Preparation of perfect, pure and uniform cochleates for drug delivery

Abstract

The current invention relates to a cochleate comprising a cationic amphiphilic material, one or more negative phospholipids, and a divalent cation. Also described is a method of producing a cochleate comprising contacting a population of liposomes comprising one or more negative phospholipids and a cationic amphiphilic material with a divalent cation source and holding such a mixture at a temperature above the transition temperature (Tm) of the one or more negative phospholipids to convert the liposomes to cochleates. Also provided is a cochleate for use in a method of treating a subject in need thereof.

Description

[0001] PREPARATION OF PERFECT, PURE AND UNIFORM COCHLEATES FOR DRUG DELIVERY

[0002] Field of Invention

[0003] The current invention relates to a cochleate. The present invention also relates to a method of producing a cochleate. The cochleates of the present invention have particular, but not exclusive, application in drug delivery. As such, there is also provided a cochleate for use in a method of treating a subject in need thereof.

[0004] Background

[0005] The listing or discussion of a prior-published document in this specification shouid not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0006] The development of micro- and nano- scale drug delivery systems has significantly advanced in recent years to address critical pharmaceutical challenges, including adverse side effects, poor bioavailability, drug instability, limited shelf life, inadequate dissolution, and inconsistent absorption (Current Medicinal Chemistry 2019, 26, 4631 ). These advanced delivery platforms offer numerous advantages, such as improved drug stability, controlled and sustained release, higher encapsulation efficiency, and enhanced drug loading capacity, all contributing to improved therapeutic outcomes. Among these systems, cochleates represent a particularly promising class of lipid-based drug carriers (Journal of liposome research 2000, 10, 523; new insights; Nagarsekar, “Cochleates: new insights into drug delivery system”, 2016, Judeh, Z. “Development of cochleate technology towards functional design and continuous production”, 2021 ; Drug development and industrial pharmacy, 2019, 45, 869). Structurally, cochleates are spiral, cylindrical assemblies formed by the tight rolling of negatively charged phospholipid bilayers such as 1 ,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and 1 ,2-dimyristoyl-sn- glycero-3-phospho-L-serine (DMPS) in the presence of multivalent cations (e.g., Ca2+, Mg2+, Zn2+), which serve as cross-linking agents (FISC advances 2015, 5, 81 188; Journal of Molecular Liquids 2021 , 335, 1 16249; Expert opinion on drug delivery, 2014, 11, 17, DOI: https: / / doi.org / 10.1517 / 17425247.2013.860131 ). This unique structure results in minimal or no internal aqueous content and imparts high mechanical rigidity, making cochleates particularly effective in protecting encapsulated drugs from degradation while enabling sustained release and improved bio-efficacy (FABAD journal of pharmaceutical sciences 2009, 34, 91 ; J. Sci. Ind. Res 2013, 2, 964). Compared to conventional nanocarriers such as liposomes and nano-emulsions, cochleates exhibit superior physicochemical stability, reduced drug leakage, and better resistance to harsh environmental conditions (Zia, Q. et al., “Novel drug delivery systems for antifungal compounds, Combating Fungal Infections: Problems and Remedy,”, 2010, 485-528; Biomedicine & Pharmacotherapy, 2018, 106, 1282). Additionally, their structural robustness contributes to improved encapsulation efficiency and precise control over drug release kinetics (Judeh, Z. “Development of cochleate technology towards functional design and continuous production”, 2021 ; Drug development and industrial pharmacy, 2019, 45, 869). This versatility of cochleates enables the encapsulation of a wide array of therapeutic agents ranging from hydrophilic and hydrophobic to neutral and charged molecules including peptides, proteins, antivirals, anticancer agents, and anti-inflammatory drugs {Advanced Drug Delivery Reviews 1998, 32, 273; Journal of liposome research 2000, 10, 523; Journal of controlled release 2002, 81 , 7). Several cochleate-based formulations have demonstrated improved delivery and therapeutic efficacy across various applications. Examples include artemisinin for sustained antimalarial delivery {Journal of Drug Delivery Science and Technology, 2019, 52, 27), raloxifene in breast cancer therapy {AAPS pharmscitech, 2016, 17. 968), fisetin for anticancer testing {Expert opinion on drug delivery, 2014, 11, 17-29, DOI: https: / / doi.org / 10.1517 / 17425247.2013.860131 ), curcumin in food and medicine industry {Foods, 2022, 11, 710), and vitamin D3 (Vit-D3) against osteoporosis {Nanomedicine: Nanotechnology, Biology and Medicine, 2020, 29, 102273).

[0007] Cochleates are primarily fabricated using one of three established methods: the trapping film method, the hydrogel method, and the dialysis method (Sopyan, I. and Gozali, D., Systematic Reviews in Pharmacy 2020, 1 1 ). Among these, the trapping film method is most widely employed due to its simplicity, reproducibility, and minimal processing requirements. Unlike the hydrogel and dialysis approaches, it does not necessitate the use of detergents or polymers, thereby making the process simple by avoiding additional purification steps for their removal {RSC advances 2015, 5, 81 188; Journal of Molecular Liquids 2021 , 335, 1 16249). The formation of cochleates is a highly nuanced process governed by the physicochemical properties of the constituent lipids and preparation parameters. Central to this process is the electrostatic interaction between the negatively charged headgroups of anionic phospholipids (e.g., DORS, DMPS) (FIG. 1 ) and divalent cations such as Caz+, Mg2+, or Zn2+. These interactions trigger lipid adhesion, membrane fusion, and structural deformation, culminating in the generation of multilamellar assemblies {Biophysical journal 2005, 89, 1812; Soft matter 2016, 12, 3797). These lamellar intermediates eventually reorganize into cochleates through a complex interplay of electrostatic forces, hydration gradients, and lipid packing transitions {International journal of pharmaceutics 2021 , 610, 121225). Throughout this transformation, a range of intermediate nanostructures such as liposomes, discs, ribbons, lamellar stacks, extended sheets, and network-like aggregates are frequently observed and well-reported (International journal of pharmaceutics 2021 , 610, 121225; Soft matter 2016, 12, 3797).

[0008] A detailed review of Field Emission Scanning Electron Microscopy (FESEM) micrographs across the literature indicates that most cochleate preparations consist of heterogeneous mixtures, wherein intermediate structures often predominate. These intermediates which are typically present as multilayered sheets, dense stacks, or interconnected networks, and in many cases, constitute a significant fraction of the observed sample. To date, no reports have unequivocally demonstrated the formation of structurally pure, well-defined cochleates devoid of such coexisting morphologies. Efforts to enhance the structural homogeneity of cochleates have focused on systematically tuning various formulation parameters, including phospholipid type, molar ratios, and concentrations; cation type / bridging agent and concentration; buffer composition and pH; as well as reaction temperature and time (European Journal of Pharmaceutics and Biopharmaceutics, 2017, 117, 270; The Journal of Physical Chemistry B, 2011 , 115, 2287; Proceedings of the National Academy of Sciences, 2019, 116, 22030; Colloids and surfaces A: physicochemical and engineering aspects 2015, 483, 187; Journal of Liposome Research 2017 , 27, 32). Despite these extensive investigations, the reproducible production of pure cochleate structures remains an unmet challenge, highlighting the need for further mechanistic insights and optimization strategies. Given their role as advanced drug delivery systems, the production of structurally uniform and compositionally pure cochleate nanoparticles is of critical importance for ensuring formulation safety, efficacy, and reproducibility. Uniformity and consistency in cochleate characteristics directly affect essential pharmaceutical parameters such as targeted delivery, controlled release kinetics, bioavailability, stability, patient compliance, precise dosing, and large-scale manufacturability. These attributes collectively determine the quality and therapeutic performance of the final product (Advanced Drug Delivery Reviews, 2018, 128, 115; Shegokar, R. and Nakach, M. “Large-scale manufacturing of nanoparticles - An industrial outlook”, Drug Delivery Aspects, Elsevier, 2020, pp. 57-77). Therefore, the development of methodologies that enable the reproducible synthesis of pure cochleates is essential for achieving precise control over their structural and functional properties, ultimately contributing to improved drug delivery outcomes and formulation quality. Concurrently, the growing interest in natural bioactive compounds derived from dietary sources reflects a paradigm shift in pharmaceutical research, driven by the limitations of synthetic or semisynthetic drugs, which are frequently associated with adverse effects such as neurological stress, stroke, and depression (J Drug DelivSci Technol, 2023, 80, 104120). Capsaicin (CAP), chemically identified as trans-8-methyl-N-vanillyl-6- nonenamide, is a leading example of such compounds. As the principal pungent component of red chili peppers, CAP has garnered attention for its extensive pharmacological potential (Polymer, 2018, 158, 223). Classified as a Biopharmaceutics Classification System (BCS) Class II molecule, CAP is characterized by low aqueous solubility and high membrane permeability, making it a valuable candidate for both nutraceutical and pharmaceutical applications (Polymer, 2018, 158, 223; Eur. J. Pharmacol., 2013, 720, 55; Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2017, 520, 62). These include pain relief (BrJ Anaesth, 2011 , 107, 490), anti-obesity (Curr. Opin. Pharmacol., 2021 , 61, 1-5; DOI: https: / / doi.Org / 10.1016 / j.coph.2021.08.012), anti-cancer (Anticancer Res., 2016, 36, 837), anti-hypertension (Nutrients, 2016, 8, 174), anti-inflammation (Cellular signalling, 2003, 15, 299) and anti-bacterial activities (Acta Pharmacologica Sinica, 2015, 36, 139). These effects are largely mediated through activation of the transient receptor potential vanilloid 1 (TRPV1) channel, a ligand-gated, non-selective cation channel widely distributed in sensory neurons, the brain, bladder, gastrointestinal tract, and vascular tissues. TRPV1 acts as a polymodal receptor that responds to various noxious stimuli, including heat, protons, endogenous lipids, and inflammatory mediators (Nutrients, 2016, 8, 174; Cellular signalling, 2003, 15, 299; Molecules, 2016, 21, 966). In the food and biomedical industries, capsaicin is also recognized for its antimicrobial efficacy against food-borne pathogens such as Bacillus cereus, Staphylococcus aureus, and Salmonella typhimurium (Colloids and surfaces B: Biointerfaces, 2011 , 87, 333). Notably, high concentrations or prolonged exposure to capsaicin result in functional desensitization of TRPV1 , a mechanism therapeutically exploited in the management of chronic pain conditions, particularly those involving thermal hyperalgesia (Nutrients, 2016, 8, 174).

[0009] Despite capsaicin’s (CAP) considerable therapeutic potential, its clinical application is limited by several pharmacological and physiological hurdles. These include gastrointestinal irritation, extensive first-pass metabolism, a short systemic half-life following intravenous administration, and poor oral bioavailability due to its hydrophobic character (Nutrients, 2021 , 13, 3995). Consequently, CAP has become an attractive candidate for advanced drug delivery systems. Conventional routes such as oral, nasal, intramuscular, and intravenous, each face challenges such as suboptimal absorption, off-target accumulation, and enzymatic degradation. In particular, oral administration is complicated by the harsh gastrointestinal pH, limited epithelial permeability, and dependency on active transport, while nasal delivery struggles with mucociliary clearance and mucus barriers, and intramuscular injections often suffer from low patient compliance (Drug Deliv Sci Technol, 2023, 80, 104120).

[0010] In response to these challenges, a variety of CAP-based drug delivery systems have been explored. These include CAP-loaded liposomes (Archives of pharmacal Research, 2015, 38, 512), CAP-nano emulsions {Materials Today: Proceedings, 2019, 18, 869), CAP-loaded methoxy polyethylene glycol)-poly(£-caprolactone) nanoparticles (CAP / NPs) {Acta Pharmacologica Sinica, 2015, 36, 139), CAP-loaded in biocompatible polymers such as PLLA (poly-L-lactic acid), poly(lactic-co-glycolic acid), and PCL (polycaprolactone ) {Journal of nanoscience and nanotechnology, 2011 , 11, 4586), CAP-loaded in nanotubes-in-microgel (Biomaterials 287, (2022) 121613), and PLGA-coated, CAP-loaded magnetic nanoparticles {Pharmaceutical Research, 2017, 34, 1255). However, despite these advancements, several of these delivery systems have critical limitations. For example, CAP-loaded liposomes suffer from low encapsulation efficiency, poor mechanical stability, and are prone to fusion, oxidation, and hydrolysis of the phospholipids {Archives of pharmacal Research, 2015, 38, 512; AAPS PharmSciTech, 2016, 17, 968). Similarly, nano-emulsions face issues related to limited drug loading capacity and instability {Current pharmaceutical design, 2017, 23, 495).

[0011] Given CAP’S broad therapeutic potential and these ongoing formulation challenges, cochleate-based delivery systems offer a promising alternative. Cochleates provide numerous advantages, including high encapsulation efficiency, enhanced stability, and controlled drug release {Int J Pharm Pharm Sci, 2010, 2, 220). Therefore, it is crucial to investigate the potential of CAP-loaded cochleates, focusing on optimizing drug encapsulation, improving controlled release properties, and ensuring the overall quality of the final formulation. Such studies are necessary to validate cochleates as a viable and effective delivery platform for CAP in pharmaceutical applications.

[0012] Application of cochleates in the delivery of amphiphilic and hydrophilic drugs is also worth exploring. However, the repulsive hydration forces may restrict the close proximity of the membranes, thus affecting the loading of amphiphilic and hydrophilic drugs. Cochleates suitable for such drug deliveries are also desirable.

[0013] It is apparent that there is a need to address the abovementioned limitations for successful preparation of pure cochleates suitable for drug deliveries of hydrophobic, amphiphilic, and hydrophilic drugs.

[0014] Summary of Invention

[0015] The current invention relates to a rapid and efficient method for producing structurally perfect, uniform, well-defined and pure cochleates that are free of intermediate structural contaminants. The cochleates may be entirely free of intermediate structural contaminants. This innovation addresses a longstanding limitation in both commercial and laboratory-scale preparations, wherein cochleate samples are invariably accompanied by undesired intermediate structures. For the first time, a mixture comprising a combination of the cationic lipid DOTAP (1 ,2-dioleoyl- 3-trimethylammonium-propane) and anionic phospholipids such as DOPS (1 ,2-dioleoyl-sn- glycero-3-phospho-L-serine, sodium salt) or DMPS (1 ,2-dimyristoyl-sn-glycero-3-phospho-L- serine) has been shown to self-assemble with divalent calcium ions (Ca2+) to rapidly yield highly uniform and pure cochleates within a significantly reduced timeframe. In addition, the current invention elucidates the underlying mechanism responsible for the formation of these structurally pure cochleates by systematically isolating and characterizing the intermediate structures involved in the transition. Notably, it was surprisingly discovered that the overall surface charge of the initial liposomal formulations comprising both cationic, anionic phospholipids and the corresponding intermediate structures plays a pivotal role in directing the morphological transformations required for the successful formation of uniform, well- defined and pure cochleates. These findings provide new mechanistic insights and lay the foundation for reproducible and scalable cochleate manufacturing with enhanced structural precision.

[0016] Given the rigid structure of cochleates and their minimal or absent aqueous core, it is crucial to explore their potential for drug encapsulation / loading. This unique architecture offers several advantages, including high physicochemical stability, reduced phospholipid oxidation, efficient drug loading, and controlled release properties. In this context, the current invention also explores the incorporation of various drugs into mixed cationic-anionic cochleate systems. The influence of DOTAP incorporation on drug encapsulation efficiency / loading, release kinetics, and the overall performance and stability of the resulting cochleates was systematically studied. This suggests that the method used to produce pure cochleates may impact critical formulation parameters such as drug loading capacity, release behaviour, structural robustness, and long-term stability. Therefore, a comprehensive investigation was carried out to elucidate how the formulation approach affects encapsulation efficiency, drug loading, drug release dynamics, and the overall quality of drug-loaded cochleates. The results validate the applicability of the current invention for practical and effective pharmaceutical delivery.

[0017] For amphiphilic and hydrophilic drugs, the current invention has addressed the limitation by using an appropriate amount of cetyltrimethylammonium bromide (CTAB) to improve the structural properties and loading of these drugs in cochleates. The current invention disclosed detailed studies on the effect of various concentrations of CTAB on the formation of DOPS cochleates. Successful encapsulation and in-vitro release of amphiphilic and hydrophilic drugs from the DOPS:CTAB cochleates were also disclosed herein. Aspects and embodiments of the invention will now be described by reference to the following numbered clauses.

[0018] 1 . A cochleate, comprising: a cationic amphiphilic material; one or more negative phospholipids; and a divalent cation.

[0019] 2. The cochleate according to Clause 1 , wherein at pH 7.4, the cochleate has a zeta potential of from about -40 mV to about -15 mV, such as from about -33 to about -20 mV, such as from about -32.75 to about -24.06 mV, such as from about -28.65 to -23.75 mV, such as from about -22.28 to about -20.1 1 mV.

[0020] 3. The cochleate according to Clause 1 or Clause 2, wherein the cochleate has a length of about 650 to about 2,000 nm, such as from about 900 to about 1 ,850 nm, such as from about 1 ,136 to about 1 ,836 nm, such as from about 1 ,241 to about 1 ,695 nm, such as from about 1 ,051 to about 1 ,403 nm.

[0021] 4. The cochleate according to any one of Clauses 1 to 3, wherein the cochleate has a width of about 80 to about 250 nm, such as from about 90 to about 210 nm, such as from about 134 to about 202 nm, such as from about 94 to about 194 nm.

[0022] 5. The cochleate according to any one of Clauses 1 to 4, wherein the cochleate has a polydispersity index (PDI) value of about 0.50 to about 1 .20, such as from about 0.70 to about 1.12, such as from about 0.72 to about 1.15, such as from about 0.865 to about 1.01 1 , such as from about 0.964 to about 1 .010, such as from about 0.865 to about 1 .007.

[0023] 6. The cochleate according to any one of Clauses 1 to 5, wherein the cationic amphiphilic material and the one or more negative phospholipids have a molar ratio of about 2:1 to about 10:1 , such as 7:3 to about 9:1 , such as about 7:3, such as about 9:1 .

[0024] 7. The cochleate according to any one of Clauses 1 to 6, wherein the cationic amphiphilic material is selected from one or more of the group consisting of cationic phospholipid, ionisable cationic lipid, cationic surfactant, and positively-charged polymer. 8. The cochleate according to Clause 7, wherein the cationic phospholipid is selected from 1 ,2-dioleoyl-3-trimethylamrnoniurnpropane (DOTAP), 1 ,2-dimyristoyl-3- trimethylammonium-propane (DMTAP), 1 ,2-distearoyl-3-trimethylammonium-propane (DSTAP), 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1 ,2-dioleyloxy-3- dimethylaminopropane (DODMA), 1 ,2-di-0-octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA), optionally wherein the cationic phospholipid is DOTAP.

[0025] 9. The cochleate according to Clause 7, wherein the ionisable cationic lipid is selected from 1 ,2-di-0-octadecenyl-3-trimethyiammonium propane (chloride salt) (DOTMA), 1 ,2- dioleoyl-3-dimethylammonium-propane (DODAP), 2-[dimethylamino]- ethyl 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DLin-MC3-DMA), C12-200 (N-[1 -(2,3-dioleyloxy)propyl]- N,N,N-trimethyiammonium methyl sulfate), 1 ,2-dioleoyl-3-trimethylammonium-propane (Al 8- DOSPA), 1 ,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1 -octylnonyl 8- [ (2- hydroxyethyl) [6-oxo-6- (undecyloxy)hexyl]amino]-octanoate (SM-102), 9,12-Octadecadienoic acid (9Z,12Z) (OF-C4-Deg-Lin), 1 ,1',1",1 '"-[(3,6-dioxo-2,5-piperazinediyl)bis(4,1 - butanediylnitrilodi-2,1 -ethanediyl)] ester, optionally the ionisable cationic lipid is 1 ,2-dioleoyl- 3-trimethylammoniumpropane (DOTAP).

[0026] 10. The cochleate according to Clause 7, wherein the cationic surfactant is selected from cetyltrimethylammonium bromide (CTAB), Benzalkonium chloride (BZK, BKC, BAK, BAC), Cetylpyridinium chloride (CPC), Stearalkonium chloride, Dodecyltrimethylammonium chloride, Lauryl Dimethyl Benzyl Ammonium Chloride, Benzethonium chloride, Dimethyldioctadecylammonium bromide, Stearylamine, Hexadecyltrimethylammonium chloride, Tetradecyltrimethylammonium bromide (TTAB; TTABr; MiTMAB), Ethylhexadecyldimethylammonium bromide, Dodecylpyridinium chloride, optionally the cationic surfactant is CTAB.

[0027] 11. The cochleate according to any one of Clauses 1 to 10, wherein the cationic amphiphilic material is 1 ,2-dioleoyl-3-trimethylammoniumpropane (DOTAP).

[0028] 12. The cochleate according to Clause 7, wherein the positively-charged polymer is selected from Poly(L-lysine) (PLL), Polyethyleneimine (PEI), Eudragit. Chitosan, Dendrimers, Poly(vinylamine), Poly(allyiamine hydrochloride) (PAH), Oligo(2-(dimethylamino)ethyl methacrylate) (Oligo-DMAEMA), Epsilon-poiy-L-lysine (short chain), Polyarginine, Trimethyl chitosan (TMC), Choline-based polyesters, Poly(imine)s (short chain), Amine-functionalized poly (carbonate), Cationic pullulan derivatives, and Poly(p-amino thioester). 13. The cochleate according to any one of Clauses 1 to 12, wherein the one or more negative phospholipids are selected from one or more of the group consisting of 1 ,2-dioleoyl- sn-glycero-3-phospho-L-serine (DORS), 1 ,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), 1 ,2-didecanoyl-sn-glycero-3-phospho-L-serine (DDRS), 1 ,2-dioctanoyl-sn-glycero- 3-phospho-L-serine (DOctPS), 1 ,2-distearoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DSPS), and 1 ,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DPPS).

[0029] 14. The cochleate according to any one of Clauses 1 to 13, wherein the divalent cation is selected from one or more of the group consisting of Ca2+, Mg2+, and Zn2+.

[0030] 15. The cochleate according to any one of Clauses 1 to 14, wherein the divalent cation is Ca2+.

[0031] 16. The cochleate according to any one of Clauses 1 to 15, wherein the cochleate further comprises an active compound.

[0032] 17. The cochleate according to any one of Clauses 1 to 16, further comprising one or more medicinal compounds.

[0033] 18. The cochleate according to Clause 17, wherein the one or more medicinal compounds are hydrophobic, hydrophilic or amphiphilic.

[0034] 19. The cochleate according to Clause 17 or Clause 18, wherein the one or more medicinal compounds are selected from one or more of the group consisting of capsaicin, silibinin, artemisinin, fisetin, raloxifene, sorafenib tosylate, irinotecan (IRT), Ibuprofen, Diazepam, Midazolam, Propofol, Haloperidol, Phenytoin, Thiopental, Fentanyl, Ketoprofen, Paclitaxel, Chloramphenicol, Insulin, Metronidazole, Doxorubicin, Ketoconazole, Cyclosporine, Itraconazole, Celecoxib, Bisoprolol , Rifampin, Curcumin, Resveratrol, Quercetin, Tocopherols (Vitamin E), Berberine, Piperine, Eugenol, Myricetin, Flavonoids, Piperlongumine, Tannins, Rutin, Thymol, Dihydromyricetin, Alpha-mangostin, Lycopene, Alkaloids (e.g., Vincristine, Vinblastine), Docetaxel, Nilotinib, Gefitinib, Imatinib (Gleevec), lndole-3-Carboxaldehyde, Chloroquine, Chlorpromazine, Propranolol, Ipratropium bromide, Tobramycin, Gentamicin, Lidocaine, Epinephrine, Cisplatin, Quinine, Ciprofloxacin, Pyridostigmine, Amitriptyline, Neomycin, Thiamine (Vitamin B1 ), Ranitidine, Pyridoxine (Vitamin B6), Betaxolol, Donepezil, Cetirizine, Amikacin, and docoshexaenic acid (DHA). 20. The cochleate according to any one of Clause 17 to 19, wherein the one or more medicinal compounds in the cochleate have an encapsulation efficiency of at least about 25%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.

[0035] 21. The cochleate according to any one of Clause 17 to 20, wherein the one or more medicinal compounds in the cochleate have a drug loading of from about 40 mg / g to about 250 mg / g, from about 50 mg / g to about 230 mg / g, from about 70 mg / g to about 215 mg / g, or from about 1 10 mg / g to about 205 mg / g.

[0036] 22. A method of producing a cochleate, the method comprising contacting a population of liposomes comprising one or more negative phospholipids and a cationic amphiphilic material with a divalent cation source and holding such a mixture at a temperature above the transition temperature (Tm) of the one or more negative phospholipids to convert the liposomes to cochleates.

[0037] 23. The method according to Clause 22, wherein the method comprises centrifuging the cochleates.

[0038] 24. The method according to Clause 22 or 23, wherein the method comprises washing the cochleates.

[0039] 25. The method according to any one of Clauses 22 to 24, wherein the method comprises freeze-drying the cochleates.

[0040] 26. The method according to any one of Clauses 22 to 25, wherein the method comprises forming the population of liposomes by: a) providing one or more negative phospholipids and a cationic amphiphilic material in a solvent; b) removing the solvent to form a mixture comprising the one or more negative phospholipids and the cationic amphiphilic material; c) hydrating the mixture to a predetermined concentration of one or both of the one or more negative phospholipids and the cationic amphiphilic material to form a hydrated mixture; and d) contacting the hydrated mixture with a divalent cation source. 27. The method according to Clause 26, wherein the solvent comprises one or both of chloroform and an alcohol, optionally wherein the alcohol is methanol.

[0041] 28. The method according to Clause 26 or 27, wherein the solvent is removed by one or both of heating and vacuum.

[0042] 29. The method according to any one of Clauses 26 to 28, wherein the step c) comprises contacting the mixture with a buffered aqueous solution, optionally wherein the buffered aqueous solution comprises phosphate-buffered saline.

[0043] 30. The method according to any one of Clauses 22 to 29, wherein the one or more negative phospholipids and the cationic material are provided in a molar ratio of from about 2:1 to about 10:1 , such as from about 3:2 to about 10:1 , such as about 7:3 to about 10:1 , such as 8:1 to 10:1 , such as around 9:1 .

[0044] 31. The method according to any one of Clauses 22 to 30, wherein the one or more negative phospholipids are selected from one or more of the group consisting of 1 ,2-dioleoyl- sn-glycero-3-phospho-L-serine (DOPS), 1 ,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), 1 ,2-didecanoyl-sn-glycero-3-phospho-L-serine (DDPS), 1 ,2-dioctanoyl-sn-glycero- 3-phospho-L-serine (DOctPS), 1 ,2-distearoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DSPS), and 1 ,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DPPS).

[0045] 32. The method according to any one of Clauses 22 to 31 , wherein the cationic amphiphilic material is selected from one or more of the group consisting of cationic phospholipid, ionisable cationic lipid, cationic surfactant, and positively-charged polymer.

[0046] 33. The method according to Clause 32, wherein the cationic amphiphilic material is selected from 1 ,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1 ,2-dimyristoyl-3- trimethylammonium-propane (DMTAP), 1 ,2-distearoyl-3-trimethylammonium-propane (DSTAP), 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1 ,2-dioleyloxy-3- dimethylaminopropane (DODMA), 1 ,2-di-0-octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA), 1 -octylnonyl 8- [ (2-hydroxyethyl) [6-oxo-6-

[0047] (undecyioxy)hexyl]amino]-octanoate (SM-102), 9,12-Octadecadienoic acid (9Z,12Z) (OF-C4- Deg-Lin), 1 , 1 ', 1 ",1 "'-[(3,6-dioxo-2,5-piperazinediyl)bis(4,1 -butanediylnitrilodi-2, 1 -ethanediyl)] ester), N,N-dioctadecyl-N,N-dimethylammonium propionate, 2-[dimethylamino]-ethyl 1 ,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DLin-MC3-DMA), 1 ,2-dioleoyl-3- trimethylammonium propane (A18-DOSPA), cetyltrimethylammonium bromide (CTAB), Benzalkonium chloride (BZK, BKC, BAK, BAC), Cetylpyridinium chloride (CPC), Stearalkonium chloride, Dodecyltrimethylammonium chloride, Lauryl Dimethyl Benzyl Ammonium Chloride, Benzethonium chloride, Dimethyldioctadecylammonium bromide, Stearylamine, Hexadecyltrimethylammonium chloride, Tetradecyltrimethylammonium bromide (TTAB; TTABr; MiTMAB), Ethylhexadecyldimethylammonium bromide, Dodecylpyridinium chloride, Poly(L-lysine) (PLL), Polyethyleneimine (PEI), Eudragit, Chitosan, Dendrimers, Poly(vinylamine), Poiyfallylamine hydrochloride) (PAH), Oligo(2- (dimethylamino)ethyl methacrylate) (Oligo-DMAEMA), Epsilon-poly-L-lysine (short chain), Polyarginine, Trimethyl chitosan (TMC), Choline-based polyesters, Poiy(imine)s (short chain), Amine-functionalized poly(carbonate), Cationic pullulan derivatives, and Poly(P-amino thioester).

[0048] 34. The method according to any one of Clauses 26, and 27 to 33 when dependent on Clause 26, wherein step a) further comprises providing an active material, such as a medicinal compound.

[0049] 35. The method according to Clause 34, wherein the active material is selected from one or more of the group consisting of capsaicin, silibinin, artemisinin, fisetin, raloxifene, sorafenib tosylate, irinotecan (IRT), Ibuprofen, Diazepam, Midazolam, Propofol, Haloperidol, Phenytoin, Thiopental, Fentanyl, Ketoprofen, Paclitaxel , Chloramphenicol, Insulin, Metronidazole, Doxorubicin, Ketoconazole, Cyclosporine, Itraconazole, Celecoxib, Bisoprolol, Rifampin, Curcumin, Resveratrol, Quercetin, Tocopherols (Vitamin E), Berberine, Piperine, Eugenol, Myricetin, Flavonoids, Piperlongumine, Tannins, Rutin, Thymol, Dihydromyricetin, Alpha- mangostin, Lycopene, Alkaloids (e.g., Vincristine, Vinblastine), Docetaxel, Nilotinib, Gefitinib, Imatinib (Gleevec), lndole-3-Carboxaldehyde, Chloroquine, Chlorpromazine, Propranolol, Ipratropium bromide, Tobramycin, Gentamicin, Lidocaine, Epinephrine, Cisplatin, Quinine, Ciprofloxacin, Pyridostigmine, Amitriptyline, Neomycin, Thiamine (Vitamin B1 ), Ranitidine, Pyridoxine (Vitamin B6), Betaxolol, Donepezil, Cetirizine, Amikacin, and docoshexaenic acid (DHA).

[0050] 36. The method according to Clause 34 or 35, wherein the one or more negative phospholipids, cationic amphiphilic material, and active material are provided in a molar ratio of from about 7:3:5 to about 7:3:10, such as about 9:1 :7.

[0051] 37. A cochleate according to any of Clauses 1 to 21 for use in a method of treating a subject in need thereof. Drawings

[0052] Certain embodiments of the present disclosure are described more fully hereinafter with reference to the accompanying drawings.

[0053] FIG. 1 depicts the structures of anionic (DOPS, DMPS) and cationic (DOTAP) phospholipids.

[0054] FIG. 2 depicts the FESEM images showing (A) DOPS: DOTAP cochleates (8:2), (B) DOPS: DOTAP (7:3), (C) DMPS: DOTAP (8:2), and (D) DMPS: DOTAP (7:3). The scale bar in the images indicate a length of 1 μm.

[0055] FIG. 3 depicts the FESEM images of liposomes prepared using various lipids and lipid mixtures: a) DOPS, b) DOPS:DOTAP, c) DMPS, and d) DMPS:DOTAP (9:1 ). The scale bar in the images indicate a length of 1 μm.

[0056] FIG. 4 depicts the morphological features of intermediate structures formed during cochleate preparation: (A) DOPS showing loosely aggregated flat sheets, (B) DOPS:DOTAP (9:1 ) with more compact and collapsed sheets, (C) DMPS showing irregular and less aggregated sheets, and (D) DMPS:DOTAP (9:1 ) with tightly packed, irregular sheets and early cochleate formation (highlighted with black arrows). The scale bar in the images indicate a length of 1 μm.

[0057] FIG. 5 depicts the FESEM images of cochleates and intermediate structures: (A) DOPS showing cochleates with residual intermediate structures (solid arrows), (B) DOPS:DOTAP (9:1 ) showing pure cochleates with wider openings (dotted arrows), (C) DMPS with aggregated cochleates showing intermediate structures, and (D) DMPS:DOTAP (9:1 ) showing pure, well-formed cochleates (solid arrows) with compact morphology. The scale bar in the images indicate a length of 1 μm.

[0058] FIG. 6 depicts the high-magnification images of cochleates illustrating the cigar-like rolling: (A) DOPS:DOTAP (9:1 ), and (B) DMPS:DOTAP (9:1 ), with rolling indicated by arrows, demonstrating the structural perfection of the cochleates. The scale bar in the images indicate a length of 200 nm.

[0059] FIG. 7 depicts the Transmission Electron Microscopy (TEM) images of (A) DOPS, (B) DOPS:DOTAP, (C) DMPS, and (D) DMPS:DOTAP cochleates showing bilayer, rolled-up cochleate structures. The scale bar in the images indicate a length of 20 nm. FIG. 8 depicts (A) Fourier Transform Infrared spectroscopy (FTIR) spectra of pure lipid and cochleate samples, and expansion of the FTIR region corresponding to the (B) phosphate group stretching, (C) -C-N- peaks of DOTAP and lipid carbonyls, and (D) alky chain stretching regions.

[0060] FIG. 9 depicts the Small Angle X-ray Scattering (SAXS) patterns of DMPS, DMPS:DOTAP, DOPS and DOPS:DOTAP cochleates indicating high order lamellar structure.

[0061] FIG. 10 depicts the differential scanning calorimetry (DSC) patterns of DOPS, DOPS: DOTAP, DMPS and DMPS: DOTAP cochleates showing transitions and thermal degradation of cochleates.

[0062] FIG. 1 1 depicts the plausible mechanism of cochleates formation in the presence / absence of cationic phospholipids.

[0063] FIG. 12 depicts the FESEM images of empty cochleates A and CAP-loaded cochleates B-H (Refer to Table 5 for the composition of the cochleates). The scale bar in the images indicate a length of 1 μm.

[0064] FIG. 13 depicts the (i) FTIR spectra of CAP and cochleates A-H, (ii) expansion of FTIR spectra region, and (iii) chemical structure of pure CAP.

[0065] FIG. 14 depicts (i) the SAXS Pattern of pure CAP and CAP-loaded cochleates A-H, and (ii) the proposed location and interaction of CAP within the lipid bilayers.

[0066] FIG. 15 depicts the High-Performance Liquid Chromatography (HPLC) chromatograms showing peaks for pure CAP (A) and CAP loaded in cochleates B-H.

[0067] FIG. 16 depicts the in-vitro release profiles: (i, ii) Pure CAP and cochleates B-H in PBS buffer, and (iii, iv) Pure CAP and cochleates B-H in in SGF and SIF.

[0068] FIG. 17 depicts the stability studies of cochleates H at: (i) different pH; and (ii) different temperature.

[0069] FIG. 18 depicts the FESEM images of cochleate, l-N. Cochleate structures are indicated by arrows. The scale bar in the images indicate a length of 1 μm. FIG. 19 depicts the SAXS patterns showing internal ultrastructure of cochleates (l-M).

[0070] FIG. 20 depicts the FESEM images of irinotecan (IRT) loaded cochleates, O-T. (Here, the arrows indicate intermediate sheet structures.) The scale bar in the images indicate a length of 1 μm.

[0071] FIG. 21 depicts the in-vitro release of IRT in (i) simulated gastric fluid (SGF) conditions, and (ii) simulated intestinal fluid (SIF) conditions.

[0072] Description

[0073] The present invention relates to a cochleate comprising a cationic amphiphilic material; one or more negative phospholipids; and a divalent cation, as well as a method of manufacturing such cochleates and such a cochleate for use in a method of treating a subject in need thereof. It has been surprisingly found that the combination of a cationic amphiphilic material one or more negative phospholipids, and a divalent cation resulting in perfectly formed cochleates. It is possible to incorporate one or more medicinal compounds, which may also be referred to as pharmacologically active compounds, to allow for drug delivery by way of the cochleates. The examples section below demonstrates how such cochleates and method of manufacturing such cochleates results in the formation of cochleate structures with high efficiency.

[0074] According to a first aspect of the present invention, there is provided a cochleate comprising a cationic amphiphilic material; one or more negative phospholipids; and a divalent cation. The combination of a cationic species is believed to neutralise at least some anionic head group of the one or more negative phospholipids. The overall charge of the cochleates remains negative, even though the negative charge is decreased by the cation and the cationic amphiphilic material, which provides the structural integrity and functional properties of the cochleates.

[0075] The word "comprising" may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word "comprising" may also relate to the situation where only the components / features listed are intended to be present (e.g. the word "comprising" may be replaced by the phrases "consists of or "consists essentially of). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word "comprising" and synonyms thereof may be replaced by the phrase "consisting of or the phrase "consists essentially of" or synonyms thereof and vice versa. The term "hydrophilic" is generally understood to describe a substance that has a high affinity for water. For example, a hydrophilic material may be one that is able to be dissolved in, be mixed with, be wetted by or absorbs water. In line with this definition, the term "hydrophilic polymer" has a high affinity for aqueous solutions.

[0076] The term "hydrophobic" is generally understood to describe a substance that repels water. For example, a hydrophobic material may include materials that do not dissolve in, be mixed with, be wetted by water or absorb an appreciable amount of water. In line with this definition, the term "hydrophobic polymer" refers to a polymer having a low affinity for aqueous solutions

[0077] The term "amphiphilic" when used herein refers to a material that displays both hydrophilic and hydrophobic properties. Typically such materials must have at least two regions - one that is hydrophilic and one that is hydrophobic, but may have more than one region of each type. Examples of amphiphilic compounds include materials such as fatty acids and lipoproteins, as well as copolymers (i.e. block copolymers) having blocks that carry hydrophilic and hydrophobic groups.

[0078] A divalent cation is a positively charged ion with a valence of 2+. Example of divalent cations, include, but are not limited, to Ca2+, Mg2+, Zn2+, and Ba2+.

[0079] The term “cochleate” may relate to structures comprising a lipid sheet, such as a lipid bilayer, which self-assembles into a spiral or shell shape.

[0080] At pH 7.4, the cochleate may have a zeta potential of from about -40 mV to about -15 mV, such as from about -33 to about -20 mV, such as from about -32.75 to about -24.06 mV, such as from about -28.65 to -23.75 mV, such as from about -22.28 to about -20.1 1 mV. Such values are believed to advantageously balance the structural and functional properties of the cochleates.

[0081] The cochleate may have a length of about 650 to about 2,000 nm, such as from about 900 to about 1 ,850 nm, such as from about 1 ,136 to about 1 ,836 nm, such as from about 1 ,241 to about 1 ,695 nm, such as from about 1 ,051 to about 1 ,403 nm. The length may be measure by any suitable means, such as, for example, optical means. It will be appreciated that cochleates may be of any suitable size / length that provides the desired function, such as that of release of an active compound, such as a drug, over time. The cochleate may have a width of about 80 to about 250 nm, such as from about 90 to about 210 nm, such as from about 134 to about 202 nm, such as from about 94 to about 194 nm. The width may be measure by any suitable means, such as, for example, optical means. It will be appreciated that cochleates may be of any suitable size / width that provides the desired function, such as that of release of an active compound, such as a drug, over time.

[0082] The cochleate has a polydispersity index (PDI) value of about 0.50 to about 1 .20, such as from about 0.70 to about 1.12, such as from about 0.72 to about 1.15, such as from about 0.865 to about 1.011 , such as from about 0.964 to about 1 .010, such as from about 0.865 to about 1.007.

[0083] The cationic amphiphilic material and the one or more negative phospholipids may have a molar ratio of about 2:1 to about 10:1 , such as 7:3 to about 9:1 , such as about 7:3, such as about 9:1.

[0084] The cationic amphiphilic material may be selected from one or more of the group consisting of cationic phospholipid, ionisable cationic lipid, cationic surfactant, and positively-charged polymer.

[0085] The cationic phospholipid may be selected from 1 ,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1 ,2-dimyristoyl-3-trimethylammonium-propane (DMTAP), 1 ,2-distearoyl-3- trimethylammonium-propane (DSTAP), 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1 ,2-dioleyloxy-3-dimethylaminopropane (DODMA), 1 ,2-di-0-octadecenyl-3-trimethyl- ammonium propane (chloride salt) (DOTMA). The cationic phospholipid may be DOTAP.

[0086] The ionisable cationic lipid may be selected from 1 ,2-di-0-octadecenyl-3 -trimethylammonium propane (chloride salt) (DOTMA), 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP) , 2- [dimethylamino]-ethyl 1 ,2-dioleoyl-sn-g!ycero-3-phosphoethanolamine (DLin-MC3-DMA), C12-200 (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate), 1 ,2- dioleoyl-3-trimethylammonium-propane (A18-DOSPA), 1 ,2-dio!eoyl-3-trimethylammonium- propane (DOTAP), 1 -octylnonyl 8- [ (2-hydroxyethyl) [6-oxo-6- (undecyloxy)hexyl]amino]- octanoate (SM-102), 9,12-Octadecadienoic acid (9Z,12Z) (OF-C4-Deg-Lin), or 1 ,1 ',1",1 '"- [(3,6-dioxo-2,5-piperazinediyl)bis(4,1 -butanediy In itrilodi-2 , 1 -ethanediyl)] ester. The ionisable cationic lipid may be 1 ,2-dioleoyl-3-trimethylammoniumpropane (DOTAP). It will be appreciated that certain compounds may fall under more than one category, such as DOTAP being a cationic phospholipid and an ionisable cationic lipid. The cationic surfactant may be selected from cetyltrimethylammonium bromide (CTAB), Benzalkonium chloride (BZK, BKC, BAK, BAC), Cetylpyridinium chloride (CPC), Stearalkonium chloride, Dodecyltrimethylammonium chloride, Lauryl Dimethyl Benzyl Ammonium Chloride, Benzethonium chloride, Dimethyldioctadecylammonium bromide, Stearylamine, Hexadecyltrimethylammonium chloride, Tetradecyltrimethylammonium bromide (TTAB; TTABr; MiTMAB), Ethylhexadecyldimethylammonium bromide, and Dodecylpyridinium chloride. The cationic surfactant may be cetyltrimethylammonium bromide CTAB. In one embodiment, the cationic amphiphilic material is 1 ,2-dioleoyl-3- trimethylammoniumpropane (DOTAP).

[0087] The positively charged polymer may be selected from Poly(L-lysine) (PLL), Polyethyleneimine (PEI), Eudragit, Chitosan, Dendrimers, Poly(vinylamine), Poly(allylamine hydrochloride) (PAH), Oligo(2-(dimethylamino)ethyl methacrylate) (Oligo-DMAEMA), Epsilon-poly-L-lysine (short chain), Polyarginine, Trimethyl chitosan (TMC), Choline-based polyesters, Poly(imine)s (short chain), Amine-functionalized poly(carbonate), Cationic pullulan derivatives, and Poly(p- amino thioester).

[0088] The one or more negative phospholipids may be selected from one or more of the group consisting of 1 ,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1 ,2-dimyristoyl-sn-glycero- 3-phospho-L-serine (DMPS), 1 ,2-didecanoyl-sn-glycero-3-phospho-L-serine (DDPS), 1 ,2- dioctanoyl-sn-glycero-3-phospho-L-serine (DOctPS), 1 ,2-distearoyl-sn-glycero-3-phospho-L- serine (sodium salt) (DSPS), and 1 ,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DPPS).

[0089] The divalent cation may be selected from one or more of the group consisting of Ca2+, Mg2+, and Zn2+. The divalent cation may be Ca2+.

[0090] The cochleate may further comprise an active compound. An active compound may be a compound which provides some physiological effect when contacted with a cell, tissue, or organism. The active compounds may be one or more medicinal compounds. Medicinal compounds may also be referred to as drugs. The term "drug" when used herein may refer to a substance useful for the treatment of or the prevention of a condition affecting a human or other animal. Said condition may be a disease, a disorder or a physiological condition. It will be appreciated that the drug may not directly affect the underlying condition, but may be used as an adjuvant with a further drug to enhance the effectiveness of the other drug. Thus, the term "drug" or “medicinal compound” herein incudes all classes of active agents, whether adjuvant or therapeutic, that may be provided to a subject through oral administration. In certain embodiments, the term "drug" may also be used herein with reference to nutraceuticals, cosmeceuticals and food-based nutrients.

[0091] The one or more medicinal compounds may be hydrophobic, hydrophilic or amphiphilic.

[0092] The one or more medicinal compounds may be selected from one or more of the group consisting of: capsaicin, silibinin, artemisinin, fisetin, raloxifene, sorafenib tosylate, irinotecan (IRT), Ibuprofen, Diazepam, Midazolam, Propofol, Haloperidol, Phenytoin, Thiopental, Fentanyl, Ketoprofen, Paclitaxel , Chloramphenicol, Insulin, Metronidazole, Doxorubicin, Ketoconazole, Cyclosporine, Itraconazole, Celecoxib, Bisoprolol, Rifampin, Curcumin, Resveratrol, Quercetin, Tocopherols (Vitamin E), Berberine, Piperine, Eugenol, Myricetin, Flavonoids, Piperlongumine, Tannins, Rutin, Thymol, Dihydromyricetin, Alpha-mangostin, Lycopene, Alkaloids (e.g., Vincristine, Vinblastine), Docetaxel, Nilotinib, Gefitinib, Imatinib (Gleevec), lndole-3-Carboxaldehyde, Chloroquine, Chlorpromazine, Propranolol, Ipratropium bromide, Tobramycin, Gentamicin, Lidocaine, Epinephrine, Cisplatin, Quinine, Ciprofloxacin, Pyridostigmine, Amitriptyline, Neomycin, Thiamine (Vitamin B1 ), Ranitidine, Pyridoxine (Vitamin B6), Betaxolol, Donepezil, Cetirizine, Amikacin, or docoshexaenic acid (DHA).

[0093] The one or more medicinal compounds in the cochleate may have an encapsulation efficiency of at least about 25%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. Encapsulation efficiency is a measure of the effectiveness of the trapping of a substance. In other words, it is a measure of how much of the one or more medicinal compounds used in the manufacture of the cochleate is ultimately carried by the cochleate.

[0094] The one or more medicinal compounds in the cochleate may have a drug loading of from about 40 mg / g to about 250 mg / g, from about 50 mg / g to about 230 mg / g, from about 70 mg / g to about 215 mg / g, or from about 110 mg / g to about 205 mg / g. As will be appreciated, the amounts of a medicinal compound (whether hydrophobic or hydrophilic) included in the cochleate will depend on the desired eventual dosage of the medicinal compound in question. Depending on the disorder, and the patient, to be treated, as well as the route of administration, the cochleates may be provided with varying therapeutically effective doses to a patient in need thereof. However, the amount administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage / severity of the disease.

[0095] According to a second aspect of the present invention, there is provided is a method of producing a cochleate, the method comprising contacting a population of liposomes comprising one or more negative phospholipids and a cationic amphiphilic material with a divalent cation source and holding such a mixture at a temperature above the transition temperature (Tm) of the one or more negative phospholipids to convert the liposomes to cochleates.

[0096] The method may comprise centrifuging the cochleates. It will be appreciated that centrifugation is one way of separating the cochleates from solution, but any other suitable separation technique may be used.

[0097] The method may comprises washing the cochleates. There may be one or more washing steps. The washing may comprise washing the cochleates in any suitable manner and with any suitable solvent to remove contaminants.

[0098] The method may comprise freeze-drying the cochleates. Freeze-drying may also be referred to a lyophilization. It will be appreciated that such a step may be accomplished using common knowledge of the person skilled in the art.

[0099] The method may comprise forming the population of liposomes by: a) providing one or more negative phospholipids and a cationic amphiphilic material in a solvent; b) removing the solvent to form a mixture comprising the one or more negative phospholipids and the cationic amphiphilic material; c) hydrating the mixture to a predetermined concentration of one or both of the one or more negative phospholipids and the cationic amphiphilic material to form a hydrated mixture; and d) contacting the hydrated mixture with a divalent cation source.

[0100] The solvent may comprise one or both of chloroform and an alcohol. Any suitable alcohol may be used. For example, the alcohol may be methanol. Combinations of two or more alcohols may be used. The solvent may be removed by one or both of heating and vacuum.

[0101] Step c) may comprises contacting the mixture with a buffered aqueous solution, optionally wherein the buffered aqueous solution comprises phosphate-buffered saline.

[0102] The one or more negative phospholipids and the cationic material may be provided in a molar ratio of from about 2:1 to about 10:1 , such as from about 3:2 to about 10:1 , such as about 7:3 to about 10:1 , such as 8:1 to 10:1 , such as around 9:1 .

[0103] The one or more negative phospholipids may be selected from one or more of the group consisting of 1 ,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1 ,2-dimyristoyl-sn-glycero- 3-phospho-L-serine (DMPS), 1 ,2-didecanoyl-sn-glycero-3-phospho-L-serine (DDPS), 1 ,2- dioctanoyl-sn-glycero-3-phospho-L-serine (DOctPS), 1 ,2-distearoyl-sn-glycero-3-phospho-L- serine (sodium salt) (DSPS), and 1 ,2-dipalmitoyl-sn-glycero -3-phospho -L-serine (sodium salt) (DPPS).

[0104] The cationic amphiphilic material may be selected from one or more of the group consisting of cationic phospholipid, ionisable cationic lipid, cationic surfactant, and positively-charged polymer.

[0105] The cationic amphiphilic material may be selected from 1 ,2-dioleoyl-3- trimethylammoniumpropane (DOTAP), 1 ,2-dimyristoyl-3-trimethylammonium-propane (DMTAP), 1 ,2-distearoyl-3-trimethylammonium-propane (DSTAP), 1 ,2-dioleoyl-3- dimethylammonium-propane (DODAP), 1 ,2-dioleyloxy-3-dimethylaminopropane (DODMA), 1 ,2-di-0-octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA), 1 -octylnonyl 8- [ (2-hydroxyethyl) [6-oxo-6- (undecyloxy)hexyl]amino]-octanoate (SM-102), 9,12- Octadecadienoic acid (9Z,12Z) (OF-C4-Deg-Lin), 1 ,1 ',1 ",1 "'-[(3,6-dioxo-2,5- piperazinediyl)bis(4, 1 -butanediylnitrilodi-2, 1 -ethanediyl)] ester), N,N-dioctadecyl-N,N- dimethylammonium propionate, 2-[dimethylamino]-ethyl 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DLin-MC3-DMA), 1 ,2-dioleoyl-3-trimethylammonium-propane (Al 8- DOSPA), cetyltrimethylammonium bromide (CTAB), Benzalkonium chloride (BZK, BKC, BAK, BAG), Cetylpyridinium chloride (CPC), Stearalkonium chloride, Dodecyltrimethylammonium chloride, Lauryl Dimethyl Benzyl Ammonium Chloride, Benzethonium chloride, Dimethyldioctadecylammonium bromide, Stearylamine, Hexadecyltrimethylammonium chloride, Tetradecyltrimethylammonium bromide (TTAB; TTABr; MiTMAB), Ethylhexadecyldimethylammonium bromide, Dodecylpyridinium chloride, Poly(L-lysine) (PLL), Polyethyleneimine (PEI), Eudragit, Chitosan, Dendrimers, Poly(vinylamine), Po!y(allylamine hydrochloride) (PAH), Oligo(2-(dimethylamino)ethyl methacrylate) (Oligo-DMAEMA), Epsilon-poly-L-lysine (short chain), Polyarginine, Trimethyl chitosan (TMC), Choline-based polyesters, Poiy(imine)s (short chain), Amine-functionalized poly(carbonate), Cationic pullulan derivatives, and Pclytp-amino thioester).

[0106] Step a) may further comprise providing an active material, such as a medicinal compound.

[0107] The active material may be selected from one or more of the group consisting of capsaicin, silibinin, artemisinin, fisetin, raloxifene, sorafenib tosylate, irinotecan (IRT), Ibuprofen, Diazepam, Midazolam, Propofol, Haloperidol, Phenytoin, Thiopental, Fentanyl, Ketoprofen, Paclitaxel, Chloramphenicol, Insulin, Metronidazole, Doxorubicin, Ketoconazole, Cyclosporine, Itraconazole, Celecoxib, Bisoprolol, Rifampin, Curcumin, Resveratrol, Quercetin, Tocopherols (Vitamin E), Berberine, Piperine, Eugenol, Myricetin, Flavonoids, Piperlongumine, Tannins, Rutin, Thymol, Dihydromyricetin, Alpha-mangostin, Lycopene, Alkaloids (e.g., Vincristine, Vinblastine), Docetaxel, Nilotinib, Gefitinib, Imatinib (Gleevec), lndole-3-Carboxaldehyde, Chloroquine, Chlorpromazine, Propranolol, Ipratropium bromide, Tobramycin, Gentamicin, Lidocaine, Epinephrine, Cisplatin, Quinine, Ciprofloxacin, Pyridostigmine, Amitriptyline, Neomycin, Thiamine (Vitamin B1 ), Ranitidine, Pyridoxine (Vitamin B6), Betaxolol, Donepezil, Cetirizine, Amikacin, and docoshexaenic acid (DHA).

[0108] The one or more negative phospholipids, cationic amphiphilic material, and active material may be provided in a molar ratio of from about 7:3:5 to about 7:3:10, such as about 9:1 :7. It has been found that the addition of the cationic amphiphilic material increases the encapsulation efficiency of hydrophilic drugs in cochleates.

[0109] In an aspect, there is provide a cochleate according to the first aspect of the present invention for use in a method of treating a subject in need thereof. It will be appreciated that the cochleate may include an effective amount of a medicinal compound. The term "effective amount" or "suitable amount" and variants thereof refer to an amount of a compound, which confers a therapeutic effect on the treated patient (e.g. sufficient to treat or prevent the disease). The effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. the subject gives an indication of or feels an effect). The amount of the medicinal compound will depend on various factors, such as the severity of the condition to be treated, the particular patient to be treated, as well as the compound(s) which is / are employed. In any event, the amount of compound in the formulation may be determined routinely by the skilled person. The current invention evaluated the influence of incorporating the cationic lipid DOTAP into anionic phospholipid systems (DOPS or DMPS) on the structural purity of the resulting final cochleates. The findings clearly demonstrate that the inclusion of DOTAP leads to the formation of high-purity cochleates, free from intermediate assemblies such as lamellar stacks, ribbons, networks, or curved sheet-like structures. High-resolution FESEM imaging confirmed the absence of these undesired morphologies, underscoring the structural homogeneity and integrity of the DOTAP-modified cochleates. Comprehensive characterization using FESEM, TEM, FTIR, zeta potential analysis, SAXS, and DSC revealed that the net surface charge and colloidal stability of precursor liposomes and intermediate phases are key determinants of cochleate purity. Mixed cationic-anionic liposomes exhibiting lower overall electrostatic charge and reduced interfacial stability undergo more rapid and efficient cochleation, resulting in structurally uniform, well-defined products. Mechanistically, DOTAP when co-administered with divalent calcium ions (Caz+) facilitates bilayer fusion by attenuating electrostatic repulsion and lowering the energy barrier for membrane deformation. This synergistic interaction with negatively charged DOPS or DMPS promotes the orderly rolling of lipid bilayers into cochleate cylinders. The resulting improvement in structural uniformity directly enhances the reliability, stability, and therapeutic applicability of cochleates as advanced drug delivery carriers.

[0110] Some advantages of the current invention over existing methods, devices, or materials may include on or more of the following.

[0111] (1 ) Perfect, uniform and pure cochleates: the disclosed preparation method provides perfect, uniform and pure cochleates without intermediate structures that are observed using the current preparation methods. Use of DOTAP provides advantages over existing methods such as rapid fusion of bilayer vesicles resulting in fast dehydration of lipid head-group and subsequent rearrangements, absence of intermediate structures in the final cochleate formulation, improved and longer cochleate morphology.

[0112] (2) Improved functional properties of the prepared cochleates: pure and uniform cochleates that are loaded with drugs provide better targeting, controlled release, bioavailability, stability, patient response, precise dosing, and manufacturability. This leads to better overall quality of the final product.

[0113] Capsaicin (CAP) is a widely studied hydrophobic drug and was chosen as a model hydrophobic drug to study the potential of cochleate-based delivery systems. Herein, CAP was successfully incorporated into cochleates formulated with various lipids and lipid combinations using the trapping film technique. This strategy disclosed in the current invention enabled the development of a refined CAP-loaded cochleate formulation with improved encapsulation efficiency and drug-loading performance. CAP-loaded cochleates confirmed high structural integrity, excellent purity, uniform morphology, efficient CAP entrapment and better CAP release. Therefore, CAP-loaded cochleates highlight the effectiveness of lipid- based cochleates in CAP delivery and provide a promising basis as a platform for the delivery of other hydrophobic therapeutics in future clinical applications.

[0114] To improve the loading of amphiphilic and hydrophilic drugs in cochleates, CTAB was added due to its ability to overcome the repulsive hydration forces and finally resulting in the close proximity of the membranes. This allows variation of the hydrophobicity of cochleates, which improves the loading of amphiphilic and hydrophilic drugs, such as irinotecan (IRT).

[0115] The cochleate formulations described in the current invention offer significant advantages as safe and effective delivery vehicles for a wide range of therapeutic agents, including antifungal and anticancer drugs, proteins, peptides, DNA, and vaccines. The disclosed preparation methods revolutionize the preparation and formulation of drug-loaded cochleates in the pharmaceutical industry. Additionally, the disclosed preparation methods are widely applicable for delivery of nutraceutical and pharmaceutical compounds with varying degree of hydrophilicity, (e.g. such as insulin, vitamin D, fisetin, raloxifene, sorafenib tosylate, thyme essential oils) to achieve better and predictable bioavailability, which is critical for therapeutic effects.

[0116] Further aspects and embodiments of the invention will now be described by reference to the following non-limiting examples.

[0117] Examples

[0118] Materials

[0119] The powdered lipids (1 ,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DORS, C(18:1 )), cholesterol, 1 ,2-dioleoyl-3-trimethylammonium propane (DOTAP, C(18:1)), and 1 ,2- dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS, C(14:0))) were procured from Avanti Polar Lipids Inc. (Alabama, USA). Solvents such as methanol and chloroform, as well as reagents including potassium hydroxide, sodium chloride, calcium chloride and phosphate-buffered saline (PBS, pH 7.4), were obtained from Sigma-Aldrich. Natural capsaicin (CAP) (CAS No.: 404-86-4), comprising approximately 55% capsaicin and the remainder dihydrocapsaicin (DHCAP), along with cetyltrimethylammonium bromide (CTAB), were also sourced from Sigma-Aldrich. Irinotecan (IRT) was purchased from Aladdin Scientific (China). All solvents, chemicals, and reagents used were of analytical grade and employed without further purification. Ultrapure water (Merck Millipore, USA) was used in all experimental procedures. Characterisation Methods

[0120] Field Emission Scanning Electron Microscopy (FESEM) Analysis

[0121] Freeze-dried cochleate powders were mounted on carbon tape and subsequently coated with a thin layer of platinum using a JFC-1600 Auto Fine Coater (JEOL, Japan) for 120 seconds at a current of 20 mA. Morphological analysis was performed using a field emission scanning electron microscope (FE-SEM; JEOL JSM-6700F, Japan) at 5 kV accelerating voltage.

[0122] Transmission Electron Microscopy (TEM) Analysis

[0123] A 5 pL aliquot of each suspension of various cochleates was applied onto 400-mesh copper grids (PELCO®, USA) and left to air-dry overnight under ambient conditions. The dried samples were subsequently analysed using a JEM-1400 Flash transmission electron microscope (JEOL, Japan) operated at 100 kV. High-resolution images were captured using a Gatan Rio9 camera system (Gatan, USA).

[0124] Determination of Zeta Potential and Polydispersity Index (PDI)

[0125] Zeta potential and polydispersity index (PDI) measurements were conducted in triplicates for various samples using Dynamic Light Scattering (DLS) on a Zetasizer Nano ZS90 (Malvern Panalytical, UK). Liposomes, intermediate structures, and cochleates suspensions were diluted with ultrapure water before zeta potential and PDI measurements were carried out. All measurements were performed in triplicates at pH 7.4.

[0126] The mean polydispersity index (PDI) and zeta potential values of the prepared cochleate formulations (A-H) were determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS, operating at 25 °C via laser Doppler micro-electrophoresis. The resulting intensity fluctuations were analysed to calculate the diffusion coefficient and particle size based on the Stokes-Einstein equation, these intensity fluctuations were used (International journal of pharmaceutics, 2021 , 610, 121225; Sartor, M., “Dynamic light scattering”, University of California, San Diego, 2003, pp. 2-21 ).

[0127] Stokes-Einstein equation:

[0128] Here, D = Translational diffusion coefficient; kb = Boltzmann constant; T = Temperature; q = Viscosity; RH = Hydrodynamic radius Measurement of Small Unilamellar Vesicles (SUVs) Using Dynamic Light Scattering (DLS) and Cochleate Size Using Imaged Software

[0129] The average hydrodynamic radius of various small unilamellar vesicles (SUVs) was measured using dynamic light scattering (DLS) on a Malvern Zetasizer Nano ZS. Measurements were conducted in triplicate at 25 °C using deionized water as the dispersant. Dimensions of cochleates were estimated by analysing microscopy images of randomly selected particles (n > 100) using the Imaged software. The average length and width of randomly selected cochleates samples (n > 100) were measured using Imaged software. In the software, calibration of image was done by correlating the dimensions of images containing FESEM images of cochleate samples from pixels to physical dimensions. Finally, measurements of around 100 cochleate structures were made (dudeh, Z. “Development of cochleate technology towards functional design and continuous production”, 2021 ).

[0130] Fourier Transform Infrared Spectroscopy (FTIR) Analysis

[0131] FTIR spectra of freeze-dried cochleate samples, were recorded using a PerkinElmer Spectrum One FTIR Spectrometer (USA) over a wavenumber range of 4000-400 cm-1. Samples were prepared by thoroughly mixing an appropriate quantity of powdered cochleates with dry potassium bromide (KBr), compressing the mixture into transparent discs, and subjecting them to spectral analysis (Journal of molecular liquids 2020, 311 , 1 13352).

[0132] Small Angle X-ray Scattering (SAXS) Analysis

[0133] SAXS measurements of the various cochleate samples were conducted using a Nano-inXider vertical SAXS / WAXS system (Xenocs SA, France), equipped with a microfocus Cu-Ka X-ray source (Genix3D, Xenocs SA, France; A = 1.54 A) and hybrid pixel detectors (Pilatus 200K, Dectris, Switzerland). All measurements were carried out at room temperature under vacuum conditions, with multiple exposures of 5 minutes each. The obtained scattering patterns were processed and analysed using XSACT software and Origin 2020b. Repeat distances (d- spacing) were calculated using Bragg’s Law: nA d = -

[0134] 2 sind where, 29 is the scattering angle and A is the X-ray wavelength.

[0135] Differential scanning calorimetry (DSC) Analysis

[0136] The thermal properties of the various cochleate samples were studied using DSC 4000 (PerkinElmer, USA). The DSC was set at a heating rate of 10°C / min spanning a range from 0°C to 260°C under a nitrogen atmosphere maintained at a flow rate of 20 mL / min (APS PharmSciTech 2016, 17, 968). Statistical Analysis

[0137] All experiments were conducted in triplicates, and the results are represented as mean values with standard deviations. Data analysis and calculations were done using Microsoft Excel (Office 365) and Origin software.

[0138] Example 1. Preparation of DOPS, DMPS, DOPS:DOTAP and DMPS:DOTAP Cochleates

[0139] Cochleates were prepared using the trapping film method as follows.

[0140] Step 1: Preparation of DOPS, DMPS, DOPS:DOTAP and DMPS:DOTAP Liposomes

[0141] Lipid powders were dissolved in a chloroform: methanol mixture (3:1 , v / v) in a round-bottom flask, and cochleate formulations were prepared as detailed in Table 1 using the trapping film method (Journal of Molecular Liquids 2021 , 335, 1 16249). The organic solvents were evaporated under reduced pressure using a rotary evaporator (BUCHI Rotavapor® R-300, Switzerland) to form a uniform thin lipid film. Residual solvents were further removed by applying vacuum for an additional hour. The resulting dry lipid film was hydrated with phosphate-buffered saline (PBS) at 60 °C for 3 hours to achieve a final lipid concentration of 5 mg / mL (6.2 mM). Small unilamellar vesicles (SUVs) were then formed by sonicating the dispersion for 20 minutes at a temperature above the lipid transition temperature (Tm), followed by extrusion through a 100 nm pore size polycarbonate membrane using a mini extruder (Avanti Polar Lipids Inc., USA) to obtain a homogeneous liposomal population. The resultant liposomal dispersion was lyophilized at -52 °C and 0.05 mbar for 24 hours using an Alpha 1-2 LD Plus freeze dryer (Martin Christ, Germany) and stored at 4 °C for subsequent use.

[0142] Table 1. Composition of liposome samples.

[0143] Entry Lipid (Molar Ratio) Molarity of DOPS Molarity of DMPS Molarity of DOTAP

[0144] (mM) (mM) (mM)

[0145] 1 DOPS (10) 6.2 NA NA

[0146] 2 DOPS:DOTAP (9:1 ) 5.58 NA 0.62

[0147] 3 DMPS (10) NA 6.2 NA

[0148] 4 DMPS:DOTAP (9:1 ) NA 5.58 0.62

[0149] NA - Not Applicable Step 2: Preparation of DOPS, DMPS, DOPS:DOTAP and DMPS:DOTAP Intermediate Structures

[0150] The preparation of intermediate structures was carried out in two sequential steps. Initially, liposomal suspensions were prepared as described in Step 1 . In the subsequent step, a CaCI2solution (6.2 mM) was introduced into the stirred liposomal suspension maintained at 60 °C using a syringe pump (New Era Pump Systems, USA) at a controlled flow rate of 10 pL / min. Upon complete addition of the CaCI2solution in a 1 :1 volume ratio with the liposome suspension, the reaction mixture was immediately transferred to a refrigerator set at 4 °C and held for 15 minutes to halt further reaction. The resulting lipid suspension was centrifuged, and the collected pellet was washed twice with ultrapure water (2 * 4 mL) using a Sigma centrifuge (Germany), followed by freeze-drying. The final product was stored at 4 °C until further use.

[0151] Step 3: Preparation of DOPS, DMPS, DOPS:DOTAP and DMPS:DOTAP Cochleates Cochleate preparation was carried out following the same protocol as described for the intermediate structures in Steps 1 and 2 above (Judeh, Z. “Development of cochleate technology towards functional design and continuous production”, 2021). However, unlike the intermediate preparation, after the complete addition of CaCI2solution (6.2 mM), the reaction was not terminated. Instead, stirring was continued for an additional 3 hours at a temperature above the lipid transition temperature (Tm), during which a characteristic white suspension formed. The resulting suspension was then centrifuged, and the pellet was washed twice with ultrapure water (2 x 4 mL), freeze-dried, and stored at 4 °C for later use.

[0152] Results and Discussions

[0153] The trapping film method was adopted in this study due to its procedural simplicity and reproducibility, allowing for enhanced control over potential process variability (Bio nanoscience 2022, 12, 274). Prior research attempting to utilize anionic phospholipids for the preparation of pure, well-defined and uniform cochleates has shown only limited success. We hypothesized that the incorporation of cationic lipids into anionic phospholipid matrices could induce favourable modifications in the self-assembly dynamics and cochleate formation process.

[0154] To investigate this hypothesis, a systematic approach was undertaken involving the preparation, isolation, and characterization of three key stages: liposomes, intermediate structures, and well-defined cochleates. Four different phospholipid systems were selected for this purpose: DOPS, DMPS, DOPS:DOTAP, and DMPS:DOTAP. Accordingly, the liposomes, intermediate structures, and cochleates of DOPS, DOPS:DOTAP, DMPS, and DMPS:DOTAP phospholipids were prepared, optimised and characterized under identical conditions (Table 1 ). It is to be noted that various ratios of anionic / cationic phospholipids were tested, and the optimal ratio was inferred to be 9:1 (Table 1 ). Other phospholipids ratios gave impure cochleates or very aggregated structures (FIG. 2).

[0155] During the liposome preparation process, thin films of DOPS, DOPS:DOTAP, DMPS, and DMPS:DOTAP phospholipids were hydrated to yield translucent dispersions. These dispersions were then subjected to sonication to facilitate the SUVs formation. To further enhance uniformity and reduce polydispersity, the resulting liposomal suspensions were passed through membrane extrusion. Field Emission Scanning Electron Microscopy (FESEM) analysis of the freeze-dried samples confirmed the successful formation of liposomes across all phospholipid compositions (FIG. 3).

[0156] Furthermore, intermediate structures were prepared by gradually adding CaCl2 (248 pL) at a flowrate of l OpL / min to an already formed translucent liposome dispersions of DOPS, DOPS:DOTAP, DMPS, and DMPS:DOTAP. Gradual addition of CaCI2solution introduces turbidity over time, suggesting a structural transition from liposomes to intermediate assemblies. To preserve these transient forms prior to their full conversion into cochleates, the reaction was promptly terminated following the complete addition of CaCI2by cooling the mixture at 4 °C for 15 minutes before proceeding with purification. At this reduced temperature, the lipids exist below their respective phase transition temperatures (Tm), thereby limiting further structural reorganization. Field Emission Scanning Electron Microscopy (FESEM) of the resulting white precipitates confirmed the presence of intermediate sheet-like structures across all tested phospholipid formulations (FIG. 4). No other intermediate structures such as stacks, networks, or ribbons have been observed.

[0157] Finally, to prepare the cochleates, the process was repeated as above, and instead of terminating the reaction, stirring was continued for 3h, whereby denser white precipitates formed as time progressed. FESEM analysis of these precipitates revealed the formation of cochleates from all phospholipids (FIG. 5).

[0158] Example 2. Morphological Analysis of the Liposomes, Intermediate Structures, and Cochleates of DOPS, DOPS:DOTAP, DMPS, and DMPS:DOTAP

[0159] The morphology of the liposomes, intermediate structures, and cochleates prepared in Example 1 was examined and discussed herein. Results and Discussion

[0160] FESEM analysis of DORS liposomes revealed a heterogeneous population comprising polydisperse, aggregated vesicles, including near-spherical and irregularly flattened morphologies (FIG. 3A). In contrast, DOPS:DOTAP liposomes exhibited predominantly spherical, smooth-surfaced vesicles with evidence of polydispersity and aggregation. Notably, some vesicles displayed flat, squarish surfaces, but no significantly flattened structures were observed (FIG. 3B). Liposomes formed from both DMPS and DMPS:DOTAP showed similar morphologies, characterized by spherical to elongated vesicles, yet demonstrated a greater degree of aggregation relative to DOPS-based systems (FIG. 3C and 3D). The observed structural distortions in all liposomal formulations may be attributed to their intrinsic softness and morphological alterations induced during freeze-drying under vacuum conditions (Sopyan, I. and Gozali, D., Systematic Reviews in Pharmacy 2020, 11 ).

[0161] FESEM images of intermediate structures prepared from DOPS, DOPS:DOTAP, DMPS, and DMPS:DOTAP show well-formed, multilayer sheets (FIG. 4). However, both DOPS and DMPS intermediate structures share similarities, presenting flat sheets loosely and irregularly aggregated with larger spaces between them (FIG. 4B and 4D). In comparison, DOPS:DOTAP and DMPS:DOTAP intermediate structures were more aggregated and appeared collapsed onto each other with significantly fewer interspaces between them (FIG. 4B and 4D vs 4A and 4D). Well-formed cochleate structures were observed along with sheets in the images of DMPS:DOTAP intermediate structures (FIG. 4D). This could be due to the reason that the smaller carbon chain of DMPS (14 carbons) as compared to longer chain of DOPS (18 carbons) and reduced overall negative charge of DMPS:DOTAP vs DOPS:DOTAP intermediate structures (see zeta potential values in Table 2 in Example 3). The flatter and collapsed appearance of intermediate structures in DOPS:DOTAP and DMPS:DOTAP, with reduced interspace compared to DOPS and DMPS structures, is attributed to the incorporation of cationic DOTAP. Here, the overall repulsive forces were reduced by the DOTAP (see zeta potential values in Table 2 in Example 3).

[0162] FESEM images of DOPS, DOPS:DOTAP, DMPS, and DMPS:DOTAP cochleates are shown in FIG. 5. The micrographs of DOPS and DMPS cochleates revealed structural similarities, exhibiting a mixture of cylindrical cochleates along with prominent intermediate forms such as stacked layers and interconnected networks. In contrast, DOPS:DOTAP and DMPS:DOTAP cochleates displayed well-defined cylindrical morphologies devoid of visible intermediate formations (see Table 3) (FIG. 1 ). Literature reports indicate that DOPS exists in a fluid phase at temperatures between 20-30 °C, with a cross-sectional molecular area of 65.3 A2, while DMPS remains in a gel phase under the same conditions, exhibiting a more condensed area of 40.8 A2(Biophysical Journal, 2004, 86, 1574). This significant reduction in molecular area for DMPS suggests stronger intermolecular interactions, likely mediated by hydrogen bonding between phosphatidylserine headgroups. Such interactions are presumed to promote tighter lipid packing, contributing to the formation of cochleates with smaller diameters in DMPS- based systems compared to those formed from DOPS. Notably, the characteristic scroll-like, rolled-up architecture of cochleates was more prominently observed in DOPS:DOTAP and DMPS:DOTAP formulations, indicating more complete and more well-ordered cochleate formation relative to the corresponding pure lipid systems (FIG. 6).

[0163] TEM micrographs of the prepared DOPS, DMPS, DOPS:DOTAP, and DMPS:DOTAP cochleate showed formation of well-rolled cochleate structures (FIG. 7).

[0164] Example 3. Zeta Potential of the Liposomes, Intermediate Structures and Cochleates of DOPS, DOPS:DOTAP, DMPS and DMPS:DOTAP

[0165] The zeta potential of particles in a dispersion was determined and are shown in Table 2.

[0166] Results and Discussions

[0167] The zeta potential trends are in complete agreement with the size and PDI trends in Tables 3 and 4 (see Example 5) and show consistent trends within and across the liposomes, intermediate structures, and cochleates (Table 2).

[0168] Table 2. Zeta potential (mV) of liposomes, intermediate structures and cochleates measured at pH 7.4.

[0169] For liposomes, intermediate structures, and cochleates, the trend in the zeta potential values for various lipids is DOPS < DMPS < DOPS:DOTAP < DMPS:DOTAP (Table 2, entries 1 -4). The introduction of positively charged DOTAP reduces the overall negative charge resulting in higher zeta potential value. Zeta potential values observed for intermediate structures and cochleates are shown in Table 2.

[0170] It was found that the zeta potential of DORS liposomes, intermediate sheets and cochleates were recorded as the -60.4 ± 1.1 mV, -45.9 ± 5.02 mV and -38.0 ± 3.09 mV, respectively. Therefore, progressive decrease in zeta potential upon calcium addition reflects the structural transformation from liposomes to lamellar intermediates and from lamellar intermediates to fully formed cochleates, supporting the occurrence of a successful wrapping process, as further corroborated by FESEM micrographs (FIG. 6). Therefore, this stepwise decline in the absolute zeta potential values from liposomes to intermediates to cochleates reflects this partial charge compensation, with the anionic character of the phospholipids continuing to dominate. This delicate charge balance is essential for maintaining the structural stability and functional performance of the cochleates.

[0171] Example 4. Size of DOPS, DOPS:DOTAP, DMPS and DMPS:DOTAP Cochleates and SUVs

[0172] The comparison of length and diameter between DOPS, DOPS:DOTAP, DMPS and DMPS:DOTAP cochleates are shown in Table 3).

[0173] Results and Discussions

[0174] DOTAP addition causes an increase in the length of the cochleates (Table 3, entries 2 and 4 vs 1 and 3).

[0175] Table 3. Size of DOPS, DOPS:DOTAP, DMPS, and DMPS:DOTAP cochleates and SUVs.

[0176] Additionally, addition of DOTAP results in an increase in width for both DOPS:DOTAP and DMPS:DOTAP as compared to DOPS and DMPS cochleates, respectively (Table 3, entries 2 and 4 vs 1 and 3, respectively). Furthermore, values in Table 3 suggests that the choice of the base anionic lipid influences the size of resulting cochleate structures due to variations in overall charge, chain length, and saturation contribute to variations in self-assembly kinetics and, consequently, the final cochleate morphology {European Biophysics Journal, 2022, 51, 205). Based on the above general trends, DMPS:DOTAP cochleates were expected to have smaller lengths and diameters than DOPS:DOTAP cochleates. This turn to be true on the addition of DOTAP also attests to the complexity of the interplay between the physicochemical characteristics of the lipids. This could be attributed to the fact that DOTAP is a highly flexible lipid which results in increased fluidity of DMPS and DOPS (IET Nanobiotechnology, 2021 , 15, 380). These observations support the trend observed for size values of SUVs (DMPS<DMPS:DOTAP<DOPS<DOPS:DOTAP) as shown in Table 3. The size of SUVs determines the final size of cochleate structures (Zarif, L. “Drug delivery by lipid cochleates”, Methods in enzymology, Elsevier, 2005, p. 314-329). Thus, the addition of DOTAP, significantly affects the size of SUVs and subsequently the cochleates (Current Medicinal Chemistry 2019, 26, 4631 ).

[0177] Cochleate size affects the final drug encapsulation efficiency, release kinetics, overall performance and stability of the delivery system (Current Medicinal Chemistry 2019, 26, 4631 ). Larger cochleates, as seen in the presence of DOTAP, may offer advantages in terms of higher drug-loading capacities or the encapsulation of larger drug molecules.

[0178] Example 5. Polydispersity Index (PDI) of Liposomes, Intermediate Structures, and Cochleates of DOPS, DOPS:DOTAP, DMPS, and DMPS:DOTAP

[0179] PDI is a crucial factor for consistent drug delivery and performance. The PDI trends for liposomes, intermediate structures (sheets), and cochleates are shown in Table 4.

[0180] Results and Discussions

[0181] PDI trend for various lipids is observed to be DOPS < DMPS < DOPS:DOTAP < DMPS:DOTAP as shown in Table 4.

[0182] Table 4. Polydispersity index (PDI) values of liposomes, intermediate structures and cochleates.

[0183] PDI

[0184] Entry Lipid Intermediate

[0185] Liposomes Cochleates

[0186] Structures

[0187] 1 DOPS 0.146 ± 0.009 0.615 ± 0.050 0.780 ± 0.340

[0188] 2 DOPS:DOTAP 0.330 ± 0.007 0.893 ± 0.156 0.936 ± 0.071

[0189] 3 DMPS 0.224 ± 0.005 0.808 ± 0.019 0.888 ± 0.127

[0190] 4 DMPS:DOTAP 0.394 ± 0.014 0.947 ± 0.092 0.987 ± 0.023 PDI values obtained for DOPS, DMPS, DOPS:DOTAP and DMPS:DOTAP liposomes are 0.146 ± 0.009, 0.224 ± 0.005, 0.330 ± 0.007 and 0.394 ± 0.014, respectively (Table 4, entries 1-4). Similarly, liposomes, intermediate structures, and cochleates made from DOPS:DOTAP lipids where the PDI values are 0.330 ± 0.007, 0.893 ± 0.156 and 0.936 ± 0.071 , respectively (Table 4, entry 2). DOTAP incorporation increases the PDI for all structures, suggesting a general impact of the cationic lipid on size heterogeneity. The increased PDI in DOTAP- containing samples may be attributed to the electrostatic interactions between cationic and anionic lipids, leading to variations in size during the self-assembly process. Apparently, lower PDI values for liposomes as compared to intermediate structures and cochleates indicate a more homogenous size distribution (Table 4). Additionally, higher PDI values of DMPS cochleates in comparison to DOPS cochleates suggested higher aggregation in DMPS cochleates and DMPS:DOTAP cochleates in comparison to DOPS cochleates and DOPS:DOTAP cochleates, respectively. These findings are important as these factors can affect the drug delivery design, emphasizing the critical role of lipid composition in tailoring the characteristics of cochleates for optimal therapeutic outcomes.

[0191] Example 6. FTIR analysis of DOPS, DOPS:DOTAP, DMPS, and DMPS:DOTAP Cochleates

[0192] We used FTIR to probe the formation of the DOPS, DOPS:DOTAP, DMPS, and DMPS:DOTAP cochleates by analysing the interaction between Ca2+and the lipid headgroups

[0193] Results and Discussions

[0194] FIG. 8 presents the FTIR spectra of pure DOPS, DMPS, and DOTAP lipids, along with those of cochleates formed from DOPS, DOPS:DOTAP, DMPS, and DMPS:DOTAP. The spectra of pure DOPS and DMPS exhibit comparable profiles, characterized by asymmetric phosphate stretching vibrations in the range of 1020-1250 cm-1. In the case of DOTAP, the -C-N- stretching associated with its ammonium group is observed at 1482 cm-1and 1489 cm-1(FIG. 8C). The CH2stretching vibrations of the alkyl chains in pure DOPS, DMPS, and DOTAP appeared within the range of 2800-3000 cm-1. FTIR analysis confirmed the successful formation of cochleates from DOPS, DOPS:DOTAP, DMPS, and DMPS:DOTAP, all of which exhibited comparable spectral profiles. Notably, the asymmetric phosphate stretching band, originally observed at 1216 cm-1in pure DOPS and DMPS, shifted to 1238 cm-1upon cochleate formation. Concurrently, the symmetric phosphate stretching band resolved into four distinct peaks at 1063 cm1, 1075 cm1, 1 102 cm1, and 1 1 12 cm1. These spectral changes are indicative of bidentate complexation between phosphate groups and Ca2+ions, accompanied by dehydration of the phosphate headgroups, thereby confirming cochleate formation (FIG. 8B) (T. Bozo, Semmelweis University, 2018; Journal of Molecular Liquids 2021 , 335, 1 16249). Additionally, the symmetric and asymmetric CH2stretching of the alkyl chains were seen at 2852 cm1and 2923 cm1, respectively (FIG. 8D), while two stretching peaks for the carbonyl ester C=O of the acyl chains were observed at 1730 cm-1and 1740 cm-1(FIG. 80). All observed peaks were sharp and well-defined, indicative of dehydrated and structurally well-organized cochleates {Biophysical journal 1993, 64, 11 13). In the FTIR spectra of DOPS:DOTAP and DMPS:DOTAP cochleates, no characteristic peaks corresponding to DOTAP or its interactions with DOPS or DMPS were detected, possibly due to the low molar ratio of DOTAP (9:1 DOPS / DMPS to DOTAP) within the formulation.

[0195] Example 7. Small Angle X-Ray Scattering (SAXS) Studies of DOPS, DOPS:DOTAP, DMPS and DMPS:DOTAP Cochleates

[0196] Small angle X-ray scattering (SAXS) provides valuable insights into the ultrastructure arrangement of the phospholipid bilayers that constitute the cochleate structure. Hence, all cochleates were subjected to SAXS to study the lamellar order in DOPS, DOPS:DOTAP, DMPS and DMPS:DOTAP cochleates (FIG. 9).

[0197] Results and Discussions

[0198] All cochleate formulations demonstrated comparable SAXS profiles, featuring intense primary reflection peaks along with weaker higher-order reflections which is a characteristic of well- organized lamellar structures. The prominent diffraction peaks correspond to the interlamellar spacing characteristic of cochleate assemblies. The calculated d-spacings were 5.14 nm for DOPS, 5.1 1 nm for DOPS:DOTAP, 4.31 nm for DMPS, and 4.33 nm for DMPS:DOTAP cochleates. These values are in close agreement with previously reported data for cochleate systems Biochemistry, 1983, 22, 2171 ; Journal of Drug Delivery Science and Technology, 2019, 52, 27; Judeh, Z. “Development of cochleate technology towards functional design and continuous production”, 2021 ; Soft matter 2016, 12, 3797; Chemistry and Physics of Lipids, 2009, 158, 46). The similarity in d-spacing values across both single and mixed lipid systems suggests a uniform lamellar stacking pattern, reflecting the successful integration of DOTAP into DOPS and DMPS bilayers. This structural arrangement, corroborated by FTIR analysis, highlights the cooperative binding of Ca2+ions to the anionic headgroups, inducing a high degree of bilayer condensation that facilitates cochleate formation. The sharp and well-defined SAXS peaks observed for all samples indicate low polydispersity and minimal structural disorder, further supporting the formation of ordered cochleate assemblies. The reduction in interlamellar spacing from DOPS (5.14 nm) to DMPS (4.31 nm) cochleates is attributed to the shorter acyl chains in DMPS (C14) relative to DOPS (C18), which also aligns with their respective particle size differences (Table 3, entries 1 and 3) (Biochemistry, 1983, 22, 2171 ). Notably, the incorporation of DOTAP into both DOPS and DMPS formulations led to an increase in particle size (Table 3, entries 2 and 4) without substantially altering the lamellar spacing, indicating that DOTAP modulates vesicle morphology without disrupting the internal structural periodicity (World J Pharm Res, 2014, 1, 1920).

[0199] Example 8. DSC Analysis of DOPS, DOPS:DOTAP, DMPS, and DMPS:DOTAP Cochleates

[0200] DSC thermograms were obtained to analyse phase transitions and interactions among DOPS, DMPS, and DOTAP in cochleates (FIG. 10).

[0201] Results and Discussions

[0202] At approximately 40 °C, DMPS with a known phase transition temperature (Tm) of 35 °C exhibited distinct thermal peaks corresponding to the transition from a lamellar gel phase to a fluid phase. In contrast, DOPS, with a much lower Tm of -11 °C, displayed no prominent transition peak in this range, as it already exists in the fluid phase under these conditions. For the mixed lipid systems, DOPS:DOTAP and DMPS:DOTAP cochleates, a broad endothermic transition cantered around 40 °C was observed. This broadening is indicative of phase coexistence, likely due to the presence of gel, ripple, and fluid phases arising from the heterogeneous composition of lipid mixtures with differing Tm values (Biophysical journal 2003, 85, 350; Advances in Condensed Matter Physics 2015, 2015, 479318). At higher temperatures (~175 °C), pure DOPS and DMPS cochleates exhibited sharp endothermic peaks, attributed to the loss of calcium ions and subsequent breakdown of the cochleate structure. In comparison, DOPS:DOTAP and DMPS:DOTAP cochleates showed a slight thermal shift of this degradation peak towards lower temperatures, suggesting altered lipid packing at the bilayer interface in the presence of DOTAP. The DSC thermograms reveal a multi-step thermal behaviour: an initial pre-transition associated with acyl chain rearrangement near the respective Tm values, a broad main phase transition near 40 °C, and a final degradation event beyond 120 °C, corresponding to the disruption of ionic interactions and release of bound calcium. The incorporation of DOTAP appears to modulate the structural and thermal properties of the bilayers, likely due to its cationic nature enhancing electrostatic interactions and influencing bilayer packing. These results confirm the successful integration of DOTAP into the cochleate bilayer, which in turn modifies the phase behaviour, thermal stability, and calcium-binding dynamics of both DOPS and DMPS-based systems, as evidenced by the distinct profiles observed in the DSC thermograms.

[0203] Example 9. Plausible Mechanism of Cochleates Formation in the Presence of Cationic Phospholipids

[0204] The suggested mechanism of cochleates formation using mixed cationic-anionic phospholipids is shown in FIG. 11 .

[0205] Discussions

[0206] At the liposomal stage, pure anionic phospholipids such as DOPS and DMPS possess negatively charged phosphate headgroups that generate strong electrostatic repulsions both within the bilayer and between adjacent vesicles. This is reflected in their high zeta potential values. These repulsive forces contribute to enhanced colloidal stability, as supported by both zeta potential and PDI values, when compared to their cationic-anionic counterparts (Tables 2 and 4). However, upon incorporation of the cationic lipid DOTAP, the surface charge is partially neutralized, leading to a reduction in electrostatic repulsion and a corresponding decrease in liposomal stability. (Tables 2 and 4). The addition of calcium ions induces electrostatic bridging between Caz+and multiple anionic phosphate groups on DOPS or DMPS, promoting liposomal aggregation and destabilization (Langmuir, 2014, 30, 1 1704; The Journal of Membrane Biology, 2000, 176, 67; Scientific Reports, 2016, 6, 38035; Langmuir, 2000, 16, 1473). This process disrupts the vesicular architecture and drives the formation of intermediate planar sheet-like structures through self-assembly, facilitated by partial charge neutralization.

[0207] FESEM analysis confirmed the presence of exclusively sheet-like intermediate structures across all formulations (FIG. 4), which retained the electrostatic characteristics of their parent liposomes. Sheets derived from pure DOPS and DMPS liposomes appeared loosely packed and widely spaced, consistent with strong residual repulsion. In contrast, DOPS:DOTAP and DMPS:DOTAP sheets were more compact and closely associated, reflecting reduced repulsion due to the presence of DOTAP. These structural observations align with the zeta potential and PDI values reported in Table 2, Table 4, and FIG. 4, respectively. Upon calcium- mediated interaction, the intermediate sheets from pure anionic liposomes transitioned inefficiently into cochleates, producing heterogeneous structures such as stacked layers, curved sheets, ribbons, and networks. This poor cochleation efficiency is attributed to the higher repulsive forces and structural rigidity of the intermediates. Conversely, the mixed anionic-cationic systems (DOPS:DOTAP and DMPS:DOTAP) underwent rapid and efficient cochleate formation, yielding more uniform and pure structures due to their lower repulsion and reduced stability. The cationic DOTAP molecules play a crucial role in overcoming the electrostatic barriers by interacting with the anionic phospholipids, thereby promoting membrane fusion. This interaction reduces the energetic cost of bilayer contact and facilitates the formation of fusion intermediates such as stalks. Concurrently, calcium binding condenses the cis leaflet, creating mechanical asymmetry that drives trans-leaflet interaction. This asymmetry facilitates fusion pore formation and the rolling of planar sheets into tightly packed cochleate cylinders {Proceedings of the National Academy of Sciences, 2020, 117, 18470).

[0208] Example 10. Preparation of Empty Cochleate, A and Drug-Loaded Cochleates, B-H

[0209] Based on the success in the Examples above, it is a logical step to investigate their potential for capsaicin (CAP) delivery. This is especially significant given that the previous examples demonstrated the preparation of the purest cochleates free of intermediate structures, such as sheets, stacks, or liposomes. However, since the cochleates in previous examples were empty (no drug loaded / encapsulated), it is essential to thoroughly investigate the impacts to drug encapsulation, controlled release, and the overall quality of the final drug-loaded cochleate formulations, so as to validate its suitability for practical pharmaceutical use.

[0210] Cochleates A-H were prepared using the trapping film method as follows.

[0211] Preparation of Drug-Loaded Cochleates

[0212] Lipids were dissolved in 4 mL of a chloroform: methanol mixture (3:1 , v / v) by stirring at room temperature in a round-bottom flask. Either individual lipids or lipid mixtures, as detailed in Table 5, were used for cochleate preparation. For empty cochleates (A), no capsaicin (CAP) was added. In formulations B-H, the specified amount of CAP was first dissolved in 1 mL of methanol prior to lipid solubilization. The organic solvent mixture was evaporated under reduced pressure using a rotary evaporator, and the resulting thin lipid film was further vacuum-dried for 1 hour to eliminate residual solvents. The dried film was then hydrated with phosphate-buffered saline (PBS, pH 7.4) to achieve a final lipid concentration of 5 mg mL-1(6.2 mM). Small unilamellar vesicles (SUVs) were formed by sonicating the dispersion for 15 minutes in a bath sonicator (ULTRASONIC LC 30), followed by extrusion through a 100 nm polycarbonate membrane using a mini extruder (Avanti® Polar Lipids Inc.). Cochleate formation was initiated by the controlled addition of 100 mM CaCI2solution (in PBS) to the SUV dispersion at a rate of 10 pL-min-1using a syringe pump, under continuous stirring at 80 °C and 250 rpm for 5 hours. This process resulted in the formation of a dense white suspension. The suspension was centrifuged, and the pellet was washed twice with ultrapure water (2 x 4 mL), freeze-dried, and stored at 4 °C for further use. All steps involving hydration, sonication, and extrusion were performed above the phase transition temperature (Tm) of the respective lipids to ensure uniform vesicle formation.

[0213] Table 5. Preparation of empty and CAP-loaded cochleates A-H using different lipids, lipid molar ratios, and CAP molar ratios.

[0214] Example 11. Characterisation of Empty Cochleate A and Drug-Loaded Cochleates B-H by Field Emission Scanning Electron Microscopy (FESEM)

[0215] The structure and morphology of the prepared cochleates A-H (Table 5) were visualized using FESEM (FIG. 1 .

[0216] Results and Discussions

[0217] FESEM images of all cochleate formulations revealed well-defined structures, with the characteristic cigar-shaped morphology clearly visible, confirming successful cochleate formation. However, variations were observed in terms of purity, degree of aggregation, surface features, and structural distinctiveness among the different samples, as summarized below in Table 6.

[0218] Table 6. Summary of the structure and morphology of cochleates A-H as inferred from the FESEM monographs (FIG. 12).

[0219]

[0220] The variations observed in Table 6 can be attributed to the lipid-to-CAP ratio, the type of lipid used, and the nature of lipid mixtures. These factors played a significant role in determining the morphology and structural integrity of the resulting cochleates. For instance, comparing cochleates B, C, and D which are prepared with DOPS:CAP ratios of 10:3, 10:7, and 10:10, respectively, reveals that the 10:7 ratio (cochleate C) produced the most well-formed structures with minimal aggregation and negligible presence of intermediate structures. Based on this, a lipid-to-CAP ratio of 10:7 was adopted in all subsequent formulations to optimize structural quality. Cochleates prepared with DMPS (e.g., sample E) exhibited more aggregation and lower purity compared to those prepared with DOPS (e.g., sample D), as shown in (FIG. 13). Notably, cochleate H, formulated using a combination of anionic lipids (DOPS and DMPS) and the cationic lipid DOTAP, displayed exceptionally uniform and well- defined structures. This formulation produced the most distinct cochleate morphology, with no observable intermediate structures such as sheets, stacks, liposomes, or ribbons. To our knowledge, the FESEM image of cochleate H represents the highest structural clarity and separation achieved among all CAP-loaded formulations. These observations are consistent with earlier results from the preparation of empty cochleates using anionic / cationic lipid mixtures, which also yielded highly pure structures. The superior structural quality of cochleate H is attributed to the cationic nature of DOTAP, which synergistically enhances calcium- mediated self-assembly and bilayer fusion. The positively charged DOTAP headgroups interact with the negatively charged DOPS / DMPS phospholipids, reducing electrostatic repulsion, lowering the energy barrier for membrane fusion, and promoting hydrophobic interactions, nucleation, and fusion stalk formation (The Journal of Physical Chemistry B, 2011 , 115, 2287; Proceedings of the National Academy of Sciences, 2020, 117, 18470). During cochleate H formation, DOTAP facilitates lipid fusion by minimizing energetic constraints, while calcium ions further condense the cis leaflet, generating mechanical asymmetry across the bilayer. This asymmetry drives trans-leaflet contact and promotes the transformation of sheet-like intermediates into tightly rolled cochleate cylinders.

[0221] Importantly, the high structural purity observed in cochleate H confirms that CAP encapsulation does not hinder cochleate formation, indicating that drug presence does not compromise morphological integrity. These findings further reinforce the conclusion drawn from previous examples: the net charge of the lipid bilayer is the critical determinant governing cochleate purity.

[0222] Based on Table 6 and FIG. 12, cochleates H characterized by their organized, rod-like structure and reduced aggregation, exhibit an ideal morphology for drug delivery, likely enhancing stability and enabling controlled, predictable CAP release compared to cochleates B-G.

[0223] Example 12. Characterisation of Empty Cochleate A and Drug-Loaded Cochleates B-H Using Fourier Transform Infrared (FTIR) Spectroscopy

[0224] The structure and morphology of the prepared cochleates A-H (Table 5) were seen using FESEM (FIG. 12). The FTIR spectra of pure CAP, empty cochleates A, and CAP-loaded cochleates B-H were also recorded for comparison and to characterize their structures (FIG. 13).

[0225] Results and Discussions

[0226] For cochleates A-H, the interaction between Ca2+ions and the phosphate (PO2“) groups of the lipids resulted in a shift of the asymmetric phosphate stretching band from 1216 cm-1to 1236 cm-1. Additionally, the symmetric phosphate stretching region exhibited splitting into four distinct peaks at 1063 cm1, 1075 cm1, 1 102 cm1, and 1 112 cm1(FIG. 13, (ii)). These spectral changes indicate bidentate complexation of calcium with the phosphate headgroups and dehydration effects, which influence the torsional angles of the P-0 ester bonds. Additional characteristic peaks were observed at 1730 cm1. corresponding to ester C=O stretching vibrations, and at 3430 cm-1, attributed to N-H stretching. Furthermore, symmetric and asymmetric CH2stretching vibrations of the lipid alkyl chains appeared at 2852 cm-1and 2923 cm1, respectively. These FTIR findings are consistent with previous reports confirming cochleate formation (Biophysical journal, 1993, 64, 11 13; Biochemistry, 1983, 22, 6318).

[0227] The FTIR spectrum of pure capsaicin (CAP) displayed distinct peaks corresponding to aliphatic C-H stretching vibrations of the long hydrocarbon tail at 2869 cm-1. Peaks at 1512 cm-1and 1555 cm-1were attributed to aromatic ring vibrations associated with the vanillyl moiety of CAP (FIG. 13, (iii)). Additional characteristic bands were observed at 1350 cm-1, corresponding to C-N stretching of the amide bond, and at 810 cm-1, representing out- of-plane C-H bending of the aromatic ring, in agreement with literature (Int J Multidiscip Curr Res, 2016, 4, 1145). In the CAP-loaded cochleates FIG. 13, (ii), changes in the intensity of the 1512 cm-1and 1555 cm-1bands were noted across formulations A-H, suggesting interactions between the vanillyl group of CAP and the lipid carbonyl groups. This interaction likely alters the electronic environment of the aromatic ring, as supported by previous studies (Biophysical journal, 2015, 108, 1425). Notably, in cochleate H, a pronounced broadening of the 3430 cm-1peak assigned to N-H stretching vibrations was observed, indicating a higher degree of hydrogen bonding between CAP and the lipid matrix. This enhanced hydrogen bonding is likely facilitated by the presence of DOTAP, due to the fact that cationic headgroups promote favourable electrostatic and hydrogen-bonding interactions. Similar effects have been reported by Poustforoosh et al., who demonstrated that DOTAP-containing bilayers increase hydrogen bonding between encapsulated drugs and lipid components (Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2024, 687, 133547).

[0228] Example 13. Small Angle X-Ray Scattering (SAXS) Analysis of Empty Cochleate A and Drug-Loaded Cochleates B-H

[0229] The SAXS pattern of cochleates A-H and pure CAP are shown in FIG. 14 (i).

[0230] Results and Discussions

[0231] Cochleates A-H displayed consistent SAXS diffraction profiles, each featuring a strong primary reflection peak accompanied by weaker higher-order reflections, characteristic of well- organized lamellar structures. The position of the first-order peak in each pattern corresponds to the interlamellar repeat distance (d-spacing), calculated to be 5.1 1 nm (A), 5.1 1 nm (B), 5.11 nm (C), 5.1 1 nm (D), 4.37 nm (E), 4.94 nm (F), 5.02 nm (G), and 5.11 nm (H), respectively. Distinct Bragg reflection peaks at approximately 2.53 nm and 1.69 nm were observed in the SAXS profiles of DOPS-based cochleates. The variations in d-spacing between DOPS cochleates (A-D) and DMPS cochleate E can be attributed to differences in the acyl chain lengths of the respective phospholipids, with DORS (Ci8) having longer chains than DMPS (C14). These findings are consistent with previously reported values by Shuddhodana et al (Journal of Molecular Liquids 2021 , 335, 1 16249) and Nagarsekar et al (Nagarsekar, “Cochleates: new insights into drug delivery system”, 2016). In the CAP-loaded cochleates (B-H), a notable reduction or complete disappearance of the diffraction peak at 20 ~ 5.97°, corresponding to crystalline CAP, was observed. This suggests a loss of crystallinity upon encapsulation, likely due to interactions between CAP and the lipid bilayer (Pharmaceutics, 2020, 12, 463). These results imply that CAP, which exists as aggregated crystals prior to encapsulation, becomes molecularly dispersed within the lipid matrix after incorporation (Biophysical journal, 2015, 108, 1425). Mechanistically, the phenolic group of CAP is believed to interact with the polar headgroups of the phospholipids, while its hydrophobic alkyl tail inserts into the nonpolar core of the bilayer. This suggests that CAP localizes at the interfacial region of the lipid bilayer, contributing to its stable incorporation and altered crystallinity within the cochleate matrix. (FIG. 14, (ii)) (The Journal of Physical Chemistry B, 2015, 119, 12086).

[0232] Example 14. Qualitative analysis of CAP in Drug-Loaded Cochleates B-H

[0233] The presence of CAP in cochleates B-H was qualitatively tested using HPLC to ensure successful loading.

[0234] Detection of CAP in the Prepared Cochleates B-H Using Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC)

[0235] Qualitative analysis of capsaicin (CAP) in cochleates B-H was conducted using high- performance liquid chromatography (HPLC; Agilent 1260 Infinity Series) to confirm successful drug loading. Chromatographic separation was carried out on a ZOFIBAX Eclipse Plus C-18 column with a mobile phase consisting of acetonitrile: water (80:20, v / v) containing 0.1% formic acid. The flow rate was maintained at 0.8 mL / min, with an injection volume of 10 pL and a total run time of 10 minutes. Detection was performed at 281 nm using a diode array UV detector (DAD). To extract CAP, the cochleates were disrupted using 200 pL of 100 mM EDTA prepared in 2% NaHCO3(pH > 10), followed by the addition of 2 mL methanol. The resulting solution was filtered through a 0.22 μm syringe filter prior to HPLC analysis.

[0236] Results and Discussions

[0237] It was observed that the chromatogram of pure CAP showed the two expected peaks of capsaicin at a retention time of 4.29 minutes and dihydrocapsaicin at a retention time of 4.83 minutes (FIG. 15) (Materials Science and Engineering: C, 2018, 93, 70). Following cochleate disruption using EDTA, as described previously, HPLC analysis confirmed the presence of CAP with retention times agreeing that of the standard, thereby verifying successful drug loading. Additional peaks observed at approximately 2.9 and 3.0 minutes were attributed to residual EDTA used during the cochleate disassembly process.

[0238] Example 15. Analysis of Length, PDI, and Zeta Potential of Empty Cochleate A and Drug-Loaded Cochleates B-H

[0239] Table 7 shows the particle size, PDI, and zeta potential of cochleates A-H.

[0240] Results and Discussions

[0241] Table 7. Length, PDI, and zeta potential values of cochleates A-H measured at pH 7.4.

[0242] Cochleate Length (nm) PDI Zeta Potential (mV)

[0243] A 916 ± 254 0.805 ± 0.12 -25.8 ± 1.91

[0244] B 976 ± 277 0.615 ± 0.05 -34.2 ± 2.22

[0245] C 957 ± 260 0.765 ± 0.20 -27.3 ± 4.22

[0246] D 1 107 ± 367 0.829 ± 0.158 -23.6 ± 0.96

[0247] E 899 ± 127 0.561 ± 0.024 -32.7± 1 .45

[0248] F 1262 ± 431 0.549 ± 0.041 -31 .3 ± 0.45

[0249] G 1 188 ± 479 0.766 ± 0.405 -38.8 ± 1.21

[0250] H 1486 ± 350 0.880 ± 0.131 -21 .7 ± 1.59

[0251] Length values were obtained using ImageJ software and PDI and zeta potential values were obtained by DLS.

[0252] As shown in Table 7, the length of the cochleates is largely influenced by the type and combination of lipids used in their formulation. Cochleates prepared with DOPS alone (A-D) exhibited greater lengths than those formed with DMPS alone (E), but shorter than those prepared using lipid mixtures such as DOPS / DMPS (F), DOPS / DMPS / cholesterol (G), and DOPS / DMPS / DOTAP (H). These differences can be explained by variations in acyl chain length, lipid charge, and bilayer integration behaviour. Lipids with longer acyl chains such as DOPS (C18:1 ) compared to DMPS (C14:0) produced longer cochleate structures, consistent with previous reports (Journal of Drug Delivery Science and Technology, 2019, 52, 27-36). Notably, cochleates H, containing DOTAP in combination with DOPS and DMPS, exhibited the greatest length. This enhancement is likely due to the intrinsic flexibility of DOTAP, which increases membrane fluidity and promotes more efficient bilayer fusion and self-assembly during cochleate formation. This interpretation aligns with findings reported by Mazaheri- Tehrani, who demonstrated that surface charge and membrane flexibility significantly influence lipid interactions and assembly behaviour (Mazaheri-Tehrani, G., “ Effect of surface charge and rigidity of liposomes on their interaction with gold nanoparticles", 2024, Loughborough University).

[0253] The polydispersity index (PDI) values for cochleates A-H ranged from 0.549 to 0.88, indicating considerable variation in particle size distribution among the different formulations. Lower PDI values observed in cochleates E (0.561 ) and F (0.549) suggest a higher degree of uniformity and better homogeneity. In contrast, higher PDI values in cochleates D (0.829) and H (0.88) reflect greater size heterogeneity, likely due to less controlled self-assembly. Intermediate values, such as those for cochleates B (0.615) and C (0.765), indicate a moderate level of particle size uniformity.

[0254] Zeta potential measurements ranged from -21 .7 mV to -38.8 mV, reflecting differences in surface charge across formulations. Cochleates G (-38.8 mV) and B (-34.2 mV) exhibited the most negative values, indicating stronger electrostatic repulsion and a higher density of surface charge. In contrast, cochleate H showed the least negative zeta potential (-21 .7 mV), which can be attributed to the presence of DOTAP, a cationic lipid component. The inclusion of DOTAP likely neutralizes the negative surface charge contributed by DOPS and DMPS, reducing the overall surface potential. Cochleates E (-32.7 mV) and F (-31.3 mV) exhibited moderate zeta potential values, suggesting a balance between electrostatic repulsion and structural compaction. Variations in zeta potential are influenced by factors such as calcium- mediated phosphate binding, aggregation state, particle size, and lipid composition. Specifically, DOTAP’s positive charge reduces the net negative surface charge by partially neutralizing anionic phosphate groups, as seen in cochleate H (Table 7).

[0255] Overall, these results highlight the critical role of lipid physicochemical properties such as headgroup charge, acyl chain length, and saturation in modulating self-assembly kinetics and determining the final structural attributes of cochleates. These trends are further corroborated by morphological differences observed in FESEM analysis. Example 16. Encapsulation Efficiency and Loading of CAP in Cochleates B-H

[0256] The encapsulation efficiency and CAP loading in cochleates B-H (Table 8) were optimized by varying the lipid composition and lipid-to-CAP molar ratio (Table 5 in Example 10).

[0257] Quantification of the Encapsulation Efficiency and Drug Loading of CAP in the Drug-Loaded Cochleates B-H

[0258] Following the complete formation of cochleates as described in Example 10, the CAP-loaded cochleates were separated from the suspension via centrifugation at 6000xg for 10 minutes. The resulting supernatant was analysed for unencapsulated / free CAP using UV-Vis spectrophotometry (Thermo Evolution™ 300), with absorbance measured at 281 nm against an external standard of pure CAP (The Journal of Physical Chemistry B, 2011 , 115, 2287). The resulting CAP-loaded cochleate pellets were washed and resuspended in ultrapure water to remove buffer components, then freeze-dried under vacuum to obtain buffer-free white cochleate powder. For encapsulation analysis, 2 mg of the freeze-dried cochleates were disrupted by adding 200 pL of 100 mM EDTA, followed by 2 mL of methanol. The resulting lipid vesicle suspension was further treated with 800 pL of ultrapure water and 1 mL of ethanol. The final mixture was filtered through a 0.22 μm syringe filter, and the filtrate was analysed at 281 nm to determine the amount of encapsulated CAP, using the same spectrophotometric method. (The Journal of Physical Chemistry B, 2011 , 115, 2287). The CAP encapsulation (%) and CAP loading (mg CAP / mg of cochleates) were calculated as follows (Eq. 1 and Eq. 2).

[0259] CA . >P > Encap 100 Eq. . r 1

[0260] C ~AAIP-. - Loading i E-q. 2

[0261] Results and Discussions

[0262] Table 8. Encapsulation efficiencies (%) and drug loading of CAP in cochleates B-H. Cochleate Encapsulation Efficiency (%) CAP Loading (mg / g cochleates)

[0263] E 73.08 ± 4.46 146.16 ± 8.92

[0264] F 61.25 ± 3.21 122.50 ± 6.43

[0265] G 71.76 ± 2.76 143.53 ± 5.52

[0266] H 93.35 ± 2.57 196.12 ± 5.40

[0267] *N.A.: Not applicable since cochleates A are empty cochleates.

[0268] Cochleates B-D, formulated with DORS lipids at increasing DOPS:CAP molar ratios (10:3, 10:7, and 10:10), demonstrated that optimal drug loading occurs at a 10:7 ratio (cochleate C), achieving the highest encapsulation efficiency (57.06 ± 0.13%) and CAP loading (128.38 ± 0.30 mg / g). This suggests that a balanced lipid-to-drug ratio is critical for maximizing entrapment. Further increasing CAP concentration, as in cochleate D, led to reduced efficiency and loading, likely due to saturation of the lipid bilayer’s capacity to incorporate CAP. Replacing DOPS with DMPS at the same 10:7 ratio (cochleate E) significantly enhanced both encapsulation efficiency (73.08 ±4.46%) and drug loading (146.16 ± 8.92 mg / g), which may be attributed to more favourable CAP-lipid interactions and improved membrane integration, as previously discussed in Example 13. However, the combination of DOPS and DMPS (cochleate F) resulted in a decline in both parameters, potentially due to structural heterogeneity within the mixed lipid bilayer that interferes with efficient drug entrapment. Incorporation of cholesterol (cochleate G) further decreased encapsulation performance compared to cochleate E. This contrasts with prior reports where cholesterol enhanced bilayer stability and drug retention {Progress in lipid research, 2008, 47, 319). The observed reduction here may be due to cholesterol-induced modulation of bilayer fluidity, which impairs CAP incorporation into the membrane (J. Mol. Liq., 2020, 311 , 113352). Strikingly, cochleate H comprising DOPS, DMPS, and the cationic lipid DOTAP exhibited the highest encapsulation efficiency (93.35 ±2.57%) and CAP loading (196.12 ± 5.40 mg / g) among all formulations. This superior performance is attributed to the synergistic effects of lipid composition, particularly DOTAP's role in enhancing membrane fluidity and self-assembly, as well as the high structural uniformity and purity observed in FESEM images. Collectively, these findings underscore the critical role of lipid selection including charge, chain length, and membrane organization in governing cochleate structure and drug encapsulation behaviour. Notably, the CAP loading achieved with cochleate H surpasses reported values for lipid-polymer hybrid nanoparticles (91.0%) (J. Mol. Liq., 2020, 311, 113352) (J. Controlled Release, 2013, 170, 51 ), liposomal nano-formulations (82%), and magnetic nanoparticles (89%) {Pharmaceutical Research, 2017, 34, 1255), positioning it as a highly promising drug delivery system. Example 17. In-Vitro Release Studies of CAP from CAP-Loaded Cochleates B-H

[0269] The in vitro release profiles of pure capsaicin (CAP) and CAP-loaded cochleates (B-H) were evaluated in phosphate-buffered saline (PBS, pH 7.4), simulated gastric fluid (SGF, pH 1.2), and simulated intestinal fluid (SIF, pH 6.8), as shown in FIG. 16.

[0270] In-Vitro CAP Release from the CAP-Loaded Cochleates B-H

[0271] CAP release was studied using a dialysis bag diffusion system under sink conditions. For each formulation, 1 mg of pure CAP or an equivalent amount of CAP-loaded cochleates (containing 1 mg / mL CAP) was placed in a cellulose dialysis membrane (MWCO 14,000 Da). The bags were immersed in 9 mL of PBS (pH 7.4) supplemented with 3 mL ethanol to ensure CAP solubility and maintain sink conditions (Mater. Sci. Eng. C, 2018, 93, 70). The receptor medium was maintained at 37 °C with constant stirring at 100 rpm (Materials Science and Engineering: C, 2018, 93, 70). At predetermined time points over 94 hours, 1 mL aliquots were withdrawn and replaced with an equal volume of fresh PBS to maintain constant volume and sink conditions (Journal of Molecular Liquids 2021 , 335, 116249). CAP content in the collected samples was quantified by UV-Vis spectrophotometry at 281 nm, as previously described.

[0272] In addition to PBS, release studies were conducted in SGF and SIF to evaluate CAP release under gastrointestinal conditions. SGF was prepared using 0.2% (w / v) NaCI solution adjusted to pH 1 .2 with 1 .0 M HCI, while SIF was prepared by adjusting 0.9% (w / v) KH2PO4solution to pH 6.8 using 0.2 M KOH. Release procedures in SGF and SIF followed the same protocol as described for PBS. The percentage of CAP released at each time point was calculated using Equation 3: Eq. 3n

[0273] Results and Discussions

[0274] Pure capsaicin (CAP) exhibited an immediate and rapid release profile, characteristic of poorly water-soluble drugs under sink conditions. In PBS (FIG. 16, (i)), over 85-90% of CAP was released within the first 4 hours, reaching >95% by 5 hours. This pronounced burst release is typical for unencapsulated hydrophobic molecules due to their rapid diffusion into the medium. In contrast, CAP-loaded cochleates B-D, formulated with increasing DOPS:CAP molar ratios (10:3, 10:7, and 10:10), exhibited significantly slower and more sustained release profiles. Cochleate B released approximately 20-35% of CAP within the first 20 hours, with cumulative release reaching 65-70% by 94 hours. Cochleate C (10:7), which had the highest encapsulation efficiency, showed a slightly higher release of about 35-40% at 20 hours and 70-75% by 94 hours. Interestingly, cochleate D (10:10) exhibited the slowest release among the three, with -38% at 20 hours and 65-70% at the endpoint. The slower release observed in all cochleate formulations is attributed to the encapsulating lipid matrix, which acts as a diffusion barrier and modulates CAP release kinetics. Cochleates E-H, which included modifications to the lipid composition such as substitution with DMPS, or inclusion of cholesterol and DOTAP exhibited distinct release behaviours in PBS (FIG. 16, (ii)), Cochleate E released -45% of CAP by 20 hours and 70-75% by 94 hours. Cochleate F showed a slightly faster release (45-50% at 20 hours), levelling off to 65-70% at 94 hours. The inclusion of cholesterol in cochleate G resulted in a slower release profile (35-40% at 20 hours, 60-65% at 94 hours), potentially due to reduced membrane permeability. Cochleate H, which incorporated DOTAP along with DOPS and DMPS, demonstrated the most prolonged release: only 35% was released at 20 hours, and 59-60% by 94 hours. This may be attributed to the highly ordered and compact structure of cochleate H, which likely impedes CAP diffusion.

[0275] In physiologically relevant conditions such as simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 6.8), release trends closely mirrored those observed in PBS (FIG. 16, (iii) and (iv)), Pure CAP continued to exhibit rapid burst release in both media, with -98% released within 5 hours. In contrast, the cochleate formulations (B-H) showed sustained and controlled release. For example, cochleate B released 40-45% of CAP at 4 hours and reached 75-80% by 94 hours, while cochleate H released a similar amount (-40-45%) at 4 hours but only 70-75% by the endpoint. These controlled release profiles across different pH conditions highlight the stability of the cochleate matrix and the protective effect of encapsulation.

[0276] The differences in release rates among the formulations can be attributed to the physicochemical properties of the lipids used such as acyl chain length, degree of saturation, and headgroup charge which influence membrane packing and permeability {European Biophysics Journal, 2022, 51, 205). Similar controlled-release trends have been reported for other lipophilic drugs encapsulated in cochleate or lipid-based systems, such as amikacin {Journal of Molecular Liquids, 2021 , 335, 116249), Amphotericin b {International Journal of Pharmaceutics, 2021 , 603, 120688), Paclitaxel {International Journal of Pharmaceutics, 2020, 586, 1 19482) and Fisetin {AAPS pharm SciTech, 2016, 17, 968). In summary, encapsulation of CAP within cochleates effectively reduced the initial burst release and enabled prolonged drug release. While pure CAP showed rapid diffusion and complete release within hours, cochleate formulations offered a more controlled and sustained release, which is advantageous for maintaining therapeutic drug levels and minimizing potential side effects associated with rapid absorption and metabolism. Example 18. Stability Studies of CAP-Loaded Cochleates H under Various pH and Temperature Conditions

[0277] Cochleate H was selected for stability evaluation based on its superior characteristics, including highest structural purity, minimal aggregation, optimal drug loading, and encapsulation efficiency (EE%) (%) (Table 8), as well as its sustained drug release profile.

[0278] Stability Studies of CAP-Loaded Cochleate H

[0279] To assess pH-dependent stability, CAP-loaded cochleate H powder equivalent to 1 mg of CAP was dispersed in PBS solutions adjusted to pH 3.3, 5.3, and 7.4, and incubated at 37 °C for 20 hours. After incubation, the amount of released CAP was quantified using UV-Vis spectrophotometry at 281 nm, as previously described above.

[0280] For thermal stability evaluation, CAP-loaded cochleate H (equivalent to 1 mg of CAP) was incubated in PBS (pH 7.4) at four different temperatures: 4 °C, 25 °C, 37 °C, and 55 °C for 20 hours. Post-incubation, CAP content was measured spectrophotometrically at 281 nm to assess the extent of drug retention under varying thermal conditions.

[0281] Results and Discussions

[0282] The in-vitro release profile of CAP from cochleate H in PBS at 37 °C over a 20-hour period demonstrated a clear pH-dependent behaviour (FIG. 17 (i)). As the pH increased from 3.3 to 5.3 to 7.4, the release of CAP progressively decreased. Under acidic conditions, protonation of the negatively charged phospholipid headgroups reduces their affinity for calcium ions, thereby weakening the lipid-calcium interactions that stabilize the cochleate structure. This destabilization leads to the loosening of the compact bilayer arrangement and facilitates CAP release into the surrounding medium (Asian journal of pharmaceutical sciences, 2015, 10, 81 ). Moreover, acidic pH conditions are known to alter lipid phase behaviour by promoting a transition from the gel phase to a more fluid bilayer state, further enhancing drug diffusion (Chemistry and physics of lipids, 1991 , 57, 293-307). Together, these effects account for the accelerated CAP release observed at lower pH values.

[0283] Thermal stability studies of cochleate H revealed a temperature-dependent increase in CAP release, as shown in FIG. 17 (ii). As incubation temperature rose from 4 °C to 55 °C, the integrity of the lipid bilayer was progressively compromised. Elevated temperatures induce a phase transition from the ordered gel phase to a disordered fluid phase, increasing bilayer fluidity and permeability (J Liposome Res, 2014, 24, 290). This structural disruption weakens the cohesive lipid-calcium network that maintains cochleate stability, thereby enhancing CAP diffusion.

[0284] These findings underscore the critical influence of both pH and temperature on the structural integrity and release behaviour of CAP-loaded cochleates. Based on the observed release kinetics, the most suitable storage condition for maintaining the stability and controlled release properties of cochleate H is at 4 °C under neutral pH conditions (Foods, 2022, 11, 710; Food Chem., 2022, 386, 132692; Colloid J., 2013, 75, 26; Polymer, 2018, 158, 223).

[0285] Example 19. Preparation and Morphological Analysis of DOPS:CTAB Cochleates

[0286] DOPS:CTAB cochleates were prepared using the trapping film method across varying DOPS:CTAB molar ratios, as outlined in Table 9. For comparison, conventional DOPS cochleates without CTAB (DOPS:CTAB = 10:0) were also prepared to serve as a control and benchmark to evaluate the impact of CTAB on cochleate structure and morphology. All formulations, including the CTAB-modified cochleates (l-M), were subjected to small-angle X- ray scattering (SAXS) analysis to investigate their internal lamellar organization. The corresponding SAXS profiles are shown in FIG. 18.

[0287] Preparation of DOPS:CTAB Cochleates

[0288] DOPS (Avanti Polar Lipids Inc.) was dissolved in a chloroform: methanol mixture (3:1 , v / v), and the lipid compositions were adjusted according to Table 9. The organic solvents were removed using a rotary evaporator to form a uniform thin lipid film, which was further vacuum- dried for 30 minutes to eliminate residual solvents. The dried film was then hydrated with phosphate-buffered saline (PBS, pH 7.4) containing the cationic surfactant CTAB (Sigma- Aldrich), and the mixture was incubated at (75 ± 5) °C for 3 hours. Small unilamellar vesicles (SUVs) were generated by sonicating the hydrated suspension for 20 minutes. Subsequently, 248 pL of 100 mM CaCI2(prepared in PBS) was added dropwise at a rate of 10 L / min under continuous stirring. The mixture was stirred for 6 hours at (70 ± 5) °C to facilitate cochleate formation. All preparation steps including hydration, sonication, and calcium addition were performed above the phase transition temperature (Tm) of DOPS to ensure proper membrane fluidity and fusion behaviour. Following cochleate formation, salts were removed by centrifugation or dialysis, and the resulting suspension was freeze-dried. The final product was stored at 4 °C as a dry powder for further analysis. Table 9. Composition of cochleates (l-N).

[0289] DOPS:CTAB Molarity of DOPS Molarity of

[0290] Sample (Molar Ratio) (mM) CTAB (mM)

[0291] Cochleates 1 10:0 6.2 N.A.*

[0292] Cochleates J 9:1 5.58 0.62

[0293] Cochleates K 8:2 4.96 1.24

[0294] Cochleates L 7:3 4.34 1 .86

[0295] Cochleates M 6:4 3.72 2.48

[0296] Cochleates N 4:6 2.48 3.72

[0297] *N.A.- Not applicable.

[0298] Morphological Analysis Using Field Emission Scanning Electron Microscopy (FESEM)

[0299] The surface morphology of cochleate formulations, l-N was examined using Field Emission Scanning Electron Microscopy (FESEM). Freeze-dried cochleate powders were sputter- coated with a thin layer of platinum using a JFC-1600 Auto Fine Coater to enhance conductivity. Imaging was performed on a JEOL JSM-6701 F FESEM operated at an accelerating voltage of 5 kV.

[0300] Internal Ultra-Structural Analysis Using Small-Angle X-Ray Scattering (SAXS)

[0301] To investigate internal lamellar organization, SAXS analysis was carried out on powdered cochleates, l-N using a Nano-inXider vertical SAXS / WAXS system (Xenocs SA), equipped with a microfocus Cu-Ka X-ray source (Genix3D, Xenocs SA). Data acquisition was performed using hybrid pixel detectors (Pilatus 200K, Dectris, Switzerland). All measurements were conducted at 25 °C under vacuum conditions.

[0302] Results and Discussions

[0303] In cochleate I, characteristic cochleate structures were observed, along with intermediate forms such as sheets and partially folded bilayers, as indicated by red arrows in FIG. 18) In contrast, the DOPS:CTAB cochleates (J-N) exhibited notable differences in the degree of sheet rolling and cochleate formation. Cochleates, J-M showed progressive improvement in structural morphology, with more defined and compact cochleate formations. However, no discernible structures were observed in cochleate N, indicating a disruption in the selfassembly process. These variations are likely attributable to changes in the DOPS:CTAB molar ratio, highlighting the critical role of CTAB concentration in modulating the structural organization and morphology of the resulting cochleates (FIG. 18).

[0304] The primary reflection peak observed in the SAXS patterns of cochleates, l-M corresponds to a d-spacing of approximately 5.04 nm, consistent with previously reported values for DOPS- based cochleates. This intense primary peak was accompanied by sharp and regularly spaced Bragg reflections at 2.52 nm and 1.70 nm (FIG. 19), indicating a highly ordered, multilamellar bilayer structure with tight lipid packing. Although the fully extended length of a DOPS molecule is approximately 27 A, the structural parameters of lipid assemblies such as liposomes, bilayers, or cochleates are influenced more significantly by the fluidity of the acyl chains and the hydration state of the lipid headgroups. As such, the bilayer thickness and interlamellar spacing are critical determinants of cochleate architecture.

[0305] For cochleates l-M, the measured d-spacing, bilayer thickness (dB), and water layer thickness (dW) were approximately 50.4 A, 34 A, and 16.8 A, respectively. In comparison, literature values for DOPS systems report d-spacing of ~50 A, bilayer thickness of ~38 A, and water layer thickness of ~12 A. Based on these values, the calculated interlamellar water thickness (dW = d-dB), inclusive of the hydrated calcium-bridging regions, was approximately 13 A. The minor deviations observed in d-spacing and water layer thickness relative to reported values may be attributed to slight differences in the degree of headgroup dehydration. It is important to note that "dehydration" here refers specifically to the removal of loosely bound or bulk water, while a limited number of water molecules remain associated with the lipid- Caz+complex to maintain structural integrity.

[0306] Example 20. Preparation and Morphological Analysis of Irinotecan (IRT)-Loaded DOPS:CTAB Cochleates

[0307] We prepared the conventional IRT loaded DOPS cochleates (O-Q) and novel IRT loaded DOPS:CTAB cochleates (R-T) by the trapping film method. FESEM images of the prepared cochleates were taken by following the protocol disclosed in Example 19.

[0308] Preparation and Characterization of IRT Loaded Cochleates Using Novel Method and Comparison to Conventional IRT Loaded Cochleates

[0309] To prepare the formulations, DOPS or DOPS:CTAB and irinotecan (IRT) were co-dissolved in

[0310] 4 ml_ of chloroform in a round-bottom flask according to the specified compositions in Table 10 The solvent was evaporated under vacuum using a Buchi R-210 rotary evaporator to form a uniform thin lipid film, which was further vacuum-dried for 1 hour to remove residual solvents. The dried film was hydrated with 4 mL of PBS buffer (pH 7.4) to achieve a final lipid concentration of approximately 6.2 mM. The resulting dispersion was sonicated for 20 minutes to form small unilamellar vesicles (SUVs). Subsequently, 250 pL of 100 mM CaCI2stock solution was added to the SUV suspension (4 mL) at a controlled rate of 10 pL / min using a syringe pump, under vigorous stirring, until a final CaCI2concentration of 6.2 mM was achieved. The mixture was stirred continuously for 6 hours at (70 ± 5) °C to facilitate cochleate formation. The resulting suspension was centrifuged at 6000 x g for 10 minutes. The obtained pellet was washed twice with 4 ml_ of distilled water, freeze-dried, and stored at 4 °C for further characterization and use.

[0311] Table 10. Composition of IRT-loaded cochleates O-T.

[0312] DOPS:CTAB:IRT Molarity of DOPS Molarity of CTAB IRT oc ea es (Molar Ratio) (mM) (mM) (mM)

[0313] O 10:0:5 6.2 N.A.* 3.1

[0314] P 10:0:7 6.2 N.A.* 4.34

[0315] Q 10:0:10 6.2 N.A.* 6.2

[0316] R 7:3:5 4.34 1.86 3.1

[0317] S 7:3:7 4.34 1.86 4.34

[0318] T 7:3:10 4.34 1.86 6.2

[0319] *N.A. - Not Applicable

[0320] Results and Discussions

[0321] FESEM images of the IRT-loaded cochleates, presented in FIG. 20 indicate that irinotecan (IRT) concentration plays a critical role in determining the final morphology of the cochleate structures. Notably, cochleates prepared using the DOPS:CTAB lipid system (R, S, T) exhibited superior morphological features compared to those prepared with DOPS alone (O, P, Q). Cochleates R demonstrated well-formed, discrete structures with clearly defined boundaries, indicating successful cochleate formation. In contrast, cochleates O exhibited a mixture of sheet-like intermediates and needle-shaped cochleates, suggesting incomplete or less efficient assembly. Additionally, while both cochleates P and S showed typical cochleate structures, they differed in aggregation levels, with P displaying more aggregation than S. At higher IRT concentrations (6.2 mM), both cochleates Q and T revealed an increased presence of sheet-like structures and fewer rolled cochleates, suggesting compromised formation efficiency. This is likely due to saturation effects at high drug-to-lipid molar ratios (10:10 for DOPSJRT and 7:3:10 for DOPS:CTAB:IRT), which may disrupt membrane stability and hinder the transition from intermediate sheets to fully rolled cochleates. Overall, these FESEM observations support the effectiveness of the novel DOPS:CTAB formulation in enhancing cochleate morphology and optimizing IRT encapsulation, particularly at moderate drug loading levels.

[0322] Example 21. Loading, Encapsulation Efficiency (%EE) and Yield of IRT in Cochleates O-T

[0323] To study the effect of CTAB addition in cochleates on the loading, %EE and yield of IRT, these parameters were determined and analysed.

[0324] Determination of Encapsulation Efficiency (% EE) and IRT Loading in the Various Cochleates To determine the amount of irinotecan (IRT) encapsulated in cochleates O-T, 2 mg of freeze- dried IRT-loaded cochleate powder was treated with 100 pL of 100 mM EDTA (prepared in 2% NaHCO3, pH >10) to disrupt the cochleate structure, followed by the addition of 3 mL of methanol. The resulting solution was filtered through a 0.22 μm syringe filter, and the filtrate was analyzed by UV-Vis spectrophotometry at 358 nm to quantify IRT content.

[0325] IRT encapsulation efficiency (%), drug loading (mg IRT per mg cochleate), and formulation yield were calculated using standard equations as described below.

[0326] (Eq. 4, Eq. 5, Eq. 6),

[0327] Results and Discussions

[0328] Initial studies focused on formulating conventional DOPS-based cochleates (O-Q) with varying DOPSJRT molar ratios (10:10, 10:7, and 10:5) to evaluate irinotecan encapsulation efficiency (%EE), drug loading (DL), and formulation yield. As shown in Table 11 , entries (i-iii), these formulations achieved %EE values of 34.36 ± 1.76%, 59.30 ± 1.47%, and 47.74 ± 2.62%, respectively, with corresponding drug loading of 91.34 ±4.68, 197.69 ±4.92, and 200.82 ± 11 .05 mg IRT / g of cochleate. Given the need for high encapsulation and drug loading in cochleate-based delivery of amphiphilic drugs like IRT, we identified DOPS:CTAB (7:3 molar ratio) as the optimal lipid system based on superior morphology (FIG. 20) and high formulation yield (Table 11 ). Using this optimized lipid composition, DOPS:CTAB cochleates (R-T) were prepared with varying IRT molar ratios to assess the influence of CTAB on formulation performance.

[0329] The DOPS:CTAB:IRT cochleates exhibited a marked improvement in both encapsulation and loading parameters. At molar ratios of 7:3:10, 7:3:7, and 7:3:5, %EE values of 66.98 ± 3.10%, 97.26 ±2.06%, and 82.61 ± 2.49% were achieved, respectively. Corresponding drug loading values were 202.07 ± 9.36, 363.48 ± 7.71 , and 383.50 ± 11.60 mg IRT / g of cochleates (Table 1 1 , entries (iv-vi)). This significant enhancement in encapsulation and loading can be attributed to the inclusion of CTAB, a cationic surfactant with a single C16 alkyl chain which likely modulates bilayer hydrophobicity and improves drug-lipid interactions. Unlike DOPS, which possesses two C18 acyl chains, the incorporation of CTAB introduces structural flexibility and may facilitate greater solubilization and integration of IRT within the cochleate matrix. Notably, all formulations (O-T), regardless of composition, achieved high yields exceeding 95%, demonstrating the robustness of both conventional and CTAB-modified cochleate preparation methods (Table 1 1).

[0330] Table 11. Loading, %EE and yield (%) of IRT in IRT-loaded cochleates l-N.

[0331] IRT Loaded IRT Loading

[0332] Entry %EE Yield (%)

[0333] Cochleates (mg IRT / g Cochleates)

[0334] I 0 91 .34 ± 4.68 34.36 ± 1.76 98.18 ± 1 .97 ii P 197.69 ± 4.92 59.30 ± 1.47 97.44 ± 0.68 iii Q 200.82 ± 1 1.05 47.74 ± 2.62 98.99 ± 0.71 iv R 202.07 ± 9.36 66.98 ± 3.10 97.66 ± 1 .71 v S 363.48 ± 7.71 97.26 ± 2.06 98.99 ± 0.71 vi T 383.50 ± 1 1.60 82.61 ± 2.49 95.14 ± 3.43 Example 22. In-Vitro Release Studies of IRT

[0335] The in vitro release profiles of pure irinotecan (IRT), DOPSJRT cochleates, and DOPS:CTAB:IRT cochleates were evaluated in simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SI F, pH 6.8), as presented in FIG. 21 .

[0336] In-Vitro Release of IRT from Cochleates

[0337] To assess drug release behaviour, DOPS:IRT cochleate P and DOPS:CTAB:IRT cochleate S were studied using a dialysis bag diffusion method. SGF was prepared by adjusting 0.2% (w / v) NaCI solution to pH 1 .2 using 1 .0 M HCI, while SIF was prepared by adjusting the pH of 0.9% (w / v) KH2PO4solution to 6.8 using 0.2 M KOH.

[0338] Each dialysis bag (cellulose membrane, MWCO 14,000 Da) was loaded with either 2.5 mg of pure IRT or cochleate formulations J and M containing an equivalent of 2.5 mg IRT. The bags were immersed in 30 ml_ of either SGF or SIF and maintained at 37 °C under constant stirring (100 rpm). Samples (100 pL) were withdrawn at predetermined time intervals over 96 hours, and each withdrawal was immediately replaced with an equal volume of fresh buffer to maintain sink conditions. Collected samples were analysed for IRT content using UV-Vis spectrophotometry, as described previously. The percentage of IRT released was calculated using Equation 7 provided below:

[0339] Pure irinotecan (IRT) exhibited rapid and complete release (-100%) within 5-6 hours under both simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) conditions. In SGF (FIG. 21 , (i)), DOPSJRT cochleates demonstrated an initial burst release of approximately 65% within the first 2 hours, followed by a sustained release reaching >95% within 48 hours. In contrast, the DOPS:CTAB:IRT cochleates displayed a more controlled release profile, with -60% released in the initial 2 hours and a gradual release reaching approximately 95% over 96 hours.

[0340] Under SIF conditions (as shown in FIG. 21 , (II)), DOPS:IRT cochleates released around 80% of IRT over 96 hours, indicating a moderately sustained release. However, the DOPS:CTAB:IRT cochleates exhibited a slower and more controlled release, with 75-80% released over the same period. The improved release control in the CTAB-modified cochleates is likely due to electrostatic interactions between the cationic surfactant (CTAB) and the anionic headgroups of DOPS, which may enhance bilayer integrity and restrict drug diffusion into the external medium. These findings are consistent with previously reported release profiles for other hydrophobic and amphiphilic drugs such as amikacin, amphotericin B, paclitaxel, and fisetin, where cochleate formulations provided sustained release and minimized burst effects.

[0341] In summary, encapsulating IRT in cochleates effectively reduced its rapid initial release and promoted sustained release over time. Between the two cochleate systems, DOPS:CTAB:IRT formulations offered superior release control, likely due to additional ionic interactions that stabilized the bilayer and modulated drug diffusion. Such a release profile is advantageous for enhancing the therapeutic efficacy of IRT by reducing peak plasma concentrations and associated side effects linked to rapid drug release and metabolism.

Claims

Claims:1 . A cochleate, comprising: a cationic amphiphilic material; one or more negative phospholipids; and a divalent cation.

2. The cochleate according to Claim 1 , wherein at pH 7.4, the cochleate has a zeta potential of from about -40 mV to about -15 mV, such as from about -33 to about -20 mV, such as from about -32.75 to about -24.06 mV, such as from about -28.65 to -23.75 mV, such as from about -22.28 to about -20.1 1 mV.

3. The cochleate according to Claim 1 or Claim 2, wherein the cochleate has a length of about 650 to about 2,000 nm, such as from about 900 to about 1 ,850 nm, such as from about 1 ,136 to about 1 ,836 nm, such as from about 1 ,241 to about 1 ,695 nm, such as from about 1 ,051 to about 1 ,403 nm.

4. The cochleate according to any one of Claims 1 to 3, wherein the cochleate has a width of about 80 to about 250 nm, such as from about 90 to about 210 nm, such as from about 134 to about 202 nm, such as from about 94 to about 194 nm.

5. The cochleate according to any one of Claims 1 to 4, wherein the cochleate has a polydispersity index (PDI) value of about 0.50 to about 1 .20, such as from about 0.70 to about 1.12, such as from about 0.72 to about 1.15, such as from about 0.865 to about 1.011 , such as from about 0.964 to about 1 .010, such as from about 0.865 to about 1 .007.

6. The cochleate according to any one of Claims 1 to 5, wherein the cationic amphiphilic material and the one or more negative phospholipids have a molar ratio of about 2:1 to about 10:1 , such as 7:3 to about 9:1 , such as about 7:3, such as about 9:1 .

7. The cochleate according to any one of Claims 1 to 6, wherein the cationic amphiphilic material is selected from one or more of the group consisting of cationic phospholipid, ionisable cationic lipid, cationic surfactant, and positively-charged polymer.

8. The cochleate according to Claim 7, wherein the cationic phospholipid is selected from 1 ,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1 ,2-dimyristoyl-3-trimethylammonium- propane (DMTAP), 1 ,2-distearoyl-3-trimethylammonium-propane (DSTAP), 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1 ,2-dioleyloxy-3-dimethylaminopropane (DODMA),I ,2-di-0-octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA), optionally wherein the cationic phospholipid is DOTAP.

9. The cochleate according to Claim 7, wherein the ionisable cationic lipid is selected from 1 ,2-di-0-octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA), 1 ,2- dioleoyl-3-dimethylammonium-propane (DODAP), 2-[dimethylamino]-ethyl 1 ,2-dioleoyl-sn- glycero-3-phosphoethanoiamine (DLin-MC3-DMA), C12-200 (N-[1 -(2,3-dioleyloxyjpropyl]- N,N,N-trimethylammonium methyl sulfate), 1 ,2-dioleoyl-3-trimethylammonium-propane (A18- DOSPA), 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1 -octylnonyl 8- [ (2- hydroxyethyl) [6-oxo-6- (undecyloxy)hexyl]amino]-octanoate (SM-102), 9,12-Octadecadienoic acid (9Z,12Z) (OF-C4-Deg-Lin), 1 ,1',1 ",1 "'-[(3,6-dioxo-2,5-piperazinediyl)bis(4,1 - butan ediyl nitrilod i-2 , 1 -ethanediyl)] ester, optionally the ionisable cationic lipid is 1 ,2 -dioleoyl- 3-trimethylammoniumpropane (DOTAP).

10. The cochleate according to Claim 7, wherein the cationic surfactant is selected from cetyltrimethylammonium bromide (CTAB), Benzalkonium chloride (BZK, BKC, BAK, BAC), Cetylpyridinium chloride (CPC), Stearalkonium chloride, Dodecyltrimethylammonium chloride, Lauryl Dimethyl Benzyl Ammonium Chloride, Benzethonium chloride, Dimethyldioctadecylammonium bromide, Stearylamine, Hexadecyltrimethylammonium chloride, Tetradecyltrimethylammonium bromide (TTAB; TTABr; MiTMAB), Ethylhexadecyldimethylammonium bromide, Dodecylpyridinium chloride, optionally wherein the cationic surfactant is CTAB.I I . The cochleate according to any one of Claims 1 to 10, wherein the cationic amphiphilic material is 1 ,2-dioleoyl-3-trimethylammoniumpropane (DOTAP).

12. The cochleate according to Claim 7, wherein the positively charged polymer is selected from Poly(L-lysine) (PLL), Polyethyleneimine (PEI), Eudragit, Chitosan, Dendrimers, Poly(vinylamine), Poly(allylamine hydrochloride) (PAH), Oligo(2-(dimethylamino)ethyl methacrylate) (Oligo-DMAEMA), Epsilon -poly-L-lysine (short chain), Polyarginine, Trimethyl chitosan (TMC), Choline-based polyesters, Poly(imine)s (short chain), Amine-functionalized poly(carbonate), Cationic pullulan derivatives, and Poly([3-amino thioester).

13. The cochleate according to any one of Claims 1 to 12, wherein the one or more negative phospholipids are selected from one or more of the group consisting of 1 ,2-dioleoyl- sn-glycero-3-phospho-L-serine (DOPS), 1 ,2-dimyristoyl-sn-glycero-3-phospho-L-serine(DMPS), 1 ,2-didecanoyl-sn-glycero-3-phospho-L-serine (DDPS), 1 ,2-dioctanoyl-sn-glycero- 3-phospho-L-serine (DOctPS), 1 ,2-distearoyl-sn-glycero-3-phospho-L-serine (sodium sail) (DSPS), and 1 ,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DPPS).

14. The cochleate according to any one of Claims 1 to 13, wherein the divalent cation is selected from one or more of the group consisting of Ca2+, Mg2+, and Zn2+.

15. The cochleate according to any one of Claims 1 to 14, wherein the divalent cation is Ca2+.

16. The cochleate according to any one of Claims 1 to 15, wherein the cochleate further comprises an active compound.

17. The cochleate according to any one of Claims 1 to 16, further comprising one or more medicinal compounds.

18. The cochleate according to Claim 17, wherein the one or more medicinal compounds are hydrophobic, hydrophilic or amphiphilic.

19. The cochleate according to Claim 17 or Claim 18, wherein the one or more medicinal compounds are selected from one or more of the group consisting of capsaicin, silibinin, artemisinin, fisetin, raloxifene, sorafenib tosylate, irinotecan (IRT), Ibuprofen, Diazepam, Midazolam, Propofol, Haloperidol, Phenytoin, Thiopental, Fentanyl, Ketoprofen, Paclitaxel , Chloramphenicol, Insulin, Metronidazole, Doxorubicin, Ketoconazole, Cyclosporine, Itraconazole, Celecoxib, Bisoprolol, Rifampin, Curcumin, Resveratrol, Quercetin, Tocopherols (Vitamin E), Berberine, Piperine, Eugenol, Myricetin, Flavonoids, Piperlongumine, Tannins, Rutin, Thymol, Dihydromyricetin, Alpha-mangostin, Lycopene, Alkaloids (e.g., Vincristine, Vinblastine), Docetaxel, Nilotinib, Gefitinib, Imatinib (Gleevec), lndole-3-Carboxaldehyde, Chloroquine, Chlorpromazine, Propranolol, Ipratropium bromide, Tobramycin, Gentamicin, Lidocaine, Epinephrine, Cisplatin, Quinine, Ciprofloxacin, Pyridostigmine, Amitriptyline, Neomycin, Thiamine (Vitamin B1 ), Ranitidine, Pyridoxine (Vitamin B6), Betaxolol, Donepezil, Cetirizine, Amikacin, and docoshexaenic acid (DHA).

20. The cochleate according to any one of Claim 17 to 19, wherein the one or more medicinal compounds in the cochleate have an encapsulation efficiency of at least about 25%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.

21. The cochleate according to any one of Claim 17 to 20, wherein the one or more medicinal compounds in the cochleate have a drug loading of from about 40 mg / g to about 250 mg / g, from about 50 mg / g to about 230 mg / g, from about 70 mg / g to about 215 mg / g, or from about 1 10 mg / g to about 205 mg / g.

22. A method of producing a cochleate, the method comprising contacting a population of liposomes comprising one or more negative phospholipids and a cationic amphiphilic material with a divalent cation source and holding such a mixture at a temperature above the transition temperature (Tm) of the one or more negative phospholipids to convert the liposomes to cochleates.

23. The method according to Claim 22, wherein the method comprises centrifuging the cochleates.

24. The method according to Claim 22 or 23, wherein the method comprises washing the cochleates.

25. The method according to any one of Claims 22 to 24, wherein the method comprises freeze-drying the cochleates.

26. The method according to any one of Claims 22 to 25, wherein the method comprises forming the population of liposomes by: a) providing one or more negative phospholipids and a cationic amphiphilic material in a solvent; b) removing the solvent to form a mixture comprising the one or more negative phospholipids and the cationic amphiphilic material; c) hydrating the mixture to a predetermined concentration of one or both of the one or more negative phospholipids and the cationic amphiphilic material to form a hydrated mixture; and d) contacting the hydrated mixture with a divalent cation source.

27. The method according to Claim 26, wherein the solvent comprises one or both of chloroform and an alcohol, optionally wherein the alcohol is methanol.

28. The method according to Claim 26 or 27, wherein the solvent is removed by one or both of heating and vacuum.

29. The method according to any one of Claims 26 to 28, wherein the step c) comprises contacting the mixture with a buffered aqueous solution, optionally wherein the buffered aqueous solution comprises phosphate-buffered saline.

30. The method according to any one of Claims 22 to 29, wherein the one or more negative phospholipids and the cationic material are provided in a molar ratio of from about 2:1 to about 10:1 , such as from about 3:2 to about 10:1 , such as about 7:3 to about 10:1 , such as 8:1 to 10:1 , such as around 9:1 .31 . The method according to any one of Claims 22 to 30, wherein the one or more negative phospholipids are selected from one or more of the group consisting of 1 ,2-dioleoyl-sn- glycero-3-phospho-L-serine (DOPS), 1 ,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), 1 ,2-didecanoyl-sn-glycero-3-phospho-L-serine (DDPS), 1 ,2-dioctanoyl-sn-glycero-3- phospho-L-serine (DOctPS), 1 ,2-distearoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DSPS), and 1 ,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DPPS).

32. The method according to any one of Claims 22 to 31 , wherein the cationic amphiphilic material is selected from one or more of the group consisting of cationic phospholipid, ionisable cationic lipid, cationic surfactant, and positively-charged polymer.

33. The method according to Claim 32, wherein the cationic amphiphilic material is selected from 1 ,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1 ,2-dimyristoyl-3- trimethylammonium-propane (DMTAP), 1 ,2-distearoyl-3-trimethylammonium-propane (DSTAP), 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1 ,2-dioleyloxy-3- dimethylaminopropane (DODMA), 1 ,2-di-0-octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA), 1 -octylnonyl 8- [ (2-hydroxyethyl) [6-oxo-6-(undecyloxy)hexy!]amino]-octanoate (SM-102), 9,12-Octadecadienoic acid (9Z,12Z) (OF-C4- Deg-Lin), 1 ,1 ', 1 ”,1 '"-[(3,6-dioxo-2,5-piperazinediyl)bis(4,1 -butanediylnitrilodi-2,1 -ethanediyl)] ester, N,N-dioctadecyl-N,N-dimethylammonium propionate, 2-[dimethy!amino]-ethyl 1 ,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DLin-MC3-DMA), 1 ,2-dioteoyl-3- trimethylammonium-propane (A18-DOSPA), cetyltrimethylammonium bromide (CTAB), Benzalkonium chloride (BZK, BKC, BAK, BAC), Cetylpyridinium chloride (CPC), Stearalkonium chloride, Dodecyltrimethylammonium chloride, Lauryl Dimethyl Benzyl Ammonium Chloride, Benzethonium chloride, Dimethyldioctadecylammonium bromide, Stearylamine, Hexadecyltrimethylammonium chloride, Tetradecyltrimethylammonium bromide (TTAB; TTABr; MiTMAB), Ethylhexadecyldimethylammonium bromide,Dodecylpyridinium chloride, Poly(L-lysine) (PLL), Polyethyleneimine (PEI), Eudragit, Chitosan, Dendrimers, Poly(vinylamine), Poly(allylamine hydrochloride) (PAH), Oligo(2- (dimethylamino)ethyl methacrylate) (Oligo-DMAEMA), Epsilon-poly-L-lysine (short chain), Polyarginine, Trimethyl chitosan (TMC), Choline-based polyesters, Poiy(imine)s (short chain), Amine-functionalized poly(carbonate), Cationic pullulan derivatives, and Poly(p-amino thioester).

34. The method according to any one of Claims 26, and 27 to 33 when dependent on Claim 26, wherein step a) further comprises providing an active material, such as a medicinal compound.

35. The method according to Claim 34, wherein the active material is selected from one or more of the group consisting of capsaicin, silibinin, artemisinin, fisetin, raloxifene, sorafenib tosylate, irinotecan (IRT), Ibuprofen, Diazepam, Midazolam, Propofol, Haloperidol, Phenytoin, Thiopental, Fentanyl, Ketoprofen, Paclitaxel , Chloramphenicol, Insulin, Metronidazole, Doxorubicin, Ketoconazole, Cyclosporine, Itraconazole, Celecoxib, Bisoprolol, Rifampin, Curcumin, Resveratrol, Quercetin, Tocopherols (Vitamin E), Berberine, Piperine, Eugenol, Myricetin, Flavonoids, Piperlongumine, Tannins, Rutin, Thymol, Dihydromyricetin, Alpha- mangostin, Lycopene, Alkaloids (e.g., Vincristine, Vinblastine), Docetaxel, Nilotinib, Gefitinib, Imatinib (Gleevec), lndole-3-Carboxaldehyde, Chloroquine, Chlorpromazine, Propranolol, Ipratropium bromide, Tobramycin, Gentamicin, Lidocaine, Epinephrine, Cisplatin, Quinine, Ciprofloxacin, Pyridostigmine, Amitriptyline, Neomycin, Thiamine (Vitamin B1 ), Ranitidine, Pyridoxine (Vitamin B6), Betaxolol, Donepezil, Cetirizine, Amikacin, and docoshexaenic acid (DHA).

36. The method according to Claim 34 or 35, wherein the one or more negative phospholipids, cationic amphiphilic material, and active material are provided in a molar ratio of from about 7:3:5 to about 7:3:10, such as about 9:1 :7.

37. A cochleate according to any of Claims 1 to 21 for use in a method of treating a subject in need thereof.

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