Nanoarchaeosome formulation for targeted delivery applications and method of preparation thereof

Abstract

The present invention describes a nanoarchaeosomal composition and method for delivering biomolecules comprising nanoparticulate vesicles of archaeal lipids, wherein the biomolecule is encapsulated in the vesicles formed by archaeal lipids. Further, the therapeutic drug encapsulated can be a breast cancer targeting drug, quercetin. The nanoarchaeosomal composition is colloidal in nature, stable at high temperatures, and under oxidative stress conditions. Further, exhibits higher loading efficiency and bioavailability. It also exhibits lesser cytotoxicity.

Description

[0001] DESCRIPTION

[0002] TITLE OF THE INVENTION: Nanoarchaeosome Formulation for Targeted Delivery Applications and Method of Preparation Thereof

[0003] FIELD OF INVENTION

[0004] The present invention relates to the field of nanotechnology and nanotherapeutics. The current invention specifically relates to nanoarchaeosome formulations for targeted delivery applications and method of preparation thereof.

[0005] BACKGROUND

[0006] Efficient delivery of drugs is a basic requirement for disease treatment and management which can greatly affect the dosage and effect of the drug being administered.

[0007] Oral administration is a preferred option for the treatment of most types of chronic diseases, especially for soluble drugs. However, the physiology of the gastrointestinal (GI) tract, such as variable extreme pH, mucus turnover, immunologic defense, and excretory elimination, constitutes a formidable barrier to oral drug delivery.

[0008] Liposomes are self-assembled, spherical lipid bilayer vesicles enclosing an aqueous core. Because of their amphiphilic nature, they can simultaneously incorporate both hydrophobic and hydrophilic compounds in the lipid bilayer and the aqueous core. They are biocompatible, weakly immunogenic, and biodegradable. They can be used to deliver various pharmaceutical compounds, including vaccines, drugs, dyes, and diagnostic agents. However, conventionally, liposomes present several disadvantages such as instability in the biological environment, due to changes in the temperature, pH, and enzymes.

[0009] Cancer presents a significant apprehension on a globalstage, displaying a notable pervasiveness and necessitating a comprehensive approach to its management. Types of cancer, such as breast cancer, are emerging as the most prevailing manifestation of this disease and act as the primary reason for cancer-related deaths among women on a worldwidemagnitude. Although countries with abundant resources have observed a decline in the overall incidence and mortality rates of cancer, it should be noted that low-resource countries have witnessed a distressing surge in recent years. This surge can likely be attributed to evolving risk factors and disparities in access to early detection and treatment (Refer 1, Winterset.al).

[0010] Chemotherapy is a method of treatment that employs a blend of medications to either eradicate cancer cells or impede the progression of cancer cells. Cytotoxic medications (which mean "toxic to cells") are typically administered orally or through a vein (intravenously or "via the bloodstream"). Conversely, chemotherapy is a form of systemic therapy, which implies that the medications circulate in the bloodstream throughout the entirety of the body (Refer 2, Sharma et.al).

[0011] Chemotherapy drugs for cancers, especially breast cancers can cause a range of common side effects like hair loss (alopecia), nail changes, mouth sores, loss of appetite, nausea, vomiting, diarrhoea, fatigue, hot flashes, vaginal dryness, nerve damage, neutropenia, and bone marrow issues. Other potential side effects include menstrual changes, fertility issues, heart damage, neuropathy, hand-foot syndrome, chemo brain, increased risk of leukaemia, hypersensitivity reactions, and fatigue (Refer 3, Burguinet.al).

[0012] Most of the drugs approved to inhibit or treat cancers, whether used alone or in combination, have numerous side effects. Even naturally occurring compounds like bioflavonoids, such as Naringenin, Rutin, Hesperidin, Quercetin, and the like that are considered safe, have shown effectiveness against certain cancers, breast cancer or other like ailments face several limitations (Refer 4, Chaurasiaet.al).

[0013] Oral anticancer drugs may experience considerable variability in pharmacokinetics and pharmacodynamics, partly resulting from restricted bioavailability. This restriction in bioavailability arises from both pharmaceutical limitations and physiological barriers, which is one of the major limitations (Refer 5, Eisenmannet.al).

[0014] Likewise, pharmaceutically active molecules with low solubility may pose a higher risk. Factors such as pharmacokinetics, pharmacodynamics, drug distribution, protein binding, and absorption can be significantly impacted by poor solubility and instability in such cases. The limited water solubility of these molecules can restrict their pharmacological effectiveness and have various implications. As a result, cancer treatments often necessitate higher concentrations and more frequent drug administration, leading to increased treatment costs and potential side effects. Consequently, achieving effective drug delivery to the desired site remains a complex challenge (Refer 6, Bhalaniet.al). Considering these limitations, the scope of the present invention depends on meticulously formulated efficient nanoarchaeosome formulations for targeted delivery applications and method of preparation thereof.

[0015] SUMMARY

[0016] The current invention describes a nanoarchaeosomal composition for delivering biomolecules comprising nanoparticulate vesicles of archaeal lipids, wherein the biomolecule is encapsulated in the vesicles formed by archaeal lipids.

[0017] In one embodiment, the current invention encompasses a thermostable nanoarchaeosomal composition for delivering biomolecules with high efficiency and low cytotoxicity comprising nanoparticulate vesicles of archaeal lipids, wherein the biomolecule is encapsulated in the vesicles formed by archaeal lipids.

[0018] In one embodiment, synthetic or naturally occurring archaeal lipids are used.

[0019] In one embodiment, synthetic as well as naturally occurring archaeal lipids can be used for making the nanoparticulate vesicles in current invention.

[0020] In one embodiment, monopolar archaeal lipids as well as bipolar archaeal lipids can be used for the making the nanoparticulate vesicles in current invention. In one embodiment, the monopolar lipid in the current invention is a diether lipid.

[0021] In one embodiment, the bipolar lipid used in the current invention is a tetraether lipid.

[0022] In one embodiment, the archaeal lipids are Diether phosphocholine, 1, 2-di-O-phytanyl-sn- glycero-3 -phosphocholine .

[0023] In one embodiment, the nanoparticulate vesicles comprise phospholipids.

[0024] In one embodiment, the phospholipids are such as l-stearoyl-2-oleoyl-sn-glycero-3- phosphocholine (SOPC) or 1, 2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0025] In one embodiment, the biomolecule encapsulated in the nanoparticulate vesicles is selected from the group consisting of therapeutic drugs, peptides, small molecules, vaccines, nucleic acid molecules and therapeutic proteins.

[0026] In one embodiment, the composition is stable at high temperature .

[0027] In one embodiment, the composition is stable at room temperature for at least 30 days. In one embodiment, the composition is stable under oxidation stress.

[0028] In one embodiment, the size of the nanoparticulate vesicles is 40-60 nm

[0029] In one embodiment, the composition is a stable colloidal composition.

[0030] In one embodiment, the composition increases the bioavailability of the biomolecule being delivered compared to only the molecule being delivered.

[0031] In one embodiment, the composition disclosed herein exhibits 99% loading efficiency of the biomolecule.

[0032] In one embodiment, the composition disclosed herein exhibits 90-100% drug release efficiency of the biomolecule in 12- 24 h.

[0033] In one embodiment, the composition disclosed herein has low cytotoxicity towards healthy cells. In one embodiment, the therapeutic drug encapsulated is a breast cancer targeting drug.

[0034] In one embodiment, the therapeutic drug encapsulated is the breast cancer targeting drug quercetin.

[0035] In one embodiment, the nanoarchaeosome encapsulated quercetin (NAQ) exhibits inhibitory concentration (IC50) of 2.5p M towards breast cancer cells in vitro.

[0036] In one embodiment, the normal cells showed no significant cytotoxicity, but the cancer cells showed 50% of cytotoxicity at 2.5pM of nanoarchaeosome encapsulated quercetin (NAQ).

[0037] In one embodiment, the concentration of the biomolecule encapsulated in the nanoparticulate vesicles or the nanoarchaeosome encapsulated quercetin is directly proportional to the cytotoxicity percent in the cancer cells. Thus, as the concentration of the NAQ increases the cytotoxicity to cancer cells also increases or vice versa.

[0038] One embodiment of the current invention is a method of making the composition of claim 1, wherein the method comprises the steps of: a. dissolving the SOPC or DOPC (1, 2-Dioleoyl-sn-glycero-3-phosphocholine) and archaeal lipid were dissolved in a ratio of 80: 20 in chloroform solvent to reach a final concentration of 1 mg / ml and vacuum drying the suspension; b. reconstituting the dried lipid with water to form multi-lamellar vesicles (MLVs); c. sonicating the MLV suspension from step (b) at 35-45°C for 20-30 minutes to form small Unilamellar vesicles (SUVs) from the MLVs; and d. centrifuging the SUVs from step (c) at a speed range 10,000-15,000 rpm for 30-45 minutes to obtain the purified SUVs in the pellet, that is 80-95% pure. BRIEF DESCRIPTION OF FIGURES

[0039] Figure 1 illustrates physicochemical characterization of nanoarchaeosomes (NA). A) Average hydrodynamic size of NA measured by DLS method. B) SEM image of NA.

[0040] Figure 2 illustrates drug release kinetics of NAQ at neutral (pH 7.4) and acidic conditions (pH 4.5). The data represents the mean ± SD.

[0041] Figure 3 illustrates FTIR spectrum of the Nanoarchaeosomes (NA), Quercetin-loaded nanoarchaeosomes (NAQ) and Quercetin (Q) alone at 25 °C.

[0042] Figure 4 illustrates drug release kinetics of NAQ at neutral (pH 7.4) and acidic conditions (pH 4.5). The data represents the mean ± SD.

[0043] Figure 5 illustrates cell toxicity effect of NA on normal fibroblast NIH 3T3 cells at 24 hr. A) The MTT assay representing the cell viability of NA treated at various concentrations of NA (0.01, 0.025, and 0.05 mg) treated NIH 3T3 cells. The values are shown as mean ± SD (ns represents no significant). B) Optical microscopic images of (i) untreated cells, (ii) NA (0.05mg) treated cells at 20x magnifications. C). Fluorescence microscopic images of (i) untreated cells, (ii) NA (0.05mg) treated cells using AO / EB staining method at 20x magnifications (Green colour fluorescence represents normal healthy cells without any nuclear damage).

[0044] Figure 6 illustrates the actin-cytoskeleton organization of NA treated NIH 3T3 cells A) Control,

[0045] B) NA treated cells at 0.05mg with (i) Phalloidin stained (ii) Hoechst stained and (iii) merged images.

[0046] Figure 7 illustrates cytotoxic effect of NAQ (2.5pM) on normal NIH 3T3 cells. A) Graph represents the toxicity effect of NAQ (2.5pM) on NIH 3T3 cells by MTT method. B) Microscopic images of (i) untreated cells, (ii) NAQ (2.5pM) treated cells at 20 x magnifications.

[0047] C). Fluorescence microscopic images of (i) untreated cells, (ii) NAQ (2.5pM) treated cells. (Green color fluorescence indicates healthy cells without any apoptotic cellular death). The values are shown as mean ± SD (ns represents no significant difference between control and treated cells).

[0048] Figure 8 illustrates Cell cytotoxic effect of NAQ at different concentrations (0.05, 0.1, 0.4, 1, 2, 3, 4, 5, 7.5 and lOpM) on MCF-7 at 24 hrs by MTT assay. A) IC50 determination of NAQ. B) Optical images of untreated (control) MCF-7 cells. C) NAQ (2.5 pM) treated MCF-7 cells at 20 x magnifications, (arrows indicate morphological change and cell death). Data were expressed as mean ± SD.

[0049] Figure 9 illustrates Effect of NAQ on cytoskeleton disruption of MCF-7 cells. A) The upper panel shows untreated control cells and B) The lower panel represents NAQ (2.5pM) treated cells at 20x magnifications, (i) Red channel specifies cytoskeleton stained with phalloidin, and (ii) Blue channel indicates nuclei stained with Hoechst (iii) Merged image of stained cells, (arrows indicate cytoskeleton damage).

[0050] Figure 10 illustrates the effect of NAQ on ROS production of MCF-7 cells with a fluorescence microscope using DCFDA assay at 20 x magnifications. A) Untreated cells, B) NAQ (2.5pM) treated cells, C) The graph represents the fluorescence intensity of Intracellular ROS. The values indicate the means ± standard deviations of three independent tests. * represents the level of significance difference at P<0.05 as compared to the control.

[0051] Figure 11 illustrates Acridine orange (AO) and Ethidium bromide (EB) staining of MCF-7 cells after 24 hrs of A) Untreated cells, B) NAQ (2.5pM) treated cells (Yellow arrow indicates dead cells with the uptake of EB); (i) AO stained, (ii) EB stained, (iii) Merged image of cells stained with AO / EB.

[0052] Figure 12 illustrates FACS analysis of control and NAQ-treated MCF-7 cells represented as a dot plot in quadrants (QI, Q2, Q3, and Q4). The left bottom quadrant (QI) shows the percentage of live cells; the other three quadrants, Q2, Q3, and Q4, represent early apoptosis, necrosis, and late apoptosis: A) Untreated MCF-7 cells, B) NAQ-treated MCF-7 cells.

[0053] Figure 13 illustrates cell cycle profiles for A) Control (untreated) and B) NAQ (2.5 pM) treated breast cancer cells MCF-7 through flow cytometry with Propidium Iodide staining. C) The graph represents the percentages of cell cycle distribution between control and treated cells. * represents the significance level at P<0.05 compared to control.

[0054] DETAILED DESCRIPTION OF THE INVENTION

[0055] This section is intended to provide an explanation and description of various possible embodiments of the present invention. The embodiments used herein, and the various features and advantageous details thereof are explained more fully with reference to non-limiting embodiments illustrated in the accompanying drawing / s and detailed in the following description. The examples used herein are intended only to facilitate an understanding of ways in which the embodiments may be practiced and to enable the person skilled in the art to practice the embodiments used herein. Also, the examples / embodiments described herein should not be construed as limiting the scope of the embodiments herein.

[0056] For convenience, the meaning of certain terms and phrases used in here, are provided below. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0057] Unless specifically stated otherwise, a process or method comprising multiple steps may include additional steps at the beginning or end of the method or may include additional intervening steps. Also, the steps may be combined, excluded, or performed in an alternate order, as appropriate.

[0058] In the following detailed description, a reference is made to the accompanying drawings that form a part hereof, and in which the specific embodiments that may be practiced is shown by way of illustration. These embodiments are described in sufficient detail to enable those skilled in the art to practice the embodiments and it is to be understood that other changes may be made without departing from the scope of the embodiments. The following detailed description is therefore not to be taken in a limiting sense.

[0059] The current invention relates to a composition comprising nanoarchaeosomes that are used for encapsulation and delivery of therapeutic or diagnostic molecules with less toxicity .

[0060] Nanoarchaeosomes: “Nanoarchaeosomes” (NA) refers to exceedingly steady nanovesicles constituted by the indigenous ether lipids that are obtained from archaea (Refer 10, Babunagappanet.al).

[0061] Archaeosomes refer to the liposomes that are composed of, completely or partly, one or more natural polar ether lipids extracted from archaea or prepared with synthetic archaeal lipids that mimic natural archaeal lipids. These vesicles exhibit several advantages over the conventional liposomes prepared from the ester lipids found in Eukarya and bacteria, such as extreme stability under various harsh environmental conditions, and reduced membrane permeability to prevent leakage of the molecule being carried by these liposomes.

[0062] Developing a nanosized drug delivery system, such as the nanoarchaeosome, for efficient drug delivery to specific cancer sites presents challenges due to stability issues in the compositions of nanosize. The stability of nanoarchaeosome at varying temperatures and oxidative stress is a significant concern and challenge. The interaction of archaeal polar lipids and antigens with multivalent cations like Ca2+has been reported to lead to the formation of some archaeal polar lipid aggregates for drug delivery, but they appear to only stabilize the archaeal polar lipid aggregates of product size in micrometers, and at low temperatures.

[0063] Nanoarchaeosomes described herein are typically in the range of 40-60 nm, with a mono- or oligolamellar monolayer or bilayer membrane composed of pure archaeolipids or a combination of other amphipathic molecules. They differ from conventional liposomes in several aspects, including lipid stereoisomerism, reactivity to chemical and physical agents, membrane fluidity and permeability, and diverse compositions. These differences make them an appealing option for biomaterials and new lipid-based nanomedicines due to their high structural stability, ease of preparation at both laboratory and industrial scales.

[0064] The current invention discloses nanoarchaeosomes (NA) that are stable at 70°C and in acidic pH-4 conditions. The nanoarchaeosomes disclosed herein have high colloidal stability at high temperature and acidic conditions. Further, nanoarchaeosomes disclosed herein exhibit high colloidal stability and also high stability at room temperature for at least 50 days. This results also confirmed that nanoarchaeosomes disclosed herein have high colloidal stability. Nanoarchaeosome quercetin disclosed herein is also highly stable, and can be stored at 4°C for long periods of time.

[0065] Previous studies have reported that nanomaterials in the size range of less than 100 nm have a higher cellular uptake rate through endocytosis, receptor-mediated endocytosis, and passive diffusion. Therefore, the NA with a size less than 100 nm promotes its accumulation at the targeted sites and increases its half-life in the blood.

[0066] Nanovesicles as pleiotropic delivery vehicles can solubilize, encapsulate, stabilize any biomolecule, and increase its absorption via various mechanisms. The amphiphilicity of liposomal vesicles in structure allows both hydrophilic and lipophilic molecule(s) to be loaded or being encapsulated in the aqueous cavity or the hydrophobic membrane.

[0067] Bioflavonoids: Bioflavonoids, typically found in the peel of green citrus fruits, rose hips, and black currants, have been used in alternative medicine as an antioxidant to treat osteoarthritis and inflammatory conditions, enhance blood circulation and promote a healthy heart, and boost the effects of vitamin C. It is of paramount importance to take into consideration that potential adverse effects of bioflavonoids may encompass symptoms indicative of an allergic reaction, such as the eruption of hives, respiratory difficulties, and swelling of the visage, lips, tongue, or pharynx. Moreover, common side effects may encompass visual damage, fluctuations in heart rhythm, cephalalgia, vertigo, cutaneous irritation, and oedema or discomfort in the lower extremities.

[0068] Quercetin: Quercetin, a chemical compound3-(4, 5 -dimethyl thiazol-2-yl)-2 derived from plants, is believed to possess advantageous properties in terms of its ability to counteract oxidative stress and reduce inflammation. This compound, also referred to as a flavonoid, naturally occurs in various foods such as apples, onions, teas, berries, red wine, as well as botanical extracts like Ginkgo biloba and St. John's wort. Additionally, it is now available in the form of dietary supplements. Quercetin is sometimes utilized in the treatment of diverse medical conditions including cardiovascular disease, neoplastic disorders, arthritic conditions, and others. It is hypothesized that the potential utilization of Quercetin aims to alleviate, treat, or prevent ailments associated with cardiac disorders, cancer-related afflictions, arthritis, urinary tract infections, allergies, respiratory viral infections, notably COVID-19, hypertension, inflammation-related disorders, and similar medical situations.

[0069] Archaeal lipids:

[0070] Archaeal lipids refer to membrane phospholipids found in the Archaea domain, a prokaryotic domain. The lipids of Archaea and Bacteria are significantly different. Archaeal lipids consist of highly methylated isoprenoid chains that are connected to a glycerol- 1 -phosphate backbone through ether bonds, whereas bacterial phospholipids consist of straight fatty acids that are connected to an enantiomeric glycerol-3-phosphate backbone through ester bonds. The chemical structure and diversity of archaeal lipids allow them to maintain stability in extreme environmental conditions, as many archaea thrive in environments with high or low temperatures, high salinity, and extreme acidic or alkaline pH values.

[0071] Based on the molecular architecture, archaeal polar lipids are of two types — monopolar and bipolar. Both monopolar and bipolar lipids have been shown to form vesicles and other well- defined membrane architectures. Definitions

[0072] The term “biomolecule” as used herein, refers to organic molecules that are essential to the maintenance and metabolic processes of living organisms. Even though they are non-living, they play a crucial role in sustaining life. These molecules come in different sizes, ranging from small molecules like primary and secondary metabolites and hormones to large macromolecules like proteins, nucleic acids, carbohydrates, lipids, and so on. Furthermore, biomolecules include therapeutic drugs, varied peptides, small molecules, vaccines, nucleic acid molecules, functional proteins, toxoids, Phosphoric acid ions, Protein Toxins, and the like.

[0073] The term “encapsulation” as used herein, refers tothe loading of the vesicles / nanoarchaeosomal vesicles described herein, for any biomolecule that needs to be delivered or transported to a target inside the body. Either hydrophilic drug or lipophilic one can be encapsulated in these amphiphilic vesicles, or even both of them simultaneously.

[0074] This method can lead to a more potent therapeutic impact while also reducing the occurrence of unwanted side effects. Micelles are employed to improve the solubility and therapeutic activity of the drugs.

[0075] The term “Oxidative stress” as used herein, refers to a condition, when there is an imbalance between the production of reactive oxygen species (free radicals) and the ability of antioxidant defences to counteract them. This imbalance leads to an excess of reactive oxygen species in the body, which subsumes the biological system's capacity to efficiently detoxify or repair the resulting damage.

[0076] The term “colloidal composition” as used herein, refers to as commonly referred to as a colloidal dispersion, is a combination of substances that consists of particles with diameters ranging from 1 to 1000 nanometer. Despite their size, these particles can maintain an even distribution throughout the solution and do not separate or settle at the bottom.

[0077] The term “bioavailability” as used herein, pertains to the degree and speed at which the active constituent (drug or metabolite) enters the systemic circulation, thereby gaining access to the intended site of action. The bioavailability of a drug is predominantly influenced by the characteristics of the dosage form, which are partially contingent on its design and production process.

[0078] The term “drug loading efficiency” as used herein, refers to the proportion of drug present in the nanoparticle compared to the total amount of drug used in the formulation of the nanoparticles. The term “cytotoxicity” as used herein, refers to the degree of toxicity exhibited by an ingredient / constituent towards cells. A compound that is cytotoxic possesses the ability to induce cellular damage or demise, which can occur via necrosis or apoptosis.

[0079] The term “multi-lamellar vesicles (MLV)” as used herein, refers to the vesicles, that are created by the electrostatic self-assembly of Large, Unilamellar Vesicles (LUVs) and Host cell proteins (HCPs). The interactions between the vesicles, mediated by HCPs, lead to a morphological evolution as they collide and engulf each other.

[0080] The term “SUVs” as used herein, refers toas "small, unilamellar vesicles" or "Sonicated / small, Unilamellar Vesicles," are typically created through sonication with a cuphorn, bath, or probe tip sonicator.

[0081] The term “zeta potential” as used herein, refers to a value calculated by measuring electrophoretic mobility in a suspension, indicating the stability of particles. Higher zeta potential means greater electrostatic repulsion between particles, leading to increased stability. Evaluating zeta potential in nanomedicine is important as it can affect the behaviour of nanomaterials in living organisms, including their biological activity, effectiveness, and safety.

[0082] Embodiments

[0083] Cancer drugs, whether used alone or in combination, often cause many side effects. Even safe natural compounds like bioflavonoids have limited effectiveness against many cancers and other illnesses. Anticancer medications taken orally may have inconsistent effects because of limited bioavailability, caused by both pharmaceutical and physiological barriers. Moreover, low solubility in pharmaceutically active molecules can pose a higher risk and impact factors like drug distribution and absorption. The limited water solubility of these molecules can hinder their effectiveness, leading to higher treatment costs and potential side effects. Delivering drugs effectively to the desired site remains a complex challenge, and the present invention proposes efficient nanoarchaeosome formulations for targeted delivery applications in cancer treatment.

[0084] Although the embodiments herein are described with various specific embodiments, it will be obvious for a person skilled in the art to practice the embodiments herein with modifications.

[0085] The current invention describes a nanoarchaeosomal composition for delivering biomolecules comprising nanoparticulate vesicles of archaeal lipids, wherein the biomolecule is encapsulated in the vesicles formed by archaeal lipids. The vesicles further comprise other phospholipids such as l-stearoyl-2-oleoyl-sn-glycero-3 -phosphocholine SOPC.

[0086] In one embodiment, synthetic or naturally occurring archaeal lipids are used.

[0087] In one embodiment, the archaeal lipids in the current study are commercially obtained diether archaeal lipids [Sigma], wherein the monopolar lipid is a diether lipid and bipolar lipid is a tetraether lipid.

[0088] In one embodiment, the biomolecule encapsulated in the nanoparticulate vesicles shows no significant toxicity on the untreated normal cells, and thus the untreated normal cells showed 100% viability.

[0089] In one embodiment, the archaeal lipids are Diether phosphocholine, 1,2-di-O-phytanyl-sn- glycero-3 -phosphocholine .

[0090] In one embodiment, a thermostable nanoarchaeosomal composition for delivering biomolecules with high efficiency and low cytotoxicity comprising nanoparticulate vesicles of archaeal lipids, wherein the biomolecule is encapsulated in the vesicles formed by archaeal lipids.

[0091] In one embodiment, the nanoparticulate vesicles comprise phospholipids.

[0092] In one embodiment, the phospholipids in the nanoparticulate vesicles are l-stearoyl-2-oleoyl-sn- glycero-3-phosphocholine (SOPC) or 1, 2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0093] In one embodiment, any archaeal lipids can be used for making the composition comprising the nanoparticulate vesicles disclosed herein.

[0094] In one embodiment, synthetic as well as naturally occurring archaeal lipids can be used for making the nanoparticulate vesicles in current invention.

[0095] In one embodiment, monopolar archaeal lipids as well as bipolar archaeal lipids can be used for the making the nanoparticulate vesicles in current invention. In one embodiment, the monopolar lipid in the current invention is a diether lipid.

[0096] In one embodiment, the bipolar lipid used in the current invention is a tetraether lipid. In one embodiment, the biomolecule is selected from the group consisting of therapeutic drugs, peptides, small molecules, vaccines, nucleic acid molecules and therapeutic proteins.

[0097] In one embodiment, the nanoparticulate vesicles are stable at high temperature.

[0098] In one embodiment, the nanoparticulate vesicles are stable at room temperature for at least 30 days.

[0099] In one embodiment, the nanoparticulate vesicles are stable under oxidative stress.

[0100] In one embodiment, the size of the nanoparticulate vesicles is 40-60 nm

[0101] In one embodiment, the nanoparticulate vesicle is a stable colloidal composition.

[0102] In one embodiment, the nanoparticulate vesicles increase the bioavailability of the biomolecule being delivered compared to only the molecule being delivered.

[0103] In one embodiment, the nanoparticulate vesicles exhibit 99% loading efficiency of the biomolecule.

[0104] In one embodiment, the nanoparticulate vesicles exhibit 90-100% drug release efficiency of the biomolecule in 12- 24 h.

[0105] In one embodiment, the nanoparticulate vesicles has low cytotoxicity towards healthy cells.

[0106] In one embodiment, the normal cells showed no significant cytotoxicity, but the cancer cells showed 50% of cytotoxicity at 2.5pM of nanoarchaeosome encapsulated quercetin (NAQ).

[0107] In one embodiment, the concentration of the biomolecule encapsulated in the nanoparticulate vesicles or the nanoarchaeosome encapsulated quercetin is directly proportional to the cytotoxicity percent in the cancer cells. Thus, as the concentration of the NAQ increases the cytotoxicity to cancer cells also increases or vice versa.

[0108] In one embodiment, the nanoarchaeosome encapsulated quercetin (NAQ) exhibits inhibitory concentration (IC50) of 2.5pM towards breast cancer cells in vitro.

[0109] In one embodiment, a method of making the nanoarchaeosomal composition for delivering biomolecules, wherein the method comprises the steps of: a. dissolving the SOPC or DOPC (1, 2-Dioleoyl-sn-glycero-3-phosphocholine) and archaeal lipid were dissolved in a ratio of 80: 20 in chloroform solvent to reach a final concentration of 1 mg / ml and vacuum drying the suspension; b. reconstituting the dried lipid with water to form multi-lamellar vesicles (MLVs); c. sonicating the MLV suspension from step (b) at 35 -45 °C for 20-30 minutes to form small Unilamellar vesicles (SUVs) from the MLVs; and d. d) centrifuging the SUVs from step (c) at a speed range 10,000-15,000 rpm for 30- 45 minutes to obtain the purified SUVs in the pellet, that is 80-95% pure.

[0110] EXAMPLES

[0111] Example 1:

[0112] Preparation of Nanoarchaeosomes (NA): NA was synthesized as described by Babunagappan et.al (Refer 10). Briefly, the SOPC and archaeal lipid were dissolved in a ratio of 80:20 in chloroform solvent to reach a final concentration of 1 mg / ml and allowed for vacuum drying. Then, the dried lipid was reconstituted with Milli-Q water and vortexed for 5 minutes. Next, as prepared sample was sonicated at 45 °C for 30 minutes using an ultrasonic bath sonicator (BR Biochem Life Sciences). Then, the sample mixture was centrifuged (Eppendorf Centrifuge 5415C) at 13, 000 rpm for 30 minutes.

[0113] Example 2:

[0114] Characterization of Nanoarchaeosomes:

[0115] 2.1 Dynamic Light Scattering (PLS) and Zetapotential: The size and surface charge characterization of synthesized NA was evaluated using DLS (Horiba Scientific DLS) and Zeta analyzer (Horiba Scientific Zeta Analyzer). NA was diluted in 1: 100 ratios. This dispersed suspension was taken for analysis and the hydrodynamic diameter and surface charge of NA were measured.

[0116] The morphological examination and elemental analysis of the NA were performed using a Scanning Electron Microscope (S-4800, Hitachi). For this study, NA was drop-casted onto a precleaned glass slide and the sample was allowed to dry at room temperature, followed by gold sputter coated at 2.5Kv and 20 mA rate of lOnm per minute (Polaran SC7640 gold sputter). Then, morphological characterization was performed using a Scanning Electron Microscope operated at the voltage of l-5Kv. Further, elemental analysis of NA was achieved by the Energy Dispersive X-ray (EDX) method.

[0117] From, the DLS analysis observed that the size of the NA is around 53.5 ± 0.9 nm (Figure 1A). The SEM images ofNA exhibited a spherical shape with a size range of 50 ± 2 nm (Figure IB) which is in agreement with DLS results. The zeta potential was then analyzed to determine the net surface charge of the NA. The surface charge of the NA showed -55±lmV (Figure. 1A). A negative zeta value indicates the NA can exhibit high colloidal stability and less aggregation due to increased electrostatic repulsion between the colloids. Several studies stated that nanomaterials with extreme surface charges (zeta potential ranges higher than 30 mV or less than -30 mV) are highly stable and have high dispersibility in the aqueous solution. Previous studies reported that nanomaterials in the size range of less than 100 nm have a higher cellular uptake rate through endocytosis, receptor-mediated endocytosis, and passive diffusion. Therefore, the NA with a size less than 100 nm promotes its accumulation at the targeted sites and increases its half-life in the blood.

[0118] 2.2 Drug encapsulation efficiency: NAQ was prepared by adding equal amount of NA (Img / ml) and quercetin (Q) (lOOpM / ml) kept at 300 rpm for 12 hours in Thermo -Mixture (Eppendorf). After incubation, NAQ was subjected to freeze thaw method as described previous study (Refer 11, Altube et al). Then, free drug concentration was quantified at 375 nm using a UV-Visible spectrophotometer (Nanodrop one, Thermo Scientific). The drug loading efficiency (DLE) was determined using the following formula.

[0119] DLE% = Total concentration of drug - Concentration of supernatant Total concentration of drug

[0120] After the size and stability characterization, the quercetin was loaded into NA using the freezethaw method as described previously. The quercetin loading efficiency of NA was found to be 99 ± 0.2%. Further, the SEM, DLS, and surface charge were evaluated for NAQ (Figure 1). There were no significant changes in the size, shape, and stability of the NAQ compared to NA. Next, the zeta potential for NA, quercetin, and NAQ were measured and found to be -55± 1 mV, +4.66 mV, and -42.1 mV. In the observation there is significant reduction in the surface charge of the NAQ compared to NA, confirming that quercetin was effectively loaded into the NA. The high drug encapsulation efficiency was achieved by freeze-thaw method, where the ice crystals formed during freezing may induce temporary pores within the NA, which may lead to passive diffusion and encapsulation of quercetin inside NA. Further, favorable interaction with both the acyl group and a head group of phospholipid and hydrophobic -hydrophobic interaction between the quercetin and NA might contribute to higher encapsulation.

[0121] 2. 3 In vitro drug release at different pH conditions: The drug release kinetics of NAQ was determined by the dialysis membrane method by Babunagappan et.al (Refer 10). In brief, NAQ filled dialysis membrane (12-14 kDa, Himedia), was submerged in a small glass bottle containing IX phosphate buffered saline (PBS) at different pH (pH 7.4 and 4.5) with continuous stirring at 150 rpm for 24 hours. Finally, samples were collected at different time intervals of 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 hours and the sample were analyzed using the High-performance Liquid Chromatography (Shimadsu LC-2050C) technique.

[0122] Next, the drug release study was performed at pH 7.4, representing a normal bloodstream, and at acidic pH 4.5, corresponding to the tumor condition. From the drug release profile (Figure 2), the observed the quercetin release was 20 + 2.3, 35 + 3.5, 50 + 4.2, 90 + 3.2, and 100 + 3.4 % at 2, 4, 8, 12, and 24 h for acidic pH. And in neutral pH the drug release of 12 + 1.2, 22+ 2.24, 34 +3.3, 52 + 4.1 and 78 + 5.9 was observed at 2, 4, 8, 12, and 24 h. It was noted that 100% of the drugs were released within 6 hours at acidic pH while in neutral pH, it took more than 18 hours to achieve 100% quercetin release from NA. The drug release rate was higher at an acidic pH of 4.5 and this fast release of quercetin at pH 4.5 may be due to the protonation of the hydroxyl group of quercetin inducing repulsive forces, leading to increased drug release at acidic pH condition. This result also correlates well with the previous report by Ferreira et al (Refer 12), where the P- cyclodextrin delivery systems show significant quercetin release at an acidic pH.

[0123] 2.4 Fourier Transform Infrared Spectroscopy (FTIR): To confirm the entrapment of quercetin on NA, fourier transform infrared spectroscopy (FTIR) was performed (Nicolet iS5 FT-IR). The sample pellets were prepared with Kbr in 1:100 (% w / w) by a pressure mould (0.5-1 Pa). The FTIR spectra were collected in the transmission wavelength ranges of 500 - 4000 cm1. The FTIR spectrum of NA showed peak at 2925 cm'1corresponds to C-H stretch, 1741 cm'1pointed to ester bond linkage, 1464 cm'1corresponds to C=0 stretching and 1239 cm'1corresponds to PO2 anti symmetric stretching as illustrated in Figure 3. The FTIR spectrum of NA was well correlated with the previous report by Subastri et al (Refer 13). The FTIR spectrum of quercetin showed a characteristic peak at a wavelength of 3481 cm'1pointed to the OH stretching, 1378 cm'1corresponds to OH bending of the phenol, 1607 cm'1and 1560 cm'1pointed to C=C aromatic ring stretching, 1312 cm'1indicates C-H aromatic hydrocarbons bending and 1200 cm'1corresponds to CO stretching in phenol. Next, the analysis of FTIR spectrum of NAQ exhibits peaks of both NA and Quercetin, confirming the quercetin loading in NA. NAQ showed peaks at 2922 cm'1corresponding to C-H stretch, 1741 cm'1corresponding to ester bond linkage, and a spectrum of 1239 cm'1indicating the PO2 anti-symmetric stretching. A few pure quercetin bands have disappeared in the spectrum of NAQ, which specified that quercetin could be well-loaded in the lipid bilayers due to hydrophobic interactions or hydrogen bonds (Refer 14, Liu et al).

[0124] 2.5 In vitro cell culture studies: Next, the drug release study was performed at pH 7.4, representing a normal bloodstream, and at acidic pH 4.5, corresponding to the tumor condition. From the drug release profile (Figure 4), observed the quercetin release of 20 + 2.3, 35 + 3.5, 50 + 4.2, 90 + 3.2, and 100 + 3.4 % at 2, 4, 8, 12, and 24 h for acidic pH. And in neutral pH the drug release of 12 + 1.2, 22 + 2.24, 34 + 3.3, 52 + 4.1 and 78 + 5.9 was observed at 2, 4, 8, 12, and 24 h. It was noted that 100% of the drugs were released within 6 hours at acidic pH while in neutral pH, it took more than 18 hours to achieve 100% quercetin release from NA. The drug release rate was higher at an acidic pH of 4.5 and this fast release of quercetin at pH 4.5 may be due to the protonation of the hydroxyl group of quercetin inducing repulsive forces, leading to increased drug release at acidic pH condition (Refer 15, Samadi et al). This result also correlates well with the previous report by Ferreira et al.

[0012] , where the P-cyclodextrin delivery systems show significant quercetin release observed at an acidic pH.

[0125] 2.6 Biocompatibility of NA on NIH 3T3 cells: To understand the effects of NA and NAQ on normal cells, the biocompatibility assay was performed using different concentrations of NA (0.01, 0.025, and 0.05 mg) and NAQ (2.5 pM) on normal fibroblast NIH 3T3 cells. The source of NIH 3T3 cell line is from mouse NIH / swiss embryo [The cell lines obtained from NCCS (National Centre for Cell Science) Pune; Accession number is CVCL_0594]. The cell viability assay results clearly showed that the NIH 3T3 cells treated with different concentrations of NA did not show any toxicity, nuclear damage, or cell death (Figure 5A). From the microscopic image, it was observed that the NA-treated cells showed (Figure 5B i)) intact cellular morphology similar to control cells (Figure 5B ii). Next, AO / EB staining was performed, and results are shown in Figure 4C. The NA-treated cells (Figure 4C-i) emit green fluorescence, representing the viability of cells. The treated cells showed no apoptotic cell death and retained their intact nuclear structure like untreated control cells (Figure 5C ii). Further, the effect of NA on actin cytoskeleton organization of NIH 3T3 was studied, and results were shown in Figure 6A represents control cells and Figure 6B represents NA treated cells, whereas (i) implies phalloidin stained cells, (ii) represents Hoechst stained cells and (iii) represents merged cells. From the figure, control cells showed cellular morphology with normal actin cytoskeleton organization. Further, NA treated NIH 3T3 cells did not show any noticeable actin cytoskeleton disorganization. So, confirmed that the drug delivery system NA could not cause any adverse effects in normal cells. Then, next, tested the cell viability for NAQ (2.5pM) treated NIH 3T3 cells. No significant decrease in viability was noticed between control and NAQ-treated cells. Figure 7B (i & ii) corresponds to the microscopic images, and Figure 7C (i & ii) represents AO / EB staining of control and NAQ-treated NIH 3T3 cells. The microscopic and AO / EB assay results with green fluorescence intensity confirmed that NAQ (2.5pM) did not cause any significant cellular changes on NIH 3T3 and are similar to untreated cells. The results confirmed that the NAQ is biocompatible and didn’t affect any normal cells.

[0126] 2.7 In vitro anticancer effect of NAQ on breast cancer cell MCF-7:

[0127] 2.8 In vitro cytotoxicity analysis: Next, the evaluated the in vitro anticancer effect at different concentrations of NAQ at various concentrations (0.05, 0.1, 0.4, 1, 2, 3, 4, 5, 7.5, and lOpM) on MCF-7 cells through the MTT method and results were illustrated in Figure 8. The observation is that the cell viability decreased with increasing concentration of NAQ. The cell viability of 100 ± 3, 99.2 ± 4, 98.6 ± 3, 82.4 ± 4, 61.5 ± 3%, 39.6 ± 2%, 18.5 ± 1%, 4.3 ± 1%, 0.8 ± 0.01% were observed for untreated cells, NAQ treated cells at concentrations of 0.05, 0.1, 1, 2, 3, 4 5 and 10 pM (Figure 8A). Then, the inhibitory concentration (ICso) of NAQ was evaluated and found to be 2.5pM (Figure 8A). The IC50 concentration of NAQ is very low when compared to the quercetin alone treated in breast cancer cells. The IC50 concentration of quercetin alone treated breast cancer cells is found to be 88pM, which is 35 -fold higher than NAQ (Figure 8S3). The IC50 value of free cisplatin is in agreement with the previous report (Refer 16, Karimian et al). Hence, it is found that NA improved the therapeutic efficacy of the quercetin by enhancing their delivery. As previously discussed NA alone didn’t exhibit any cytotoxicity on normal fibroblast cell lines. However, to prove that the cytotoxicity effect in MCF-7 cells is only exhibited by NAQ, the cytotoxicity effect of NA at different concentrations 0.01, 0.025, and 0.05mg was assessed on MCF-7 cells. The biological origin of MCF-7 cells is pleural effusion of a 69-year- old Caucasian metastatic breast cancer. The results showed that NA-treated MCF-7 cells exhibited cell viability similar to untreated cells (Figure 6A) and no morphological changes in the cellular structure were observed (Figure 6S5B).

[0128] The morphology of the untreated cells and NAQ-treated cells at IC50 concentration after 24 hours was depicted in Figure 8. The untreated control cells showed healthy normal cell structures with intact nuclear morphology (Figure 8B). The NAQ (2.5pM) treated cells showed distinctive attributes of cell death (pointed out red arrow), including loss of cell membrane integrity, cell shrinkage, chromatin condensation and cell detachment as compared to untreated cells (Figure 8C). The optical microscopic images of the MCF-7 cells treated with NAQ of different concentrations are represented in Figure S4. With an increase in the NAQ concentrations, the MCF-7 cells exhibited morphological change with cell death. The enhanced cytotoxicity effect of NAQ on MCF-7 cells may be related to the NA’s lipophilic property, which endorses quercetin’s intracellular uptake. Further, NA markedly increased the quercetin solubility and stability on MCF-7. Next, further study was carried out at IC50 concentration of NAQ to elucidate possible modes of action in breast cancer treatment.

[0129] 2. 9 Effect of NAQ on actin-cytoskeleton organization of MCF-7: Next, to determine the effect of NAQ on actin cytoskeleton arrangements, thus performed a Phalloidin-Hoechst staining assay on MCF-7 cells. The results are illustrated in Figure 9. From Figure 9A, the observation that the untreated cells showed normal actin-cytoskeleton organization (Figure 9A i) and intact nuclear structure (Figure 9A ii). However, the NAQ-treated MCF-7 cells at IC50 concentration showed a disorganized structure of actin-microfilaments (Indicated by white arrow) and nuclei (Figure 9B i, ii and iii) compared to untreated breast cancer cells. Thus it is speculated that the cytoskeleton damage in the NAQ-treated MCF-7 cells may be due to increased oxidative stress caused by NAQ.

[0130] Commonly, a perfect redox balance is prominent for maintaining normal cellular physiology. So, a physiological quantity of ROS levels is vital for the maintenance of cytoskeleton remodeling. In contrast, any abnormalities in the ROS level induce actin filament disorganization and chromatin condensation, leading to cellular death (Refer 17 & 18, Sharma et al & Kaur et al). Generally, actin acts as a contractile protein, supporting the maintenance of cell shape, morphology and motility in normal cells. In contrast, in cancer cells, it regulates tumour cell progression through affecting cell proliferation and migration of cancer cells (Refer 19, 20, & 21, Burridge et al, Deepa et al & Kamble et al) From the results, the confirmed that NAQ, at a concentration of 2.5p M, considerably affects the actin filaments architecture of MCF-7 cells by inducing intracellular ROS.

[0131] 2.10 Effect of NAQ on intracellular ROS generation: Next, determined the intracellular ROS production in MCF-7 cells using a DCFDA staining assay. The fluorescence microscopic imaging showed that untreated cells showed less green fluorescence due to a lower ROS level (Figure 10A). The NAQ-treated MCF-7 cells showed bright green fluorescence (Figure 10B) as compared to untreated MCF-7 cells due to high intracellular ROS level (Figure 10B). Further, fluorescence intensity was quantified. Figure 10C represents the NAQ-treated MCF-7 cells induced ROS; a 2-fold increase in the ROS level was observed compared to untreated cells. ROS acts as an effective mediator in cancer cells to induce apoptotic cell death. The excess of intracellular ROS level leads to DNA damage. This result demonstrates that NAQ induces more ROS generation, leading to apoptotic-related cell death due to the pro-oxidant effect of quercetin. Biswas et al. (Refer 22) reported that quercetin exhibited pro-oxidant activity due to the autooxidation of free radicals (hydroxyl radical and semiquinone radical), which are very toxic and can interact irreversibly with different cellular components through the formation of covalent interaction with sulfhydryl or other groups producing secondary free radicals. So, it is speculated that quercetin-loaded NA may diminish the antioxidant defense and cause cell death by having superoxide anion radicals (Refer 23, Geetha et al).

[0132] 2.11 Apoptosis and necrosis effect of NAQ on MCF-7 breast cancer cells by AO / EB dual staining: AO / EB staining has been generally used to evaluate the cells undergoing apoptosis from the viable cells through morphological changes of the nucleus. To find the apoptotic effect of NAQ on MCF-7 cells, AO / EB staining was performed. From the analysis, the untreated (control) cells showed intact normal cellular configurations by emitting green fluorescence (Figure 11 A- i, ii and iii). In contrast, NAQ (2.5pM) treated breast cancer cells showed extensive cell shrinkages, nuclear condensation, membrane blebbing, and cellular damage through emitting considerable red fluorescence (Figure 1 IB- i, ii and iii). These various nuclear damages may be due to the oxidative stress induced by NAQ (Refer 24, Kocyigit et al). Hence, this observation clearly proposed that NAQ may cause programmed cell death in breast cancer cells.

[0133] 2. 12 FACS analysis of NAQ-induced apoptosis on MCF-7 breast cancer cells: Next, to gain deeper insight into the NAQ cellular mode of toxicity, FACS -based detection of apoptosis and necrosis using Annexin V-APC / PI dyes was carried out. The untreated control cells showed 98.34% viability (Figure 12A) and, upon NAQ treatment at 2.5 pM, 0.25%, 23.31%, and 48.48% of the cells exhibited early apoptosis, late apoptosis, and necrosis (Figure 12B). From the results, it was observed that NAQ could effectively suppress the growth of breast cancer cells through stimulation of necroptosis signaling pathways. Studies showed that necroptosis plays a vital role in the inhibition of the proliferation and viability of breast cancer cells (Refer 25, Ranganathan et al). Moreover, activating necrosis in tumor cells is a potential approach to evade the failure of cancer treatments due to apoptosis resistance. Hence, NAQ may provide strategies for developing effective therapeutic agents against apoptosis -resistant cancer.

[0134] 2.13 Effect of NAQ on cell cycle arrest: Next, flow cytometry cell cycle analysis was carried out to evaluate the percentage of cells that exist in each phase. Figure 13A shows around 0.8 ± 0.5, 62.8 ± 2.5, 12.8 ± 1.12, and 23.6 ± 0.25 of the cell percentages at Sub-Gl, G0 / G1, S, and G2 / M phases for control cells. In the NAQ-treated cells (Figure 13B), cell percentages of 1.9 ± 0.02, 84.7 ± 1.3, 5.1 ± 2.8, and 8.3 ± 1.26 were observed at Sub-Gl, G0 / G1, S, and G2 / M phase. The results showed that NAQ elicited significant cell cycle arrest at the G0 / G1 phase compared to the control. NAQ showed 1.35 fold increased cell arrest in the G0 / G1 phase compared to control cells (Figure 13C). Additionally, the percentage of cells in the S phase was significantly less for the cells treated with NAQ when compared to control. This finding is concord with previous report [Refer 25] on arresting the cell at G0 / G1 phase with considerable cellular apoptosis in breast cancer cells. This finding agreed with, Ranganathan et al. [Refer 26], demonstrated that Quercetin treated MCF-7 cells diminishes cell proliferation through G0 / G1 phase arrest.

[0135] The present invention emphasizes the significance of nanoarchaeosomes nanoparticles as a promising nanocarrier for cancer treatment and aids in overcoming the stumbling blocks of quercetin in cancer treatment. The colloidally stable NA to deliver bioflavonoid quercetin to anti cancer therapy. The NA improved quercetin’s loading and release kinetics. Additionally, NAQ showed excellent invitro anticancer activity in MCF-7 cells with IC50 value of 2.5 pM, which is thirty-five-fold lesser than IC50 value of free cisplatin. NAQ induced apoptosis and necroptosis-related cell death through ROS production and by efficiently arresting the cells at G0 / G1 phases. Therefore, our findings suggest that NAQ can be a potent anti -breast cancer agent. Nevertheless, advanced investigation must be carried out to evaluate the efficacy of NAQ on in vivo breast cancer model.

[0136] References

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Claims

We Claim :

1. A thermostable nanoarchaeosomal composition for delivering biomolecules with high efficiency and low cytotoxicity comprising nanoparticulate vesicles of archaeal lipids, wherein the biomolecule is encapsulated in the vesicles formed by archaeal lipids.

2. The composition of claim 1, wherein the vesicles comprise phospholipids.

3. The composition of claim 2, wherein the phospholipids are such as l-stearoyl-2-oleoyl- sn-glycero-3 -phosphocholine (SOPC) or 1, 2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC).

4. The composition of claim 1, wherein the biomolecule is selected from the group consisting of therapeutic drugs, peptides, small molecules, vaccines, nucleic acid molecules and therapeutic proteins.

5. The composition of claim 1 , wherein it is stable at high temperature .

6. The composition of claim 1, wherein it is stable at room temperature for at least 30 days.

7. The composition of claim 1, wherein it is stable under oxidation stress.

8. The composition of claim 1, wherein size of the nanoparticulate vesicles is 40-60 nm9. The composition of claim 1, wherein it is a stable colloidal composition.

10. The composition of claim 1, wherein it increases the bioavailability of the biomolecule being delivered compared to only the molecule being delivered.

11. The composition of claim 1, wherein it exhibits 99% loading efficiency of the biomolecule.

12. The composition of claim 1, wherein it exhibits 90-100% drug release efficiency of the biomolecule in 12- 24 h.

13. The composition of claim 1, wherein it has low cytotoxicity towards healthy cells.

14. The composition of claim 2, wherein the therapeutic drug encapsulated is a breast cancer targeting drug.

15. The composition of claim 12, wherein the therapeutic drug encapsulated is the breast cancer targeting drug quercetin.

16. The composition of claim 13, wherein the nanoarchaeosome encapsulated quercetin (NAQ) exhibits inhibitory concentration (IC50) of 2.5p M towards breast cancer cells in vitro.

17. A method of making the composition of claim 1, wherein the method comprises the steps of: a) dissolving the SOPC or DOPC (1, 2-Dioleoyl-sn-glycero-3-phosphocholine) and archaeal lipid were dissolved in a ratio of 80: 20 in chloroform solvent to reach a final concentration of 1 mg / ml and vacuum drying the suspension; b) reconstituting the dried lipid with water to form multi-lamellar vesicles (MLVs); c) sonicating the MLV suspension from step (b) at 35-45°C for 20-30 minutes to form small Unilamellar vesicles (SUVs) from the MLVs; and d) centrifuging the SUVs from step (c) at a speed range 10,000-15,000 rpm for 30-45 minutes to obtain the purified SUVs in the pellet, that is 80-95% pure.

18. A method of treating breast cancer in a subject in need thereof, the method comprising administering nanoarchaeosome encapsulated quercetin (NAQ).

19. The method as claimed in claim 18, wherein the amount of nanoarchaeosome encapsulated quercetin (NAQ) is administered is 2-3.5 pM.

20. The method as claimed in claim 18, wherein the nanoarchaeosome encapsulated quercetin (NAQ) has low cytotoxicity towards healthy cells.