 mN). Each possibility is a separate embodiment.

Optionally, the herein disclosed aNPs may be devoid of one or more stated material, in some embodiments, they may be devoid of a specific polymer addition thereto, or that they may be devoid of a specific polymer by depletion thereof from an aNPs sample. Each possibility is a separate embodiment.

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.

In some embodiments, the algae may include species or genus belonging, for example, but not limited to red algae (division Rhodophyta including class Florideophyceae), brown algae (including class Phaeophyceae), green algae (including division Chlorophyta), and bluegreen algae (cyanobacteria including the family Spirulinaceae). 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 1A, or from any combination thereof.

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

In some embodiment, the alga(e) comprises edible alga(e).

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

In some embodiments, the algae comprise 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.

In some embodiments, the algae comprise 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 comprise one or more species or genus of cyanobacteria.

In some other specific embodiments, the algae comprise one or more species belonging to a genus selected from any one or more of: Chondracanthus, Gracilaria, Chondrus, Undaria, Sargassum, Laminaria, Ecklonia, Macrocystis, Palmaria, Gigartina, Spirulina Arthrospira, Chlorella, and Haematococcus, or any combination thereof. Each possibility is a separate embodiment.

In some other specific embodiments, the algae comprise one or more species belonging to a genus selected from any one or more of: Chondracanthus, Gracilaria, Chondrus, Undaria, Sargassum, Laminaria, Ecklonia, Macrocystis, Palmaria, Gigartina, Spirulina Arthrospira, and Chlorella, or any combination thereof. Each possibility is a separate embodiment.

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

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).

In some embodiments, the alga(e) comprises alga(e) cells/biomass, wherein the alga(e) cells/biomass or the aNPs are substantially devoid of polymer addition thereto. In some embodiments, the alga(e) comprises alga(e) cells/biomass, wherein the alga(e) cells/biomass or the aNPs are devoid of polymer addition thereto. In some embodiments, the alga(e) consists of alga(e) cells/biomass. In some embodiments, the alga(e) consists essentially of alga(e) cells/biomass.

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, the nanoparticles (NPs) made of alga(e) (aNPs) include nonsoluble and amphiphilic molecules of the alga(e)/ alga(e) cells/biomass including glycosylated forms thereof.

In some embodiments, the nanoparticles (NPs) made of alga(e) (aNPs) include nonsoluble and amphiphilic molecules of the alga(e)/ alga(e) cells/biomass and devoid of one or more material/substance.

In some embodiments, nanoparticles (NPs) made of alga(e) (aNPs) consist essentially of non-soluble and amphiphilic components of the alga(e)/ alga(e) cells/biomass, including glycosylated forms thereof. Each possibility is a separate embodiment.

In some embodiments, the nanoparticles (NPs) made of alga(e) (aNPs) include membranes and proteins of the alga(e)/ alga(e) cells/biomass.

In some embodiments, the nanoparticles (NPs) made of alga(e) (aNPs) include membranes and proteins of the alga(e)/ alga(e) cells/biomass and devoid of one or more material/molecule .

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. The terms “NPs made of alga(e)” (“aNPs”), “algal-based NPs” (“aNPs”) may be interchangeably used.

According to some embodiments, disclosed are Nanoparticles made of alga(e) (aNPs), comprising an average particle diameter of less than about 650 nm and/or surface charge more negative than about -10 mV, wherein the aNPs are capable of adhering to mucosal epithelium tissue, and wherein the aNPs comprise membranes and proteins of the alga(e).

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, that further contribute/facilitate/improves the structure or function of the generated/obtained aNPs.

According to some embodiments, NPs made of alga(e) may be devoid of one or more specified materials or substances (i.e., devoid of addition thereto, alternatively, devoid by depletion therefrom).

As a non-limiting example, in some embodiments, aNPs made of H. pluvialis may be devoid of the lipid-soluble keto-carotenoid pigment Astaxanthin, by depletion of Astaxanthin therefrom.

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 or function of the aNPs or the method of preparing the same.

In some embodiments, 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.

In some embodiments, the alga(e) cells/biomass is essentially/substantially devoid of polymer. 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.

In some embodiments, the alga(e) cells/biomass is devoid of addition of any polymer or biopolymer, natural or synthetic, that stabilizes/facilitates/improves the structure or function of the aNPs.

In some embodiments, the alga(e) cells/biomass is essentially devoid of polymer, including, but not limited to, PLA, PLGA, PEG, PCL, alginate, gelatin, chitosan, Astaxanthin, or any combination thereof. Each possibility is a separate embodiment. In some embodiments, the polymer is purified.

In some embodiments, the nanoparticles (NPs) made of alga(e) (aNPs) may be devoid of one or more specified materials, including, for example, but not limited to, Astaxanthin.

In accordance in some embodiments, the alga(e) consists of cells/biomass, including dry, dissolved, and lysed forms of the cells/biomass. Each possibility is a separate embodiment.

In some embodiments, the nanoparticles (NPs) are made of or include one or more alga(e) species/types. In some embodiments, the nanoparticles (NPs) are made of or include one or more alga(e).

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.

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 1A above.

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. 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.

According to some embodiments, the alga(e) / alga(e) cell/biomass comprises a combination of at least about 20% (w/w), at least about 30% (w/w), at least about 40% (w/w), at least about 50% (w/w), at least about 55% (w/w), at least about 60% (w/w), at least about 65% (w/w), at least about 70% (w/w), at least about 75% (w/w), at least about 80% (w/w), at least about 85% (w/w), at least about 90% (w/w), at least about 95% (w/w), at least about 96% (w/w), at least about 97% (w/w), at least about 98% (w/w), or at least about 99% (w/w) one or more Spirulina Arthospira species, in combination with any one of the 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, and Chlorella, or any combination thereof. Each possibility is a separate embodiment.

In some embodiments, the alga(e) comprises one or more types of alga(e) selected from a group consisting of Spirulina Arthospira, Kombo, Giant Kelp, Dulse, Gigartina Red Marine, Gracilaria, Wakame, Kelp Laminaria Digitata, chlorella, and H. pluvialis, or any combination thereof. Each possibility is a separate embodiment.

In some embodiments, the alga(e) comprises one or more types of alga(e) selected from a group consisting of Spirulina Arthospira, Kombo, Giant Kelp, Dulse, Gigartina Red Marine, Gracilaria, Wakame, Kelp Laminaria Digitata, and chlorella, or any combination thereof. Each possibility is a separate embodiment.

In some embodiments, the alga(e) comprises one or more types of alga(e) selected from a group consisting of Spirulina Arthospira, Kombo, Giant Kelp, Dulse, Gigartina Red Marine, Gracilaria, and H. pluvialis, or any combination thereof. Each possibility is a separate embodiment.

In some embodiments, the alga(e) comprises one or more types of alga(e) selected from a group consisting of Spirulina Arthospira, Kombo, Giant Kelp, Dulse, Gigartina Red Marine, and Gracilaria, or any combination thereof. Each possibility is a separate embodiment. In some embodiments, the alga(e) comprises or more types of alga(e) selected from a group consisting of Spirulina Arthospira, Kombo, Giant Kelp, and Gracilaria, or any combination thereof. Each possibility is a separate embodiment.

In some embodiments, the alga(e) comprises one or more types of alga(e) selected from a group consisting of Spirulina Arthospira, Kombo, and Giant Kelp, or any combination thereof. Each possibility is a separate embodiment.

In some embodiments, the alga(e) comprises one or more types of alga(e) selected from Spirulina Arthospira Platensis and Chlorella.

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.

In some embodiments, the cyanobacteria comprise (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). Each possibility is a separate embodiment.

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). Each possibility is a separate embodiment.

In some embodiments, the alga(e) comprises Spirulina Arthospira. In some embodiments, the alga(e) comprises one or more species belonging to Spirulina Arthospira

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

In some embodiments, species belonging to Spirulina Arthrospira include but are not limited to: 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.

According to some embodiments, the alga(e) comprises at least about 50% (w/w) of one or more species belonging to Spirulina Arthospira. In some embodiments, the alga(e) or the aNPs comprises at least about 20% (w/w), at least about 30% (w/w), at least about 40% (w/w), at least about 50% (w/w), at least about 55% (w/w), at least about 60% (w/w), at least about 65% (w/w), at least about 70% (w/w), at least about 75% (w/w), at least about 80% (w/w), at least about 85% (w/w), at least about 90% (w/w), at least about 95% (w/w), at least about 96% (w/w), at least about 97% (w/w), at least about 98% (w/w), or at least about 99% (w/w) of one or more species belonging to Spirulina Arthospira, relative to other alga(e) species of the aNPs. Each possibility is a separate embodiment.

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. Each possibility is a separate embodiment. 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.

In some embodiments, the alga(e) comprises (or consists essentially of) Spirulina Arthospira Platensis. Each possibility is a separate embodiment. In some embodiments, the alga(e) consists of Spirulina Arthospira Platensis.

In some embodiments, the alga(e) comprises a combination of any one or more species belonging to Spirulina Arthospira with any alga(e), including multicellular or unicellular, edible, or non-edible. Each possibility is a separate embodiment.

In some embodiments, the alga(e) comprises a combination of any one or more species belonging to Spirulina Arthospira with Chlorella. In some embodiments, the alga(e) comprises a combination of Spirulina Arthospira Platensis with Chlorella.

According to some embodiments, the aNPs comprise 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. According to some embodiments, the average particle diameter is less than about 650 nm. In some embodiments, the average particle diameter is less than 169 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 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.

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 146 nm, or between 80 nm and 146 nm, or between about 126 nm and about 605 nm, or in the range between 80 nm and about 130 nm, or in the range between 80 nm and about 170 nm. Each possibility is a separate embodiment.

In some embodiments, the average particle diameter is in the range between 100 nm and 1000 nm, or in the range between 100 nm and about 650 nm, or in the range between 100 nm 170 nm, or in the range between 115 nm and 170 nm, or in the range between 100 nm and 160 nm, or in the range between 120 nm and 160 nm, or in the range between 100 nm and 135 nm, or in the range between 125 nm and 135 nm, or in the range between 100 nm and 130 nm, or in the range between 125 nm and 130 nm, or in the range between 120 nm and 130 nm, or in the range between 105 nm and 145 nm. Each possibility is a separate embodiment.

In some embodiments, aNPs comprising a combination of one or more species of Spirulina Arthospira and chlorella aNPs have the smallest average particle diameter of less than 160 nm or 170 nm. Each possibility is a separate embodiment. In some embodiments, the average particle diameter of aNPs comprising essentially of Spirulina Arthospira is about 126 nm.

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 -22mV, 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.

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.

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.

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.

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 Drug Delivery System (DDS), (especially, for example, Spirulina NPs that had the smallest particle size of 126 + 2 nm among the tested algae).

In addition, the negative surface charge of the aNPs is also advantageous as a negative surface charge correlates with mucoadhesiveness by forming hydrogen bonds with mucins, which can attach to negatively charged molecules via positively charged amino acids in the terminal domains. Moreover, loose mucins in-vivo (e.g., secreted by epithelial tissue into the lumen of the intestine, the nasal/buccal cavity, vaginal canal, or similar) might adhere, and coat (termed mucin biocoating) charged NPs and even neutralize their effective surface charge, (it is also surprising in this regard that Spirulina NPs, for example, had the most negative surface charge of -38 + 3 mV among the tested algae). The disclosed advantageous structural characteristics of the NPs made of alga(e), including their nano-size, negative surface charge, and spherical shape, predict plausible effective functionality as oral DDS to mucosal tissue, especially for Spirulina Arthospira aNPs.

According to some embodiments, the aNPs have a poly dispersity index (PDI) of less than 0.7. According to some embodiments, the aNPs are characterized by a poly dispersity index (PDI) of less than 0.3.

According to some embodiments, the aNPs are characterized by a polydispersity 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.

According to some embodiments, the aNPs are characterized by a polydispersity 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 0.12 and about 0.3.

According to some embodiments, the aNPs comprise a polydispersity index (PDI) of about 0.14. In some embodiments, the one or more alga(e) species comprises Spirulina Arthospira.

According to some embodiments, the aNPs comprise a protein content (mg/mL) of more than about 0.01 mg/mL.

According to some embodiments, the aNPs comprise a protein content (mg/mL) of more than about 0.01 mg/mL, more than about 0.04 mg/mL, more than about 0.07 mg/mL, more than about 0.1 mg/mL, more than about 0.15 mg/mL, more than about 0.44 mg/mL, more than about 0.68 mg/mL, more than about 0.77 mg/mL, more than about 0.8 mg/mL, more than about 0.9 mg/mL, more than about 1.0 mg/mL, more than about 1.3 mg/mL, more than about 1.6 mg/mL, more than about 2.0 mg/mL, more than about 2.2 mg/mL, more than about 2.4 mg/mL, more than about 2.6 mg/mL, more than about 2.8 mg/mL, more than about 3.0 mg/mL, more than about 3.2 mg/mL, more than about 3.4 mg/mL, more than about 3.6 mg/mL, more than about 3.8 mg/mL, more than about 4.0 mg/mL. Each possibility is a separate embodiment. According to some embodiments, the aNPs comprise a protein content (mg/mL) of between about 0.01 mg/mL and about 4.0 mg/mL.

According to some embodiments, the Spirulina Arthospira aNPs comprise a protein content of about 2.6 mg/mL.

Reference is now made to FIGs. 1A-1C and Table IB present the characterization of aNPs structural properties, including its size, shape, surface charge, protein content, and PDI.

According to other embodiments, the aNPs are capable of adhering to mucosal epithelium tissue.

According to other embodiments, the aNPs are capable of adhering to mucosal epithelium tissue at a fracture strength of more than about 200 pN/mnr when exposed to forces between 20 mN and 200 mN.

In some embodiments, the aNPs are capable of adhering to mucosal epithelium tissue at a fracture strength of more than about 200 pN/mm² when exposed to forces equal to or between 20 mN and 200 mN. Each possibility is a separate embodiment.

In some embodiments, the aNPs are capable of adhering to the mucosal epithelium tissue at a characteristic fracture strength of more than about 200 pN/mnr when human GI forces are replicated, and wherein said replicated human GI forces comprise exposure to an applied force equal to or between 20 mN and 200 mN. Each possibility is a separate embodiment.

In some embodiments, the mucosal epithelium tissue is a gastrointestinal (GI) tissue. In some embodiments, the mucosal epithelium tissue is an intestinal tissue.

In some embodiments, the aNPs are capable of adhering to mucosal epithelium tissue at a fracture strength of more than about 200 pN/mm², more than about 240 pN/mm², more than about 280 pN/mm², more than about 320 pN/mm², more than about 380 pN/mm², more than about 450 pN/mm², more than about 500 pN/mm², more than about 600 pN/mm², more than about 700 pN/mm², more than about 800 pN/mm², more than about 1000 pN/mm², more than about 1200 pN/mm², more than about 1500 pN/mm², more than about 1800 pN/mm², more than about 2100 pN/mm², more than about 2500 pN/mm², more than about 2700 pN/mm², more than about 3000 N/mrir. or more than about 3500 N/mrir. when exposed to forces equal to or between 20 mN and 200 mN. Each possibility is a separate embodiment.

In some embodiments, the aNPs are capable of adhering to mucosal epithelium tissue at a fracture strength of between about 100 p N/mnr and about 4500 p N/mnr or between about 500 p N/mnr and about 4500 p N/mnr when exposed to forces between 20 mN and 200 mN.

In some embodiments, Spirulina, Kombu, and Giant Kelp have the highest capability of adhering to mucosal epithelium tissue.

In some embodiments, the Spirulina Arthospira aNPs are capable of adhering to mucosal epithelium tissue at a fracture strength of about 1786 ± 81 or about 3127 ± 272 pN/mm2 for 20 mN and 200 mN, respectively. Each possibility is a separate embodiment.

In some embodiments, the Spirulina Arthospira aNPs exhibit superior capability of adhering to mucosal epithelium tissue, wherein the superior capability includes adhering to mucosal epithelium tissue at between about 2-fold and 10-fold stronger than the other tested aNPs.

In some embodiments, the Spirulina Arthospira aNPs are capable of adhering to mucosal epithelium tissue at between about 2-fold and 10-fold stronger than the other tested aNPs.

In some embodiments, the Spirulina Arthospira aNPs are capable of adhering to mucosal epithelium tissue at a fracture strength of between about 500 p N/mnr and about 2000 p N/mnr when exposed to forces of 20 mN.

In some embodiments, the Spirulina Arthospira aNPs capable of adhering to mucosal epithelium tissue at a fracture strength of between about 1500 pN/mnr and about 3500 pN/mnr when exposed to forces of 200 mN.

According to some embodiments, increased mucoadhesion fracture strengths comprise increased disentanglement of mucins and the formation of additional chemical bonds. In some embodiments, increased disentanglement of mucins increases the number of surface interactions between the aNPs and the mucosal epithelial layer. In some embodiments, the mucosal epithelium tissue comprises a mucosal epithelium of a vertebrate. In some embodiments, the vertebrate is selected from one or more fish, amphibians, reptiles, birds, and mammals, or any combination thereof. Each possibility is a separate embodiment.

In some embodiments, the mucosal epithelium tissue comprises a mucosal epithelium of a mammal. In some embodiments, the mammal is selected from one or more of humans, mice, pigs, sheep, or any combination thereof. Each possibility is a separate embodiment.

In some embodiments, the mucosal epithelium tissue comprises a mucosal epithelium of a human.

In some embodiments, the mucosal epithelium tissue is selected from one or more of gastrointestinal (GI) epithelium tissue, nasal epithelium tissue, buccal epithelium tissue, vaginal epithelium tissue, or any combination thereof. Each possibility is a separate embodiment.

In some embodiments, the mucosal epithelium tissue is gastrointestinal (GI) epithelium tissue.

According to some embodiments, the aNPs are capable of adhering to the mucosal epithelium tissue of a vertebrate. According to some embodiments, the aNPs are capable of adhering to the mucosal epithelium tissue of a mammal. According to some embodiments, the aNPs are capable of adhering to the mucosal epithelium tissue of a human.

In some embodiments, the mucosal epithelium tissue comprises a vertebrate. In some embodiments, the mucosal epithelium tissue comprises a mammal. In some embodiments, the mucosal epithelium tissue comprises a human.

Reference is now made to FIGs. 2A-2C and FIGs. 3A-3C related to the capability of the aNPs of adhering to mucosal epithelium tissue.

According to some embodiments, the mouse Mucin2 sequence exhibited the highest level of homology to human Mucin2. The Mucin2 sequences of a sheep and a pig are relatively lower but noteworthy similarity to human Mucin2. Reference is now made to Table 2 related to the level of homology of human Mucin2 to other mammals.

According to some embodiments, the disclosed aNPs provide advantageous and surprising structural and functional characteristics, including their spherical shape, nano-size, negative surface charge, and capability of adhering to mucosal epithelium tissue. These advantageous and surprising structural and functional characteristics of the NPs make it suitable as a drug delivery system (DDS) to mucosal tissue. Each possibility is a separate embodiment.

In some embodiments, the provided aNPs are suitable for use as a drug delivery system (DDS) to mucosal tissue, thereby facilitating local and/or systemic effects. Each possibility is a separate embodiment.

In some embodiments, the delivery, or the drug delivery system (DDS) to mucosal tissue, includes local and/or systemic delivery /effect. Each possibility is a separate embodiment.

In some embodiments, the drug delivery system (DDS) is an oral DDS targeting intestinal mucosal epithelium. In some embodiments, the delivery comprises oral delivery.

As used herein, the term “delivery” is related to the term “drug delivery system” (“DDS”) and refers to adherence of the aNPs to the mucosal epithelium tissue and/or cellular uptake of one or more active ingredients encapsulated or associated with the aNPs or attached to it. Each possibility is a separate embodiment.

In some embodiments, the delivery includes local delivery /effect. In some embodiments, the delivery includes a systemic effect.

According to some embodiments, the aNPs comprise 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). The terms “comprise”, “associated”, and “encapsulated” are interchangeably used.

In some other related embodiments, the aNPs are capable of facilitating the delivery of one or more active ingredients encapsulated therein to the mucosal epithelium tissue. In some embodiments, the delivery comprises encapsulation/association of one or more active ingredients with the aNPs including attachment of the active ingredient(s) to the aNPs membrane, adherence of the aNPs to the mucosal epithelium tissue, and cellular uptake of the one or more active ingredient(s).

In some embodiments, the delivery comprises encapsulation/association of one or more active ingredients with the aNPs including enclosure of the active ingredient(s) in the aNPs hydrophilic core, adherence of the aNPs to the mucosal epithelium tissue, and cellular uptake of the one or more active ingredient(s).

In some embodiments, the cellular uptake includes the release of the active ingredient(s). In other embodiments, the cellular uptake includes transversal of the epithelium.

In some related embodiments, the delivery of one or more active ingredients to the mucosal epithelium tissue may include adherence of the aNPs to the mucosal epithelium tissue and cellular uptake of the one or more active ingredient(s), including the release of the active ingredient(s).

In some related embodiments, the delivery of one or more active ingredients to the mucosal epithelium tissue may include adherence of the aNPs to the mucosal epithelium tissue and cellular uptake of the one or more active ingredient(s), including transversal of the epithelium of the one or more active ingredient(s).

The delivery of the active ingredient to the mucosal epithelium tissue may cause a local effect and/or systemic effect. Each possibility is a separate embodiment. In some embodiments, local delivery comprises cellular uptake by epithelial cells. In some embodiments, systemic delivery comprises cellular uptake by epithelial cells and further transversing intestinal epithelium.

According to some embodiments, aNPs having high mucoadhesive fracture strength, including those selected from Spirulina Arthospira, Kombu, and Kelp, exhibited up to about 100% cellular uptake to mucosal epithelium tissue when incubated at ratios of in the range of between 1:1 and 100:1 of aNPs : cells, respectively. Each possibility is a separate embodiment.

According to some embodiments, aNPs having high mucoadhesive fracture strength, including those selected from: Spirulina Arthospira, Kombu, Kelp and Gracilaria, exhibited up to about 100% cellular uptake to mucosal epithelium tissue when incubated at ratios of about 100:1 of aNPs : cells, respectively. Each possibility is a separate embodiment.

It is suggested that a correlation may exist between negative surface charge and mucoadhesive fracture strength and/or cellular uptake.

In some embodiments, the aNPs comprise one or more of Spirulina Arthospira, Kombu, and Kelp, or any combination thereof. Each possibility is a separate embodiment.

In some embodiments, the aNPs comprise one or more of Spirulina Arthospira, Kombu, Kelp, and Gracilaria, or any combination thereof. Each possibility is a separate embodiment.

In some embodiments, Spirulina NPs exhibited a superior ability for delivery of active ingredient(s), including mucoadhesion, cellular uptake, and release of the active ingredient(s) into human epithelial cells.

In some embodiments, the aNPs comprise or consist of Spirulina Arthospira. Each possibility is a separate embodiment.

In some embodiments, one or more active ingredient(s) is being released from the aNPs for a period of at least about 12 hours.

In some embodiments, the aNPs release the one or more active ingredient(s) for a period of at least about 3 hours, for at least about 6 hours, for at least about 9 hours, for at least about 12 hours, for at least about 1 day, for at least about 2 days, for at least about 3 days, for at least about 4 days, for at least about 5 days, for at least about 6 days, for at least about 7 days, for at least about 8 days, for at least about 9 days, or more. Each possibility is a separate embodiment.

In some embodiments, the aNPs release the one or more active ingredient(s) for a period of up to about 30 days. In some embodiments, the aNPs release the one or more active ingredient(s) for a period of up to about 14 days. In some embodiments, the aNPs release the one or more active ingredient(s) for a period of up to about 9 or 10 days.

In some embodiments, aNPs increase intestinal retention time associated with oral drug delivery.

Reference is now made to FIGs 4A-4C and Table 3 related to cellular uptake of active ingredient(s). According to some embodiments, the aNPs comprise 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). In some embodiments, the active ingredient(s) is encapsulated/associated/comprised with the aNPs.

According to some embodiments, as used herein the terms “associated with aNPs” or “encapsulated” or “comprised” 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 may be initiated when the one or more active ingredient(s) are mixed with the alga(e) nanoparticles (aNPs).

In some embodiments, the association with aNPs may improve the active ingredient’s bioavailability and stability by protecting the active ingredient from degradation, providing sustained release, enhancing retention time, and overall prolonging the therapeutic effect. Each possibility is a separate embodiment.

In some embodiments, the “encapsulated” or “associated” may refer to aNPs having/comprising the one or more active ingredient(s) completely or partially enclosed inside the aNPs core; in some embodiments the “encapsulated” or “associated” may refer to aNPs having/comprising the one or more active ingredient(s) completely or partially attached to the outer or inner surface of the aNPs membrane. Each possibility is a separate embodiment.

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). In some embodiments, “encapsulation” or “association” refers to the aNPs comprising one or more active ingredients (i.e., enclosed in the aNPs or attached to it from the internal side or from the outside). The terms “associated”, “comprised” and “encapsulated” may be used interchangeably.

According to some embodiments associating/encapsulating one or more active ingredients comprises mixing or incubating aNPs with one or more active ingredients. Each possibility is a separate embodiment. 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.

In some embodiments, one or more active ingredient(s) are mixed or incubated with the aNPs at a certain ratio.

In some related embodiments, the active ingredient(s) is encapsulated/associated to the aNPs. In some embodiments, the encapsulation/association of the aNPs with the one or more active ingredient(s) includes aNPs having the one or more active ingredient(s) enclosed therein and/or aNPs having the one or more active ingredient(s) attached thereto. Each possibility is a separate embodiments.

In some embodiments, encapsulation/association of the aNPs with the one or more active ingredient(s) includes an active ingredient(s) at least partially enclosed in the aNPs hydrophilic core. In some embodiments, encapsulation/association of the aNPs with the one or more active ingredient(s) includes an active ingredient(s) at least partially attached to the aNPs membrane from the inner side or the outer side. Each possibility is a separate embodiment.

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

In some embodiments, the term “attached” refers more specifically to the active ingredient(s) being at least partially attached to the aNPs membrane from the inner side or the outer side. Each possibility is a separate embodiment.

Association of the disclosed aNPs with Fluorescein Isothiocyanate dextran (FITC- Dextran) was demonstrated.

In some embodiments, the (one or more) active ingredient(s) comprises hydrophilic, hydrophobic, or amphipathic properties. Each possibility is a separate embodiment. In some embodiments, the (one or more) active ingredient(s) is characterized by having hydrophilic properties. In some embodiments, the delivery comprises association/ encapsulation of the one or more active ingredients with the aNPs.

In some embodiments, the aNPs are capable of associating/encapsulating active ingredient(s) having a molecular weight (MWs) of at least about 4 kDa, or at least about 250 kDa, or more. Each possibility is a separate embodiment.

In some embodiments, the (one or more) active ingredient(s) have a MW of between about 4 kDa and about 250 kDa, or between about 4 kDa and about 300 kDa. Each possibility is a separate embodiment.

In some embodiments, encapsulation includes encapsulation efficiency (EE) of at least about 15%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%. Each possibility is a separate embodiment.

In some embodiments, the (one or more) active ingredient(s) is encapsulated with efficiency (EE) of between about 15% to about 100% or between about 15% to about 60%. Each possibility is a separate embodiment.

In some embodiments, the (one or more) active ingredient(s) is carbohydrate. In some embodiments, the (one or more) active ingredient(s) is fluorescent.

Advantageously, this is indicative of the ability to efficiently encapsulate active ingredients having a range of MWs, including, for example, but not limited to, peptides, proteins, and antibodies.

Reference is now made to FIGs 5A-5B related to the encapsulation of active ingredient(s), where association of the aNPs with Fluorescein Isothiocyanate dextran (FITC- Dextran) is demonstrated.

According to some embodiments, NPs made/prepared of more than one type of alga(e) may exhibit hydridic properties.

In some embodiments, more than one type of algae includes a combination of, for example, two, three, four, or more different species of algae. Such a combination of species may yield aNPs having a ‘mixed’ structural and functional properties reflecting the contribution of each one of the algae to the ‘mixture’.

In some embodiments, the alga(e) comprises a combination of Spirulina Arthrospira and chlorella. In some embodiments, Spirulina Arthrospira and chlorella are mixed at a ratio of about 20:80. In some embodiments, Spirulina Arthrospira and chlorella are mixed at a ratio of about 50:50.

In some embodiments, the alga(e) comprises a combination of Spirulina Arthrospira and chlorella mixed at a ratio of at least about 20:80 respectively, at least about 50:50 respectively, at least about 60:40 respectively, at least about 70:30 respectively, at least about 80:20 respectively, or at least about 90:50 respectively.

In some embodiments, the alga(e) comprises a combination of Spirulina Arthrospira and chlorella mixed at a ratio in the range of between about 20:80 and about 99:1, respectively. In some embodiments, the alga(e) comprises a combination of Spirulina Arthrospira and chlorella mixed at a ratio in the range of between about 50:50 and about 99:1, respectively.

In some embodiments, the mucoadhesive fracture of hydridic NPs made of both Spirulina Arthrospira and Chlorella Vulgaris is between about 200 pN/mm² and about 1000 pN/mm² when exposed to an applied force of 20 mN.

Advantageously, hydridic NPs made of a combination of different algae are useful for yielding new sets of structural and functional properties.

Reference is made to FIG. 6 related to aNPs made of more than one type of alga(e) species.

According to some embodiments, aNPs have enhanced capability of adhering to the mucosal epithelium tissue with respect to adhering to at least one of any one of the nanocarriers made of 5% alginate, 5% gelatine, 5% chitosan, Nori algae, Chlorella, a combination of Spirulina with Chlorella, or inedible algae; and wherein the enhanced capability of adhering comprises an increase of at least 3 -fold, at least 5-fold, or at least 7-fold in fracture strength (pN/mm²). Each possibility is a separate embodiment.

Reference is now made to FIG. 7 related to aNPs’ enhanced capabilities with respect to other nanocarriers/nanoparticles, including those made of polymers. The disclosed NPs made of alga(e) may be used for the delivery of one or more active ingredients to mucosal epithelium tissue. In some embodiments, the mucosal epithelium tissue is gastrointestinal (GI) epithelium tissue.

In some embodiments, one or more encapsulated active ingredient(s) is characterized by hydrophilic, hydrophobic, or amphipathic properties. Each possibility is a separate embodiment. In some embodiments, the one or more active ingredient(s) is characterized by having hydrophilic and/or amphipathic properties. Each possibility is a separate embodiment. In some embodiments, the one or more active ingredient(s) is characterized by having hydrophilic properties.

In some embodiments, delivery of one or more active ingredients to mucosal epithelium tissue includes mucosal epithelium tissue selected from one or more of: gastrointestinal (GI) epithelium tissue, nasal epithelium tissue, buccal epithelium tissue, and vaginal epithelium tissue, or any combination thereof. Each possibility is a separate embodiment.

According to some embodiments, the active ingredient comprises one or more of a pharmaceutical/drug, a tag, a food supplement, or any combination thereof. Each possibility is a separate embodiment.

In some embodiments, the active ingredient includes a pharmaceutical. Non limiting examples of a pharmaceutical drug includes small molecules and/or a biological drug or any substance used in the diagnosis, treatment, or prevention of disease and for restoring, correcting, or modifying organic functions.

According to some embodiments, a preferred active ingredient or pharmaceutical may include a biological drug. According to some embodiments, a preferred active ingredient or pharmaceutical may include a protein-based drug.

According to some embodiments, the preferred active ingredient or pharmaceutical may include, for example, but is not limited to drugs known in the art for treating cancer and/or an inflammatory disease. Each possibility is a separate embodiment.

In some embodiments, non-limiting examples of a protein-based drug include a peptide/polypeptide, a protein, an antibody, or any combination thereof. Each possibility is a separate embodiment. Non-limiting examples of protein-based drugs include, but are not limited to Insulin to treat diabetes, rituximab or trastuzumab used in cancer therapy, replacement enzyme therapy for lysosomal storage disorders, peptide hormones like growth hormone for treating growth disorders or erythropoietin for treating anemia, and drugs (e.g., immune-related drugs) to treat inflammatory bowel disease (IBD). Reference is made to Example 9 exemplifying association of aNPs with protein-based drugs.

In some embodiments, the active ingredient or the drug comprises a biological drug. In some embodiments, the drug comprises a biological drug.

In some embodiments, the biological drug includes, for example, but is not limited to, a protein-based drug and/or a nucleic acid-based drug. Each possibility is a separate embodiment. In some embodiments, the biological drug comprises a protein-based drug.

In some embodiments, non-limiting examples of a nucleic acid-based drug include antisense oligonucleotides, such as miRNAs/siRNA.

In some embodiments, the use of aNPs as DDS, including encapsulation of biologies such as peptides, hormones, enzymes, and antibodies, may improve drug bioavailability drug stability by protecting from degradation, providing sustained release or enhanced retention time and overall prolonging the therapeutic effect.

A tag may include, for example, but is not limited to, ultrasound contrast agents or other reporter/fluorescent molecules.

According to another aspect, there is provided a composition comprising the aNPs according to any one of the preceding embodiments and a pharmaceutically acceptable carrier.

In some embodiments, the aNPs or the composition comprising the same, for use in the delivery of one or more active ingredients to mucosal epithelium tissue, wherein the delivery of the one or more active ingredients to the mucosal epithelium tissue comprises adherence of the aNPs to the mucosal epithelium tissue and cellular uptake of the one or more active ingredients; and wherein the aNPs or the composition comprise one or more active ingredient(s). Each possibility is a separate embodiment.

In some embodiments, the one or more active ingredients is released from the aNPs. In some embodiments, the aNPs or the composition comprising the same, wherein the mucosal epithelium tissue is selected from one or more of: gastrointestinal (GI) epithelium tissue, nasal epithelium tissue, buccal epithelium tissue, and vaginal epithelium tissue, or any combination thereof. Each possibility is a separate embodiment.

In some embodiments, the aNPs or the composition comprising the same, for use in treating, attenuating, and/or preventing the progression of a gastrointestinal (Gl)-disease in a subject in need thereof, and wherein the aNPs or the composition comprise one or more active ingredient(s). Each possibility is a separate embodiment.

In some embodiments, the aNPs, or the composition comprising the same, for use in treating, attenuating, and/or preventing progression of a disease in a subject in need thereof. Each possibility is a separate embodiment. In some embodiments, the aNPs or the composition comprising the same, for use in treating IBD and/or cancer. Each possibility is a separate embodiment. In some embodiments, the aNPs or the composition comprising the same, for use in treating.

In some embodiments, the aNPs or the composition comprising the same wherein the Gl-disease is selected from Inflammatory Bowel Disease (IBD) and/or cancer. Each possibility is a separate embodiment.

In some embodiments, the aNPs, or the composition comprising the same, for use in a method of treatment, wherein the administration comprises orally administering a therapeutically effective amount of encapsulated aNPs.

In some embodiments, the aNPs or the composition comprising the same for use in a method of treatment wherein the subject is human.

According to another aspect, there is provided a method for delivery of one or more active ingredients to mucosal epithelium tissue in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of the aNPs or the composition comprising the same, and wherein the delivery of the one or more active ingredients to the mucosal epithelium tissue comprises adherence of the aNPs to the mucosal epithelium tissue and cellular uptake of the one or more active ingredients, and wherein the aNPs or the composition comprise one or more active ingredient(s). Each possibility is a separate embodiment. In some embodiments, the one or more active ingredients is released from the aNPs.

According to a related aspect, there is provided a method for treating, attenuating, and/or preventing progression of gastrointestinal (Gl)-disease in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of the aNPs or the composition comprising the same, wherein the aNPs or the composition comprise one or more active ingredient(s). Each possibility is a separate embodiment.

In some embodiments, the Gl-disease is selected from Inflammatory Bowel Disease (IBD) and/or cancer.

Inflammatory bowel disease (IBD) is a group of chronic autoimmune disorders characterized by inflammation of the colon and small intestine. Non-limiting examples of IBD include the two main IBD representatives, having increasing incidence and prevalence: Ulcerative colitis (UC) and Crohn's disease (CD). In some embodiments, the Gl-disease comprises Ulcerative colitis (UC) and Crohn's disease (CD). Each possibility is a separate embodiment.

In some embodiments, the cancer comprises a tumor of the mucosal epithelium tissue, such as but not limited to cancer of the Gl-tract, the buccal tissue or vaginal tissue. Each possibility is a separate embodiment. In some embodiments, the cancer comprises Gl-cancer.

In these instances (IBD and cancer of mucosal epithelium tissue), the treatment may rely on a local effect of the active ingredient delivered by the adherence of the aNPs of the present invention to the mucosal epithelium tissue.

In other embodiments, a systemic effect of the active ingredient delivered by the adherence of the aNPs of the present invention to the mucosal epithelium tissue may enable treatment of additional diseases or cancer in remote tissues which are not in close proximity to the mucosal epithelium tissue.

In some embodiments the mucosal epithelium tissue comprises pathological un-healthy tissue/cells, damaged or ‘inflicted with a disease/condition’. In some embodiments, the mucosal epithelium tissue comprises cancerous tissue/cells. In some embodiments, the mucosal epithelium tissue comprises inflamed tissue/cells. In some embodiments the mucosal epithelium tissue comprises physiologically normal ‘healthy’ tissue/cells. According to another aspect, there is provided a method for treating, attenuating, and/or preventing progression of a disease in a subject in need thereof. According to another aspect, there is provided a method for treating a disease in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of the aNPs or the composition comprising the same, wherein the aNPs or the composition comprises one or more active ingredient(s), and wherein the treating/administering comprises delivery of the one or more active ingredients to a mucosal epithelium tissue of the subject, and wherein the delivery comprises adherence of the aNPs to the mucosal epithelium tissue and cellular uptake of the one or more active ingredients. In some embodiments, the therapeutic effect comprises a local effect on the mucosal epithelium tissue of, for example, the Gl-tract. In some embodiments, the therapeutic effect comprises a systemic effect.

In some embodiments, there is provided a method for preparing nanoparticles made of alga(e) (aNPs), which are capable of adhering to mucosal tissue.

According to yet another aspect, there is provided a method for preparing nanoparticles made of alga(e) (aNPs), comprising 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 homogenized suspension and collecting supernatant containing the aNPs; (iv) applying/loading the supernatant containing the aNPs onto a chromatography or a density gradient and subjecting it to ultracentrifugation; (v) and collecting a fraction comprising nonsoluble and amphiphilic alga(e) components; thereby isolating aNPs comprising non- soluble and amphiphilic membranes and membrane proteins; wherein the aNPs are capable of adhering to mucosal tissue. Each possibility is a separate embodiment.

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.

In some embodiments, the obtained/isolated nanoparticles comprise membranes and proteins of the obtained alga(e) cells/biomass.

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

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

In some embodiments, the method farther comprises a step of depleting one or more substance or polymer. In some embodiments, the obtained nanoparticles comprise membranes and proteins of the obtained alga(e) cells/biomass, and devoid by depletion of one or more substances/polymers.

In some embodiments, the alga(e) cells/biomass is devoid of addition of a polymer thereto, 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.

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.

In some embodiments, the alga(e) cells/biomass is 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 dry, dissolved, and lysed forms of the cells/biomass.

In some embodiments, the alga(e) cells/biomass is devoid of external addition of any polymer, natural or synthetic, that stabilizes/contributes/facilitates/improves the structure or function of the aNPs.

In some embodiments, the alga(e) cells/biomass is devoid of the addition of any polymer, including, but not limited to, PLA, PLGA, PEG, PCL, alginate, gelatin, and chitosan, Astaxanthin, or any combination thereof. Each possibility is a separate embodiment. 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.

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.

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.

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.

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.

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

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. 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% duty cycle. In some embodiments, sonication comprises transducer at a range between 45% - 55% duty cycle.

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.

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.

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.

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.

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, Giant Kelp, Dulse, Gigartina Red Marine, Spirulina Arthrospira and Chlorella, or any combination thereof. Each possibility is a separate embodiment. In some embodiments, the alga(e) comprises one or more alga(e) selected from Spirulina Arthospira, Kombo, Giant Kelp, and Gracilaria, or any combination thereof. Each possibility is a separate embodiment.

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

In some embodiments, the alga(e) comprises at least about 50% (w/w) of one or more species belonging to Spirulina Arthrospira.

In some embodiments, the alga(e) comprises at least about 20% (w/w), at least about 30% (w/w), at least about 40% (w/w), at least about 50% (w/w), at least about 55% (w/w), at least about 60% (w/w), at least about 65% (w/w), at least about 70% (w/w), at least about 75% (w/w), at least about 80% (w/w), at least about 85% (w/w), at least about 90% (w/w), at least about 95% (w/w), at least about 96% (w/w), at least about 97% (w/w), at least about 98% (w/w), or at least about 99% (w/w), of one or more species belonging to Spirulina Arthospira including to Spirulina Arthospira Platensis, relative to other species. Each possibility is a separate embodiment.

In some embodiments, the alga(e) consists of Spirulina Arthrospira. In some embodiments, the alga(e) comprises 100% (w/w) one or more species belonging to Spirulina Arthospira including to Spirulina Arthospira Platensis, relative to other species.

In some embodiments, species belonging to Spirulina Arthospira include, for example, but are not limited to: Arthospira Platensis, Arthospira Maxima, Arthospira ardissonei, Arthospira erdosensis, Arthospira fusiformis, Arthospira indica, Arthospira innermongoliensis, Arthospira jenneri, Arthospira massartii, or any combination thereof, each possibility is a separate embodiment.

In some embodiments, one or more species belonging to Spirulina Arthospira comprises Spirulina Arthospira Platensis. In some embodiments, one or more species belonging to Spirulina Arthospira consists essentially of Spirulina Arthospira Platensis.

According to some embodiments, disclosed are Nanoparticles made of alga(e) (aNPs), comprising an average particle diameter of less than 146 nm and/or surface charge more negative than about -30 mV, wherein the aNPs are capable of adhering to mucosal epithelium tissue.

According to some embodiments, the aNPs comprise an average particle diameter of about 126 nm and/or surface charge of about -38 mV, wherein the aNPs are capable of adhering to mucosal epithelium tissue; in some embodiments the alga(e) comprises Spirulina Arthospira Platensis.

According to some embodiments related to the method of preparing aNPs, the method further comprising a step of associating/encapsulating one or more active ingredients with the aNPs, wherein said encapsulating comprises mixing the obtained aNPs with one or more active ingredients.

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

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-centrifugation.

In some embodiments, the encapsulating comprises encapsulation efficiency (EE) of between about 15% and about 100%, or between about 15% and about 85%, or between about 15% and about 70%, or between about 30% and about 60%, or between about 35% and about 55%. Each possibility is a separate embodiment.

In some embodiments, the encapsulating comprises encapsulation efficiency (EE) of at least about 15%, at least about 35%, at least about 55%, at least about 75%, at least about 95%. Each possibility is a separate embodiment.

In some embodiments, the ultracentrifugation comprises a centrifugal force of at least about 25,000 g, at least about 50,000 g, at least about 100,000 g, at least about 150,000 g, or at least about 200,000 g. In some embodiments, the ultracentrifugation comprises a centrifugal force of between 25,000 g and about 400,000 g, or between 50,000 g and about 350,000 g, or between about 50,000 g and 300,000 g. Each possibility is a separate embodiment.

In some embodiments, homogenization comprises sonication. According to yet another aspect, there are provided nanoparticles made of algae (aNPs), obtained or obtainable by the method of preparing aNPs.

In some embodiments, the aNPs comprising an average particle diameter of less than about 650 nm and/or surface charge more negative than about -9 mV (or +9 mV), wherein the aNPs are capable of adhering to mucosal epithelium tissue. Each possibility is a separate embodiment.

In some embodiments, the aNPs are spherical. In some embodiments, the aNPs are spherical and have a hydrophilic core.

Reference is now made to FIG. 8 related to the method of preparing NPs made of alga(e) (aNPs).

In some embodiments, the aNPs comprise a plurality of different membrane proteins of the alga(e). In some embodiments, the aNPs comprise two or more membrane proteins selected from any one of the membrane- associated proteins demonstrated in Example 8.

According to some embodiments, the aNPs comprise 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 comprise 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.

In some embodiments, the aNPs comprise about 229 membrane associated proteins. In some embodiments, the plurality of membrane-associated proteins is selected from those demonstrated in Example 8.

Reference is made to Example 8 presenting characterization of aNPs protein content. 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.

EXAMPLES

Materials and Methods

Materials - Dulbecco's Modified Eagle Medium (DMEM), L-Glutamine (L-Glu), and Penicillin-Streptomycin (P/S) were purchased from Sartorius, Israel. Phosphate -buffered saline (PBS), Fetal Bovine Serum (FBS), Bradford reagent, trypsin, 40 kDa Fluorescein Isothiocyanate dextran (FD40), sucrose, and bovine serum albumin (BSA) were purchased. The edible algae used herein were purchased.

Preparation of Algal NPs (aNPs) - to prepare aNPs (see FIG. 8), each algae powder 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.

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.

Mucoadhesion Measurement of aNPs - mucoadhesion measurements were conducted using a texture analyzer instrument. Initially, 50 pL of the NPs solution was spread on a glass plate and dried for 30 minutes. Then, the small intestine of mice, pigs, or sheep was washed in PBS and carefully placed on top of a flat pin head (A=40.7 mm²) so the lumen side faced outwards. The pin was mounted on the texture analyzer probe (capacity of 5 N, by Shimadzu), which was then lowered at a speed of 1.0 mm/s until the tissue came in contact with the aNPs with an applied force of 20 mN and 200 mN for 420 seconds. The contact forces were chosen according to a study conducted to measure the peristaltic forces inside the intestines of a lamb. These forces were measured via an encapsulated prototype with a force sensor, where it was found that the range of peristaltic forces in the small intestine was between 0-180 mN. As mentioned, the mucoadhesive fracture strength was derived from the peak force required to separate two layers.

Association/Encapsulation of Fluorescent Molecule in aNPs - for these experiments, six algae were selected - three with the most significant mucoadhesive forces and three exhibiting the lowest mucoadhesive forces. In some experiments, 3 different FITC-dextran having molecular weights (MWs) of 4 kDa, 40 kDa, and 250 kDa were associated/encapsulated. To evaluate the cellular uptake in Caco-2 cells, FD40 was encapsulated in the chosen aNPs. 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 and sonicated 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 FD40-loaded aNPs were harvested as described earlier. 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 FD40 and analyzed using a fluorometer at excitation and emission wavelengths of 490 nm and 525 nm, respectively. The EE achieved for the tested aNPs ranged from 30 to 55%.

Caco-2 Uptake of aNPs - to prepare the cell culture medium, 450 mL of DMEM was thoroughly mixed with 50 mL of FBS, 5 mL each of L-Glu, and 5 mL of P/S. The cellular uptake of the chosen aNPs was evaluated with Caco-2 cells - the prominent GI in vitro model.

First, Caco-2 cells were seeded in 24-well plates and incubated at a concentration of 2xl0⁵ cells/mL for 48 hours at 37°C in 1 mL of cells medium. Then, the cells were counted using an automated cell counter, and the number of NPs was determined using the DLS instrument. Following these measurements, the ratios 1:1 and 100:1 (aNPs/Caco-2 cells, respectively) were used for each tested alga. Caco-2 cells were incubated with free FD40 at an equivalent amount as encapsulated in the aNPs and used as a control. After three hours of incubation, the wells were carefully washed twice with PBS to remove free FD40 and aNPs. Subsequently, 300 pL of trypsin was added to each well and incubated for five minutes in the incubator to detach the cells. Then, the cell suspension was transferred to an Eppendorf test tube with 1 mL of medium. Cells were obtained by centrifugation at 500 g for 12 minutes and were resuspended in a solution of PBS containing 0.5% BSA. Later, the samples were transferred to 96-well microplates and analyzed under the Fluorescence-Activated Cell Sorter (FACS) instrument to test the degree of internalization of Caco-2 cells to algae NPs.

Protein BLAST Analysis - the mucin2 glycoprotein serves as the primary constituent of the outer mucus layer and imparts viscoelastic properties to mucus. The human mucin2 protein sequence was retrieved from the Entrez repository with the accession number AZL49145.1 in FASTA format. The mouse, pig, and sheep sequences were obtained from the Protein Basic Local Alignment Search Tool (BLAST) (accessions are shown in Table 2). Later, the Protein BLAST was used to search for similarities against the sequences mucin2 from mice, pigs, and sheep. The presented score value was used to assess the degree of similarity between the different mucin sequences.

Statistical Analysis - the data were analyzed using Prism version 9 (by GraphPad). The normality of the data was assessed using the Shapiro-Wilk test. To confirm the homogeneity of variances, the Brown-Forsythe test was used. A one-way ANOVA (two-tailed) was employed to examine differences among multiple groups using the Bonferroni post hoc test. The data represented in the study is mean and standard deviation (SD) of n > 3. All statistical analyses were conducted at a significance level of a = 0.05.

Example 1 - characterization of aNPs structure

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)). Astaxanthin was also used as a starting material for processing by the same method of preparation, serving as a control NPs for the herein described exemplification. The 13 edible aNPs and Astaxanthin based-NPs were comprehensively characterized for their structure and function.

At first, aNPs structural properties were evaluated. The evaluation of their structure included analyses of their size, polydispersity index (PDI), protein content, surface charge (i.e., zeta potential), and shape. The results of the structural evaluation are presented in Table IB below and in FIGs.

1A-1C.

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

* note: Astaxanthin NPs are not alga(e) nanoparticles (aNPs). Astaxanthin is a lipid- soluble keto-carotenoid pigment extracted from H. pluvialis. To leave no doubt, contrary to edible aNPs made of Haematococcus pluvialis as presented in Table 1A, Astaxanthin NPs consist of lipid-soluble keto-carotenoid pigment, and therefore are not encompassed by the scope of the disclosed aNPs of the invention. The Astaxanthin NPs refers to NPs prepared from the lipid-soluble keto-carotenoid pigment, known in the art as Astaxanthin, by using Astaxanthin as starting material and processing thereof by the same method of preparation of aNPs.

As can be seen in Table IB, 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 60.8xl0⁹ NPs/(mLxg), while the protein content/concentration ranged from 0.01 to 3.24 mg/mL. Spirulina Arthospira Platensis NPs are characterized by having the smallest particle size of 126 ± 02 nm (average particle diameter; see also FIG. 1A) and the lowest PDI of 0.14. Furthermore, Spirulina NPs had the highest relative concentration with 60.8 ± 2.9 xlO⁹ NPs/(mLxg).

Additionally, there were differences in protein content between the different algae when the aNPs with the highest protein content were harvested from Astaxanthin (i.e., 3.24 ± 0.02 mg/mL), followed by Spirulina with 2.61 ± 0.81 mg/mL. In all other aNPs, the protein concentration was lower than 1 mg/mL.

Advantageously and surprisingly, 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 Drug Delivery System (DDS).

Also, the shape of Spirulina NPs was visualized using transmission electron microscopy (TEM). As can be seen in FIG. IB, Spirulina NPs are spherical.

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 drug delivery, PDK0.3, and preferably PDK0.2, is considered mono/homodisperse.

Furthermore, Spirulina NPs displayed the highest initial concentration of 60.8 ± 2.9 xlO⁹ NPs/(mL*g), indicating a substantial yield and cost-effective production process.

Finally, the surface charge is another crucial structural property in predicting the effectiveness of an oral DDS. Thus, the zeta potentials were measured in DDW for the harvested aNPs.

As depicted in FIG. 1C, the zeta potential values observed for the aNPs range from -38 to -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. A negative surface charge correlates with mucoadhesiveness by forming hydrogen bonds with mucins, which can attach to negatively charged molecules via positively charged amino acids in the terminal domains. Moreover, loose mucins in-vivo (e.g., secreted by epithelial tissue into the lumen of the intestine, the nasal/buccal cavity, vaginal canal, or similar) might adhere, and coat (termed mucin biocoating) charged NPs and even neutralize their effective surface charge.

Thus, it is postulated that Spirulina NPs would have the highest mucoadhesion amongst the tested aNPs since they showed the highest negative surface charge.

In conclusion, the disclosed advantageous structural characteristics of the edible aNPs, especially of Spirulina NPs, including their spherical shape, nano- size, and negative surface charge, predict plausible functionality for NPs as DDS.

Example 2 - aNPs are capable of adhering to mucosal epithelium tissue

To evaluate the functionality of the aNPs as a DDS to mucosal tissue, and specifically as oral DDS to mucosal tissue, the mucoadhesion fracture strengths of the 14 produced edible aNPs were measured ex-vivo. The measurements were performed utilizing the small intestines of mice, pigs, and sheep using a texture analyzer. The obtained results are presented in FIGs 2A-2C, respectively.

Advantageously, as can be seen in FIG. 2A (left and right), Spirulina Arthrospira Platensis NPs exhibited the highest mucoadhesion fracture strength of 1786 ± 81 and 3127 ± 272 pN/mm2 for 20 mN and 200 mN, respectively. These forces were between about 2-fold and 10-fold stronger than the other tested aNPs. Kombu NPs also exhibited high mucoadhesion of 2691 ± 509 pN/mm² at the applied force of 200 mN (statistically like Spirulina NPs). On the other hand, Wakame (170 ± 47 pN/mm²) and Gracilaria (338 ± 42 pN/mm²) aNPs exhibited the lowest mucoadhesion fracture strength towards the intestines of mice for both applied forces.

Generally, the measured mucoadhesive fracture strength increases when the applied force increases from 20 mN to 200 mN (FIG. 2A; left vs. right ). It is postulated that the increase in mucoadhesion fracture strengths may stem from the disentanglement of mucins and the formation of additional chemical bonds. The increased applied force may augment the contact surface area - by the disentanglement of mucins - thus increasing the number of surface interactions between the aNPs and the intestinal epithelial layer.

Next, the intestines of pigs were used to measure the mucoadhesion fracture strength of the harvested aNPs.

Advantageously, as can be seen in FIG. 2B (left and right) for pig's intestines, Spirulina NPs exhibited the highest mucoadhesion fracture strength of 1539 ± 112 and 1901 ± 100 pN/mm² for 20 mN and 200 mN, respectively. These forces were about 2-fold and 5-fold stronger than observed for the tested aNPs. Dulse and Gigartina Red Marine (1800 ± 83 and 1609 ± 158 pN/mm², respectively) NPs also exhibited high mucoadhesion at the applied force of 200 mN (statistically similar to Spirulina NPs). On the other hand, Sargassum NPs showed the lowest mucoadhesion towards the intestines of pigs for both applied forces, 295 ± 52 and 427 ± 109 pN/mm² for 20 mN and 200 mN, respectively.

As observed for mice intestines, a similar trend was detected herein for pigs' intestines. Enhancing the applied force from 20 mN to 200 mN (FIG. 2B; left vs. right) increased the measured mucoadhesive force. This observation further supports the hypothesis of an increase in contact area when the applied force increases.

Lastly, the intestines of sheep were used to measure the mucoadhesion fracture strength of the harvested aNPs.

Advantageously, as shown in FIG. 2C (left and right) for sheep intestines, Spirulina NPs exhibited the highest mucoadhesion fracture strength towards the intestines of sheep of 828 ± 66 and 1386 ± 46 pN/mm² for 20 mN and 200 mN, respectively. These forces were between about 1.5-fold and 3.5-fold stronger than the rest of the tested aNPs. Notably, Kombu and Astaxanthin (1008 ± 201 and 822 ± 230 pN/mm², respectively) aNPs also exhibited high mucoadhesion at the applied force of 200 mN (statistically like Spirulina NPs). Conversely, Sargassum (243 ± 37 pN/mm²) and Wakame (335 ± 52 pN/mm²) aNPs exhibited the lowest mucoadhesion fracture strengths towards the intestines of sheep for both applied forces. Finally - as observed with mice and pig's intestines - for all the tested aNPs, the measured mucoadhesion fracture strength was also increased when the applied force was increased from 20 mm to 200 mN (FIG. 2C; left vs. right). To further analyze mucoadhesiveness and compare the fracture strengths of the different aNPs at the three ex vivo intestinal animal models, six aNPs were chosen - three aNPs with the highest mucoadhesion (Spirulina, Kombu, and Kelp) and three with the lowest mucoadhesion (Sargassum, Gracilaria, and Chondracanthus Chamissi).

As illustrated in FIGs. 3A-3B, notable variations in mucoadhesion were observed among the ex vivo intestinal models for the highest and lowest aNPs. Figure FIG. 3A highlights the algae with the most robust mucoadhesive properties, with the mouse intestinal model yielding the highest results at 200 mN of about 3400 pN/mm², followed by pig's having about 2000 pN/mm²) and sheep's intestines having about 1500 pN/mm² (red bars; I.-III., respectively). At 20 mN, no significant difference was observed between mucoadhesion in mice and pigs, but both outperformed sheep intestines (blue bars; I.-III., respectively). In contrast, the findings in FIG. 3B are not as definite regarding aNPs with weaker mucoadhesive properties both at 20 mN and 200 mN (blue and red bars; I.-III., respectively).

Advantageously and surprisingly, Spirulina NPs consistently exhibited superior mucoadhesion at 20 mN and particularly at 200 mN (FIG. 3A. I.; blue and red bars, respectively). Meanwhile, other aNPs demonstrated varying mucoadhesion degrees (FIG. 3A. II.-III. and FIG. 3B. I.-III.).

In conclusion, the disclosed advantageous functional characteristics of the edible aNPs, especially of Spirulina Arthrospira, Kombo, Kelp, and even more specifically of Spirulina NPs, including its capability of adhering to mucosal epithelium tissue, predict plausible functionality for NPs as DDS to mucosal tissue, specifically of oral DDS.

Example 3 - homology of human Mucin2 glycoprotein

To evaluate which intestinal animal model is the most suited for predicting mucoadhesion in humans, a comparative analysis was made between sequences of the glycoprotein Mucin2, which is the main component of mucus. The comparison included sequence alignment (using the protein blast tool) between human Mucin2 (a protein consisting of 5130 amino acids (aa) to Mucin2 of the animals of the hereinabove tested ex vivo models - mice, pigs, and sheep intestines. The level of homology of human Mucin2 to Mucin2 of mice, pigs, and sheep can be seen in Table 2 below.

Table 2: sequence alignment of Mucin2 glycoprotein from a mouse, sheep, and a pig against the sequence of human Mucin2.

Animal Sequence ID Length [aa] Identities Positives Gaps [%] Query Score

[%] [%] Cover [%]

Mouse NP_076055.4 4576 80 87 0 43 2532

Sheep XP_042093899.1 5972 49 63 3 87 1495

Pig XP_020938146.1 5759 49 63 3 60 1474

First, it is important to define the specific sequence alignment metrics of the Identities and Positives parameters mentioned in Table 2. Identities refer to the count of positions where the amino acids in compared sequences match precisely. Positives denote positions where the aligned amino acids exhibit similar properties (i.e., both are hydrophobic/acidic, etc.). As shown in Table 2, the mouse Mucin2 sequence displayed more identities and positives than the sheep and pig sequences, indicating the high resemblance of Mucin2 from a mouse to a human.

Moreover, insertions cause “gaps” in the alignment, resulting in missing or incomplete information known as insertion-deletion mutations (indels). The mouse sequence of Mucin2 had no gaps, whereas the sheep and pig sequences displayed 3% gaps, indicating slight structural divergence in those regions. Additionally, query cover refers to the proportion of the query sequence (human Mucin2) that aligns with the compared sequence. Higher coverage implies a more significant overlap, potentially highlighting functional regions. Here, the sheep sequence exhibited the highest coverage (87%), the pig had 60%, and the mouse had the lowest (43%).

Nevertheless, the final score presented in Table 2 considers all the parameters and quantifies the extent of similarity between the compared sequences. The mouse sequence exhibited the highest score (2532), suggesting a substantial likeness to human Mucin2. The sheep and pig sequences had similar final scores (1495 and 1474, respectively), signifying relatively lower but noteworthy similarity to human Mucin2.

It was observed that the intestines of mice showed the highest similarity to human Mucin2, suggesting they are most suited for studying mucoadhesive properties of materials for oral delivery. In further support of this conclusion, it was previously found that mice are preferred for measuring mass transport across the mucosal layer of the intestines since their intestinal resident time, and mucus thickness align well with human conditions.

Example 4 -aNPs are capable of facilitating cellular uptake of active ingredient(s) to human mucosal intestinal

High mucoadhesion force does not guarantee successful drug delivery, including orally. A DDS also needs to facilitate the absorbance of the drug by the epithelial tissue, including its cellular uptake for local effect and further transversal of the intestinal epithelium into the bloodstream for system effect.

To assess whether high mucoadhesion force correlates with enhanced cellular uptake, the same six aNPs mentioned hereinabove Example 2 as having the highest and lowest mucoadhesion force, were evaluated for their ability to deliver a high molecular weight molecule to human mucosal tissue. Namely, the three aNPs with the highest mucoadhesion (Spirulina Arthospira Platensis, Kombu, and Kelp) and the three with the lowest mucoadhesion (Sargassum, Gracilaria, and Chondracanthus Chamissi) were encapsulated or at least partially enclosed in the aNPs core with the 40 kDa hydrophilic Fluorescein Isothiocyanate dextran (FITC-Dextran / FD40) and measured for their ability to perform cellular uptake into human intestinal epithelium Caco-2 cells. Caco-2 cells were exposed to the six aNPs encapsulating FD40 for three hours and then analyzed via FACS for their fluorescent content.

The result can be seen in FIGs. 4A-4B and Table 3 below show the efficiency of the cellular uptake of FD40 at aNPs: Caco-2 cells ratios of 1:1 (FIG. 4A) and 100:1 (FIG. 4B).

Table 3: Mean Fluorescence Intensity of FITC (MFIF) and percentage of cellular uptake into Caco-2 cells of six tested aNPs associated with FD40 and incubated at aNPs to Caco-2 cells ratios of 1:1 and 100:1, respectively.

The three aNPs having high mucoadhesion (c) exhibited 100% cellular uptake into Caco-2 cells when incubated at ratios of 1:1 and 100:1. Conversely, the aNPs with low mucoadhesive forces (Sargassum, Gracilaria, and Chondracanthus Chamissoi) showed only about 0.60%-0.65% cellular uptake into Caco-2 cells when incubated at a 1:1 ratio. Interestingly, when incubated at a 100:1 ratio, Gracilaria NPs showed a 100% cellular uptake, while Sargassum and Chondracanthus remained ineffective. These results are indicative of a correlation that exists between mucoadhesive force and cellular uptake by Caco-2 cells.

Further, the Mean Fluorescence Intensity of FITC (MFIF) in the exposed cells was also calculated and used for further analysis (Table 3). Herein, the measured MFIF of the highly mucoadhesive aNPs was significantly higher than observed of the aNPs with low mucoadhesion.

Surprisingly, Spirulina NPs exhibited an almost 3-fold increase in MFIF when incubated in a 100:1 ratio compared to 1:1 (73629 vs. 207996, respectively), further demonstrating Spirulina NPs' exceptional and advantageous potential as oral DDS. This was not observed for Kombu and Kelp NPs, where there was a decrease of 20-30% in MFIF when the incubation ratio was increased to 100: 1.

It is therefore postulated that this might result from P-glycoprotein (Pgp) efflux as it was found that the Pgp efflux transporter is expressed in Caco-2 cells.

Finally, a release profile of 40kDa FITC-dextran (FD40) encapsulated by Spirulina NPs was determined, in PBS solution. Spirulina NPs exhibited release of the encapsulated FITC- dextran active ingredient into a solution for at least 1 day and up to 9 days (FIG. 4C)

Overall, Spirulina, Kombu, Kelp, and Gracilaria, but especially Spirulina NPs, exhibited a superior ability for delivery of active ingredient(s) associated therewith or at least partially enclosed therein, including mucoadhesion, cellular uptake, and release of the active ingredient(s) into human epithelial cells. Example 5 - Spirulina NPs encapsulating a range of molecular weights (MWs) FITC-dextran

To evaluate the ability of algal-based NPs to encapsulate/associate with active ingredients having a range of molecular weights (MWs), the encapsulation efficiency (EE) of the hydrophilic Fluorescein Isothiocyanate dextran (FITC-Dextran) was tested by associating/encapsulating FITC-Dextran having different MWs ranging from 4 kDa to 250 kDa with the aNPs.

Encapsulation includes mixing/incubating aNPs with FITC-Dextran, as illustrated in FIG. 5A.

As can be seen in FIG. 5B, FITC-Dextran having MW of 4 kDa, 40 kDa, and 250 kDa were all successfully encapsulated, or at least partially enclosed in the core of Spirulina Arthrospira Platensis aNPs with an EE of between 15% to 60%.

Notably, the highest EE of about 55% for FITC-Dextran was achieved for the 40 kDa molecule (FD40).

Advantageously, this is indicative of the ability to efficiently encapsulate/associate active ingredients having a range of MWs, including, for example, but not limited to peptides, proteins, and antibodies, as well as nucleic acid-based drugs such as, but not limited to, DNA vectors and miRNAs/siRNA.

Example 6 - aNPs made of a combination of Spirulina and chlorella exhibit hydridic properties

Next, it was tested whether NPs made/prepared of mor than one alga(e) would exhibit hydridic properties. It is plausible that aNPs made of more than one type of alga (for example, two or three different species of algae) may yield aNPs having a ‘mixed’ structural and functional properties reflecting the contribution of each one of the algae to the ‘mixture’.

This assumption was tested by evaluating the capability of aNPs to adhere to mucosal epithelium tissue. aNPs made of a combination of Spirulina Arthrospira Platensis and chlorella were prepared by mixing both algae at a ratio of 50:50. As can be seen in FIG. 6, the mucoadhesive fracture of hydridic NPs made of both Spirulina Arthrospira Platensis and chlorella Vulgaris (about 450 pN/mnr) is an intermediate that lays within the range (for example, in the current example, approximately an average) set by each Spirulina Arthrospira (about 750 pN/mm²) or chlorella (about 250 pN/mm²).

In accordance, it is reasonable to assume that this result represents a whole set of hydridic characteristics, including both structural and functional properties, such as, but not limited to, hydridic size, hydridic surface charge, hydridic uptake capabilities, and similar.

In other words, while the herein disclosed non-limiting example of a combination of Spirulina Arthrospira and chlorella resulted in aNPs having less mucoadhesive force, the same aNPs may have other (structurally or functionally) ‘enhanced’ properties/capabilities that may make this combination suitable for use as a DSS, for example, this combination may have enhanced cellular uptake.

To conclude, hydridic NPs made of a combination of different algae may be advantageous for yielding new sets of structural and functional properties.

Example 7 - Spirulina aNPs show superior capability of adhering to mucosal epithelium tissue

A comparison was made between edible aNPs made of Spirulina Arthrospira and other nanoparticles/nanocarriers made of a combination of Spirulina Arthrospira Platensis with Chlorella Vulgaris, Chlorella Vulgaris only (CNPs), Nori algae, an inedible alga, or of either one of the polymers alginate, gelatine, and chitosan.

The capability of these nanocarriers of adhering to mucosal epithelium tissue was evaluated by measuring its fracture strength to intestinal tissue of mice.

As can be seen from FIGs. 7A-7B the edible aNPs made of Spirulina exhibited enhanced capability of adhering to the mucosal epithelium tissue with respect to adhering of at any one of the nanocarriers made of 5% alginate, 5% gelatine, 5% chitosan, Nori algae, Chlorella Vulgaris, a combination of Spirulina Arthrospira Platensis with Vulgaris Chlorella, or inedible algae; and wherein the enhanced capability of adhering comprises an increase of at least 3-fold, at least 5-fold, or at least 7-fold in fracture strength (pN/mm²). Example 8 - Characterization of Spirulina aNP’s protein content

To characterize the protein content of Spirulina Arthrospira NPs liquid chromatography mass-spectrometry (LC-MS/MS) analysis was performed.

Methods - 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 and FIG. 8) 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.

Results -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’.

Example 9 - aNPs can be associated with a range of molecular weights (MWs) protein-based drugs

The use of aNPs as drug delivery systems (DDS), including the association or encapsulation of biologies such as peptides, hormones, enzymes, and antibodies, may improve drug bioavailability and drug stability by protecting the drug from degradation, providing sustained release, enhancing retention time, and overall prolonging the therapeutic effect.

To evaluate the ability of algal-based NPs to be encapsulated/associated with biologies, particularly with protein-based drugs having different molecular weights (MWs) or different physicochemical properties (for example, peptides, proteins or antibodies having hydrophilic and/or amphipathic properties), the encapsulation efficiency (EE) of several protein-based drugs is measured using one or more of the herein disclosed edible aNPs, including Spirulina Arthrospira Platensis NPs.

Moreover, the ability of these aNPs encapsulated with protein-based drugs to deliver the drugs to mucosal epithelium tissue is evaluated by measuring their capability to adhere to the tissue and facilitate uptake of the active ingredients.

Non-limiting examples of therapeutic proteins having a MW in the range of between about 5 kDa and about 300 kDa that may be associated with aNPs to enhance their therapeutic efficacy include insulin, coagulation factor VIII, iduronidase, antithrombin III, vascular endothelial growth factor receptor 1, tumor necrosis factor, antibodies such as infliximab, adalimumab, trastuzumab, bevacizumab, and rituximab, according to some embodiments.

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

1. Alga(e) derived nanoparticles (aNPs), comprising non-soluble and amphiphilic alga(e) components, wherein the aNPs have an average particle diameter in the range of between 100 nm and about 650 nm and/or surface charge ranging between about -10 mV and -45 mV, and wherein the aNPs are capable of adhering to mucosal epithelium tissue and wherein the aNPs are capable of facilitating delivery of one or more active ingredients associated with the aNPs to the mucosal epithelium tissue.

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

3. The aNPs of claim 1 or 2, wherein the aNPs have a spherical shape .

4. The aNPs any one of claims 1-3, wherein the alga(e) or the aNPs are substantially devoid of polymer addition thereto.

5. The aNPs of any one of claims 1-4, wherein the aNPs are capable of adhering to mucosal epithelium tissue at a fracture strength of more than about 200 pN/mnr when exposed to forces between 20 mN and 200 mN.

6. The aNPs of any one of claims 1-5, wherein the delivery of the one or more active ingredients to the mucosal epithelium tissue comprises adherence of the aNPs to the mucosal epithelium tissue and cellular uptake of the one or more active ingredients.

7. The aNPs of any one of claims 1-6, wherein the alga(e) comprises one or more edible alga(e).

8. The aNPs of any one of claims 1-7, wherein the alga(e) comprises one or more alga(e) species belonging to a genus selected from Chondracanthus, Gracilaria, Chondrus, Undaria, Sargassum, Laminaria, Ecklonia, Macrocystis, Palmaria, Gigartina, Spirulina Arthrospira, Chlorella, and Haematococcus, or any combination thereof.

9. The aNPs of claim 8, wherein alga(e) comprises one or more alga(e) species belonging to a genus selected from Spirulina Arthrospira, Laminaria, and Gracilaria, or any combination thereof.

10. The aNPs of any one of claims 1-9, wherein the alga(e) comprises one or more species belonging to Spirulina Arthrospira.

11. The aNPs of any one of claims 1-10, wherein the alga(e) comprises a combination of Spirulina Arthrospira with one or more additional alga(e) species.

12. The aNPs of claim 11, wherein the alga(e) comprises at least about 20% (w/w) of the one or more species belonging to Spirulina Arthrospira relative to other alga(e) species.

13. The aNPs of claim 12, wherein the alga(e) comprises at least about 50% (w/w) of one or more species belonging to Spirulina Arthrospira relative to other alga(e) species.

14. The aNPs of any one of claims 8-13, wherein the one or more species belonging to Spirulina Arthospira comprises Spirulina Arthrospira Platensis.

15. The aNPs of any one of claims 1-14, comprising an average particle diameter in the range between about 100 nm and 160 nm and/or a surface charge in the range between about -30 mV and about -45 mV.

16. The aNPs of any one of claims 11-15, wherein the combination of Spirulina Arthrospira with one or more additional alga(e) comprises a combination of Spirulina Arthrospira with Chlorella.

17. The aNPs of any one of claims 1-16, comprising a polydispersity index (PDI) of less than 0.7.

18. The aNPs of any one of claims 1-17, wherein the mucosal epithelium tissue is selected from one or more of: gastrointestinal (GI) epithelium tissue, nasal epithelium tissue, buccal epithelium tissue, and vaginal epithelium tissue, or any combination thereof.

19. The aNPs of any one of claims 1-18, comprising enhanced capability of adhering to the mucosal epithelium tissue with respect to adhering of at least one of any one of nanocarriers made of: 5% alginate, 5% gelatine, 5% chitosan, Nori algae, Chlorella, a combination of Spirulina with Chlorella, or an inedible alga; and wherein the enhanced capability of adhering comprises an increase of at least 3 -fold.

20. The aNPs of any one of claims 1-19, comprising one or more active ingredient(s) associated with the aNPs.

21. The aNPs of any one of claims 1-20, wherein the one or more active ingredient comprises a pharmaceutical drug, a tag, and a food supplement, or any combination thereof.

22. The aNPs of any one of claims 1-21, wherein the active ingredient comprises a proteinbased drug.

23. The aNPs of any one of claims 1-22, wherein the aNPs release the one or more active ingredient(s) for a period of at least about 12 hours.

24. A composition comprising the aNPs according to any one of claims 1-23, and a pharmaceutically acceptable carrier.

25. The aNPs according to any one of claims 1-23 or the composition according to claim 24, for use in delivery of one or more active ingredients to mucosal epithelium tissue of a subject in need, wherein the aNPs or the composition comprises one or more active ingredient(s), and wherein the delivery of the one or more active ingredients to the mucosal epithelium tissue comprises adherence of the aNPs to the mucosal epithelium tissue and cellular uptake of the one or more active ingredients.

26. The aNPs or the composition comprising the same for use according to claim 25, wherein the mucosal epithelium tissue is selected from one or more of: gastrointestinal (GI) epithelium tissue, nasal epithelium tissue, buccal epithelium tissue, and vaginal epithelium tissue, or any combination thereof.

27. The aNPs or the composition comprising the same, for use according to claim 25 or 26, wherein the delivery of the of one or more active ingredients to mucosal epithelium tissue comprises local and/or systemic effects.

28. The aNPs according to any one of claims 1-23 or the composition according to claim 24, for use in treating, attenuating, and/or preventing progression of a gastrointestinal (Gl)-disease in a subject in need thereof, wherein the aNPs or the composition comprising the same comprise one or more active ingredient(s), and wherein the treating, attenuating, and/or preventing progression of a gastrointestinal (Gl)-disease in a subject in need thereof comprises delivery of one or more active ingredients to the GI mucosal epithelium tissue of the subject, and wherein the delivery of the one or more active ingredients to the mucosal epithelium tissue comprises adherence of the aNPs to the mucosal epithelium tissue and cellular uptake of the one or more active ingredients.

29. The aNPs or the composition for use according to claim 28, wherein the GI disease is selected from Inflammatory Bowel Disease (IBD) and/or cancer.

30. The aNPs, or the composition for use according to any one of claims 25-29, administrated orally at a therapeutically effective amount.

31. A method for delivery of one or more active ingredients to mucosal epithelium tissue, in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of the aNPs according to any one of claims 1-23 or the composition of claim 24, wherein the aNPs or the composition comprising the same comprise one or more active ingredient(s), and wherein the delivery of the one or more active ingredients to the mucosal epithelium tissue comprises adherence of the aNPs to the mucosal epithelium tissue and cellular uptake of the one or more active ingredients.

32. A method for treating, attenuating, and/or preventing progression of gastrointestinal disease in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of the aNPs according to any one of claims 1-23 or the composition comprising the same of claim 24, wherein the aNPs or the composition comprising the same comprise one or more active ingredient(s).

33. A method for preparing alga(e) nanoparticles (aNPs), comprising 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;

(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; 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 and/or surface charge in a range of between about -10 mV and about -45 mV, and wherein the aNPs are capable of adhering to mucosal epithelium tissue.

34. The method of claim 33, wherein the homogenization of the alga(e) cells/biomass comprises sonication.

35. The method of claim 34, wherein insonation comprises a transducer at a range between 50% - 70% amplitude and/or at a range between 40% - 60% duty cycle.

36. The method of any one of claims 33-35, wherein 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

37. The method of any one of claims 33-36, wherein 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.

38. The method of any one of claims 33-37, wherein the ultracentrifugation comprises a centrifugal force of at least about 50,000 g.

39. The method of any one of claims 33-38, wherein the alga(e) comprises one or more alga(e) species belonging to a genus selected from Chondracanthus, Gracilaria, Chondrus, Undaria, Sargassum, Laminaria, Ecklonia, Macrocystis, Palmaria, Gigartina, Spirulina Arthrospira, Chlorella, and Haematococcus, or any combination thereof.

40. The method of claim 39, wherein alga(e) comprises one or more alga(e) species belonging to a genus selected from Spirulina Arthrospira, Laminaria, and Gracilaria, or any combination thereof.

41. The method of any one of claims 33-40, wherein the alga(e) comprise one or more species belonging to Spirulina Arthrospira.

42. The method of any one of claims 33-41, wherein alga(e) comprises a combination of Spirulina Arthospira with one or more additional alga(e) species.

43. The method of claim 42, wherein the alga(e) cells/biomass comprise at least about 20% (w/w) Spirulina Arthospira species relative to other alga(e) species.

44. The method of any one of claims 39-43, wherein the one or more Spirulina Arthospira species comprises Spirulina Arthospira Platensis.

45. The method of any one of claims 33-44, wherein the obtained aNPs have a polydispersity index (PDI) of less than 0.3.

46. The method of any one of claims 33-45, further comprising a step of associating one or more active ingredients, wherein said associating comprises mixing the obtained aNPs with one or more active ingredients.

47. The method of any one of claims 33-46, wherein the obtained aNPs have an average particle diameter of less than 169 nm and/or surface charge more negative than about - 10 mV.

48. The method of any one of claims 33-47, wherein the obtained aNPs comprise a plurality of different membrane proteins of the alga(e).

49. The method of any one of claims 33-48, wherein the obtained alga(e) cells/biomass are devoid of polymer addition thereto.

50. Alga(e) nanoparticles (aNPs), obtained or obtainable by the method of any one of claims 33-49.

51. Alga(e) nanoparticles (aNPs), comprising non-soluble and amphiphilic alga(e) components, wherein the aNPs have an average particle diameter in the range between 100 nm and 169 nm and/or surface charge ranging between -10 mV and -45, and wherein the aNPs are capable of adhering to mucosal epithelium tissue and wherein the aNPs are capable of facilitating delivery of one or more active ingredients to the mucosal epithelium tissue, and wherein the alga(e) comprises one or more species belonging to Spirulina Arthrospira, preferably the one or more species comprises Spirulina Arthospira Platensis.