Compounds, compositions and methods for delivery of bioactive agents

Abstract

The present disclosure relates to a lyotropic liquid crystal lipid carrier that can be used to deliver an active agent. The disclosure also relates to cosmetic or pharmaceutical compositions comprising the lipid carriers, and methods of using the lipid carriers or compositions thereof to treat, prevent or diagnose diseases, disorders or conditions, such as cancer or bacterial or fungal infections.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Australian Provisional Patent Application No. 2022901069 filed on April 22, 2022, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates to the field of nanomedicine. Specifically, the present disclosure relates to lipid carriers or nanoparticles for delivering bioactive agents. Disclosed herein are carriers (such as nanoparticles) comprising synthetic lipids, methods for preparing the same, and uses of the nanoparticles for formulating and delivering pharmaceutical or cosmetic agents. Background Art

[0004] Any reference to background art herein should not be construed as an admission that such art constitutes common general knowledge in Australia or elsewhere.

[0005] Pharmaceutical and cosmetic agents can be difficult to deliver due to reasons such as poor solubility in aqueous media, low cell permeability, chemical and / or biological instability, rapid enzymatic and metabolic degradation, elimination from the body, and poor safety and tolerability. Many of these bioactive agents would be safer and more effective if they could be selectively administered. For example, chemotherapeutic drugs are often extremely toxic to both cancer cells and normal cells, and in most cases there is no way to bias systemic delivery to cancer cells to reduce off-target effects.

[0006] There are different strategies to overcome the challenges associated with the delivery of bioactive agents, such as new dosage forms, alternative forms (salts / complexes) of medicaments, prodrugs or other conjugates of drugs or alternative routes of administration. Other examples include development of nano-delivery systems such as liposomes, polymer micelles, microemulsions and self-emulsifying drug delivery systems, nano-dispersions, inorganic nanoparticles, solid lipid nanoparticles, inclusion complexes and nano-delivery systems based on chemical conjugation. The development of these technologies and their application to a wider range of bioactive agents are constantly making progress.

[0007] It is desirable to provide additional carriers for delivering pharmaceutical and cosmetic agents and other bioactive agents such as nucleic acids. It is also desirable to develop new carriers that can preferentially deliver bioactive agents to disease or infection sites. Such carriers will be useful for disease therapy and diagnosis, as well as formulation of pharmaceutical and personal care agents. Summary of the invention

[0008] The present disclosure provides compounds, compositions, and methods that can be used in any biological system where it may be desirable to deliver a material that is protected from or otherwise separated from the system until specific conditions exist.

[0009] Specifically, the present disclosure provides compounds, compositions and methods for improving the delivery of bioactive agents. Among them, the present application provides compounds, compositions and methods for preparing and using novel delivery agents that are stable in circulation and can undergo structural changes under appropriate physiological conditions (e.g., pH), and the structural changes allow the delivery efficiency of the bioactive agent to be controlled.

[0010] The compounds, compositions and methods provided by the present disclosure promote the effective delivery of active agents by providing active agents encapsulated in lipid carriers that are stable at ambient pH in circulation and undergo structural changes under appropriate physiological conditions (e.g., reduced pH) that increase the release of the active agent in a local environment with those physiological conditions. Examples of such local environments with reduced pH include tumors, sites affected by certain bacterial and fungal infections, and subcellular organelles.

[0011] Non-limiting examples of active agents that can be delivered using the compounds, compositions and methods of the present disclosure include both hydrophilic and lipophilic agents, including therapeutically or cosmetically active molecules, small molecule drugs, peptides, proteins, hormones, vitamins, antibodies, oligonucleotides, siRNA, DNA, mRNA, and imaging agents.

[0012] By utilizing the structural phase changes of lipid carriers under appropriate physiological conditions (e.g., reduced pH), the release of encapsulated active agents from lipid carriers can be controlled and engineered for targeted delivery of active agents, thereby improving efficacy and reducing off-target side effects of the active agents.

[0013] In a first aspect, a lyotropic liquid crystal (LLC) lipid vector is provided, comprising a structural lipid and an amino lipid, wherein the structural lipid is a lyotropic liquid crystal phase forming lipid, the amino lipid has an amide linker, and the LLC lipid vector is suitable for undergoing a mesophase transition when exposed to a decrease in pH.

[0014] In an embodiment, the structured lipid is capable of forming a cubic mesophase structure. In an embodiment, the structured lipid comprises a hydrophobic tail group selected from the group consisting of oleyl, linoleoyl, linolenoyl, phytanoyl, farnesoyl or an extended aliphatic hydrophobic, optionally selected from oleyl, linoleoyl and phytanoyl. In an embodiment, the structured lipid is glyceryl monooleate (glyceryl monooleate).

[0015] In embodiments, the amino lipid is present in an appropriate wt% of the total lipid content of the LLC lipid carrier to provide the mesophase transition upon exposure to a pH drop. In embodiments, the amino lipid is about 5% to about 50 wt% of the total lipid content of the LLC lipid carrier.

[0016] In an embodiment, the amino lipid has the structure of formula (I):

[0017] Cyc-LR

[0018] (I)

[0019] Wherein Cyc is a nitrogen heterocycle or a heteroaryl;

[0020] L is an amido linker; and

[0021] R is a C10 to C44 carbon chain.

[0022] In an embodiment, the amino lipid has the structure of formula (Ib):

[0023]

[0024] wherein Cyc is a 5-membered or 6-membered nitrogen heterocyclic group or heteroaryl group, the nitrogen heterocyclic group or heteroaryl group is optionally selected from the group consisting of pyridyl, pyrimidinyl, piperidinyl, piperazinyl, morpholinyl and pyrazinyl;

[0025] L is an amido linker;

[0026] R is a C10 to C44 carbon chain, optionally a C12 to C24 alkyl or alkenyl group, which is optionally interrupted by one or more heteroatoms; and

[0027] n is an integer of 1 to 6.

[0028] In an embodiment, R is selected from the group consisting of oleyl, linoleoyl, linolenoyl, phytanoyl and farnesoyl.

[0029] In an embodiment, the amino lipid is selected from the group consisting of:

[0030]

[0031] wherein R is as defined above.

[0032] In an embodiment, the LLC lipid carrier further comprises a stabilizer, optionally wherein the stabilizer is a stabilizing polymer, optionally a nonionic triblock copolymer. In an embodiment, the stabilizer is present in an amount of about 10wt% of the total lipid content of the LLC lipid carrier. The stabilizer is Pluronic F127 (Poloxamer 407) or Poloxamer 80.

[0033] In embodiments, the LLC lipid vector is in the form of lyotropic liquid crystal (LLC) nanoparticles.

[0034] In embodiments, said mesophase transition upon exposure to a decrease in pH is a transition caused by a decrease in pH to below pH 7.

[0035] In embodiments, said mesophase transition upon exposure to a decrease in pH is a transition caused by a decrease in pH of 1 to 4 pH units.

[0036] In embodiments, the mesophase transition upon exposure to a decrease in pH is a transition to a mesophase having an interfacial curvature lower than the interfacial curvature of the mesophase occupied by the lipid vector prior to the decrease in pH.

[0037] In embodiments, the mesophase transition upon exposure to a decrease in pH is a transition to a mesophase corresponding to a lower CPP value.

[0038] In embodiments, the mesophase transition upon exposure to a drop in pH is a transition from a hexagonal phase to a cubic phase.

[0039] In embodiments, the mesophase transition upon exposure to a drop in pH is a transition from one mesophase to another, the transition following the mesophase transition sequence of L2→H2→Q2→L3. In embodiments, the mesophase transition is H2→Q2.

[0040] In embodiments, the LLC lipid vector comprises a hexagonal lyotropic liquid crystal phase structure at a pH of about pH 7 or higher, optionally about pH 7.4 or higher.

[0041] In embodiments, the LLC lipid vector comprises a cubic lyotropic liquid crystal phase structure at a pH of about pH 4.0-pH 7.0.

[0042] In embodiments, the LLC lipid vector comprises a hexagonal lyotropic liquid crystal phase structure at a pH of about pH 7 or higher, optionally about pH 7.4 or higher, and a cubic lyotropic liquid crystal phase structure at a pH of about pH 4.0-pH 7.0.

[0043] In an embodiment, the LLC lipid carrier further comprises an active agent, optionally wherein the active agent is pharmaceutically active or cosmetically active. In an embodiment, the active agent is selected from the group consisting of peptides, proteins, enzymes, small molecule drugs and nucleic acids, optionally wherein the active agent is selected from the group consisting of radionuclides, imaging agents, polymers, antibiotics, fungicides, metal-containing nanoparticles, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, antianxiety agents, hormones, growth factors, steroid agents, gene expression regulators, knockdown agents, siRNA, RNAi agents, mRNA, DNA, dicer substrates, miRNA, shRNA, antisense oligonucleotides, aptamers and microbial toxins. In an embodiment, the active agent is a topoisomerase I inhibitor, and the topoisomerase I inhibitor is optionally selected from camptothecin, irinotecan and SN-38.

[0044] In embodiments, the LLC lipid carrier has a water content of between about 20 wt % and about 60 wt %.

[0045] In a second aspect, a lyotropic liquid crystal (LLC) lipid nanoparticle composition is provided, which is a dispersion of the LLC lipid carrier according to the first aspect in a polar medium.

[0046] In a third aspect, a cosmetic composition is provided, comprising the LLC lipid carrier according to the first aspect or the LLC lipid nanoparticle composition according to the second aspect and a cosmetically acceptable carrier, diluent and / or excipient.

[0047] In a fourth aspect, a pharmaceutical composition is provided, comprising the LLC lipid carrier according to the first aspect or the LLC lipid nanoparticle composition according to the second aspect and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0048] In an embodiment, the cosmetic composition according to the third aspect or the pharmaceutical composition according to the fourth aspect is formulated as a composition for injection, topical administration or subcutaneous administration.

[0049] In a fifth aspect, a method for delivering an active agent to a biological system is provided, the method comprising the step of administering to the biological system the LLC lipid carrier according to the first aspect, the LLC lipid nanoparticle composition according to the second aspect, or the composition according to the third aspect or the fourth aspect.

[0050] In embodiments, delivery of the active agent to the biological system is regulated by the response of the LLC lipid carrier to a predefined pH range. In embodiments, the response comprises a mesophase structural transition of the LLC lipid carrier, optionally wherein the phase transition is a transition from a hexagonal phase structure to a cubic phase structure of the LLC lipid carrier. In embodiments, the mesophase structural transition occurs at a pH of about pH 7.0-pH 4.0, optionally about pH 7.0-pH 5.5. In embodiments, the response provides preferential release of the active agent in the biological system at the predefined pH range.

[0051] In a sixth aspect, a method for controlled release of an active agent is provided, the method comprising the following steps: forming the LLC lipid carrier according to the first aspect, the LLC lipid nanoparticle composition according to the second aspect, or the composition according to the third aspect or the fourth aspect, and applying the LLC lipid carrier, the LLC lipid nanoparticle composition, or the composition to a biological system comprising a target area with a predefined pH, thereby achieving preferential release of the active agent at the target area.

[0052] In embodiments, the LLC lipid carrier undergoes a mesophase structural transition in response to the predefined pH. In embodiments, the mesophase structural transition is a transition from a hexagonal phase structure to a cubic phase structure of the LLC lipid carrier.

[0053] In a seventh aspect, a method for treating or preventing a disease, disorder or condition in a mammal is provided, the method comprising administering to the mammal a therapeutically effective amount of the LLC lipid carrier according to the first aspect, the LLC lipid nanoparticle composition according to the second aspect, or the composition according to the third aspect or the fourth aspect.

[0054] In an eighth aspect, an LLC lipid carrier according to the first aspect, an LLC lipid nanoparticle composition according to the second aspect, or a composition according to the third aspect or the fourth aspect for use in treating or preventing a disease, disorder or condition is provided.

[0055] In the ninth aspect, provided is a use of the LLC lipid carrier according to the first aspect, the LLC lipid nanoparticle composition according to the second aspect, or the composition according to the third aspect or the fourth aspect in the preparation of a medicament for treating a disease, disorder or condition.

[0056] In embodiments, the disease, disorder or condition is cancer or a bacterial infection or a fungal infection.

[0057] In the tenth aspect, a method for diagnosing a disease, disorder or condition in a mammal is provided, the method comprising the following steps: administering the LLC lipid carrier according to the first aspect, the LLC lipid nanoparticle composition according to the second aspect, or the composition according to the third aspect or the fourth aspect to the mammal or a biological sample obtained from the mammal to facilitate diagnosis of the disease, disorder or condition in the mammal, wherein the active agent is a labeled active agent.

[0058] In embodiments, the disease, disorder or condition is cancer or a bacterial infection or a fungal infection.

[0059] In an eleventh aspect, an amino lipid of formula (Ib) is provided:

[0060]

[0061] wherein Cyc is a 5- or 6-membered nitrogen heterocyclic radical or heteroaryl selected from the group consisting of piperidinyl, piperazinyl, pyrrolinyl, pyrrolidinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, pyrrolinyl, pyrazolinyl, thiazolinyl, thiomorpholinyl, tetrahydropyridinyl, dihydropyridinyl, tetrahydropyrimidinyl, pyrimidinyl, pyridinyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, oxadiazolyl, pyrazinyl, tetrazolyl, thiazolyl, isoxazolyl, isothiazolyl, isoxazolone, triazolyl, oxadiazolyl, thiadiazolyl, and pyridazinyl;

[0062] L is an amido linker;

[0063] R is a C10 to C44 carbon chain, optionally a C12 to C24 alkyl or alkenyl group, which is optionally interrupted by one or more heteroatoms; and

[0064] n is an integer of 1 to 6.

[0065] In an embodiment, Cyc is selected from the group consisting of pyridinyl, pyrimidinyl, piperidinyl, piperazinyl and pyrazinyl.

[0066] In an embodiment, R is selected from the group consisting of oleyl, linoleoyl, linolenoyl, phytanoyl and farnesoyl.

[0067] In an embodiment, the amino lipid is selected from the group consisting of:

[0068]

[0069] wherein R is as defined above.

[0070] The various features and embodiments of the present invention mentioned in the above sections are applicable to other sections after necessary modifications where appropriate. Therefore, the features specified in one section can be combined with the features specified in other sections where appropriate.

[0071] Additional features and advantages of the present invention will become apparent from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] While it should be understood that various embodiments disclosed herein may be used, in the following, several examples are described herein with reference to the following figures:

[0073] Figure 1 is a schematic diagram of the pH-dependent phase transition of the lipid carriers described in this application for drug delivery in tumor therapy.

[0074] Figure 2 The chemical structures of exemplary synthetic amino lipids are shown.

[0075] Figure 3 is a series of SAXS derived partial lipid-water phase diagrams obtained by adding increasing amounts of amino lipids to MO dispersed with F-127 at 25°C across the pH range of 3.0 to 10. The highlighted box represents the key phase transition from hexagonal to cubic phase. A) Partial phase diagram of nanoparticles obtained by incorporating OAPy-4, B) partial phase diagram of nanoparticles with OAPy-2, C) partial phase diagram of nanoparticles with OAMo-1, and D) partial phase diagram of nanoparticles with OAPi-1. The amount of amino lipid added to the formulation is determined by R AL (Also referred to as R MO ) represents, which is defined as the wt / wt ratio of amino lipids to total lipids. N / D is an undetermined and unrecognizable phase. Recognizable phases include bicontinuous cubic phase (Im3m is the original cubic phase, Pn3m is the double diamond cubic phase, and Ia3d is the gyroscopic cubic phase), sponge phase (L3), reverse micelle phase (L2), hexagonal phase (H2) and lamellar phase (L α ).

[0076] Figure 4 is a graphical representation of the effects of synthesized amino lipids and pH on the mesophase structure of MO nanoparticles.

[0077] Figure 5 A) By using OAPy-4 as MO Representative one-dimensional SAXS spectra of nanoparticles prepared by doping MO with 0.4. a) Results at 37°C, B) Results at room temperature.

[0078] Figure 6is a graphical representation of the particle size and polydispersity index (PDI) of lipid nanoparticles loaded with SN-38. The measurements were averaged from triplicates and the results are reported as mean + / - standard deviation.

[0079] Figure 7 is a partial SAXS phase image of lipid nanoparticles of SN-38 and amino lipids loaded with different wt% SN-38. The highlighted box indicates the key phase transition from hexagonal mesophase structure to cubic mesophase structure.

[0080] Figure 8 is a graphical representation of drug loading determined for lipid nanoparticles of SN-38 and amino lipids loaded with different wt% SN-38. Measurements were averaged from triplicates and results are reported as mean + / - standard deviation.

[0081] Fig. 9 is a graphical representation of the release of SN-38 drug from lipid nanoparticles prepared using the amino lipid OAPy-4 (N-(pyridin-4-ylmethyl)oleamide). The nanoparticles were suspended in 1000 mL of release medium in PBS buffer at pH 7 and pH 5. The SN-38-loaded nanoparticles were hexosomes at neutral pH and cubosomes at pH 5. At predetermined time intervals (60 min, 120 min, 240 min, and 420 min), 10 μL samples were withdrawn from the dialysis tubing and diluted with 990 μL of HPLC solvent. The HPLC method quantitatively measured the SN-38 remaining in the dialysis tubing. The experiment was performed in triplicate on three independently prepared SN-38-loaded formulations, and all values ​​are presented as mean ± SD (n = 3). DETAILED DESCRIPTION

[0082] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0083] Unless otherwise noted, all publications discussed and / or cited herein are incorporated herein in their entirety.

[0084] Unless the context requires otherwise, singular terms shall include plural and plural terms shall include singular. Throughout this disclosure, unless otherwise specifically stated or the context requires otherwise, references to individual steps, material compositions, step groups, or material composition groups shall be deemed to cover one and a plurality of (i.e., one or more (one or more)) steps, material compositions, step groups, or material composition groups in these steps. Therefore, as used herein, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" include plural aspects. For example, reference to "one" includes single and two or more; reference to "a" includes single and two or more; reference to "the" includes single and two or more, etc.

[0085] Those skilled in the art will appreciate that, in addition to the disclosure specifically described, the disclosure herein may be changed and modified. It should be understood that the disclosure includes all such changes and modifications. The disclosure also includes all examples, steps, features, methods, compositions, formulations and processes individually or collectively mentioned or indicated in this specification, as well as any and all combinations of any two or more of the steps or features.

[0086] The term "and / or", for example, "X and / or Y" should be understood to mean "X and Y" or "X or Y", and should be considered to provide clear support for both meanings or either meaning.

[0087] Unless otherwise indicated, the terms "first," "second," and the like are used herein merely as labels and are not intended to impose ordinal, positional, or hierarchical requirements on the items to which these terms refer. Furthermore, reference to a "second" item does not require or preclude the presence of a lower-numbered item (e.g., a "first" item) and / or a higher-numbered item (e.g., a "third" item).

[0088] As used herein, the phrases “at least one of” or “one or more of”, when used with a list of items, means that different combinations of one or more of the listed items may be used and only one of the items in the list may be required. The item may be a specific object, thing, or category. In other words, “at least one of” means that any combination of items or numbers of items in the list may be used, but not all items in the list may be required. For example, “at least one of item A, item B, and item C” may mean item A; item A and item B; item B; item A, item B, and item C; or item B and item C. In some cases, “at least one of item A, item B, and item C” may mean, for example, but not limited to, two of item A, one of item B, and ten of item C; four of item B and seven of item C; or some other suitable combination.

[0089] It should be understood that, for the sake of clarity, certain features described herein in the context of separate embodiments may also be provided in a single embodiment in combination. Conversely, for the sake of brevity, various features described in the context of a single embodiment may also be provided individually or in any sub-combination.

[0090] Throughout this specification, various aspects and compositions of the present disclosure may be presented in a range format. The range format is included for convenience and should not be interpreted as a fixed limitation on the scope of the present disclosure. Therefore, unless otherwise stated, the description of the range should be deemed to have explicitly disclosed all possible sub-ranges and individual numerical values ​​within the range. For example, unless an integer is required or the context implies an integer, the description of a range such as from 1 to 5 should be deemed to have specifically disclosed sub-ranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 5, 3 to 5, etc.; and individual and partial numbers within the stated range, for example, 1, 2, 3, 4, 5, 5.5 and 6. This applies regardless of the breadth of the disclosed range. When specific values ​​are required, these values ​​will be indicated in the specification.

[0091] In this patent specification, the terms "comprises," "comprising," "includes," "including," or similar terms are intended to mean a non-exclusive inclusion, such that a method or composition comprising a list of elements includes not only those elements but may also include other elements that are not listed.

[0092] “Consisting of is meant to include and be limited to whatever follows the phrase “consisting of.” Thus, the phrase “consisting of indicates that the listed elements are required or necessary, and other elements may not be present.

[0093] "Consisting essentially of is meant to include any elements listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified in the present disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or necessary, but other elements are optional and may be present or absent depending on whether they affect the activity or action of the listed elements.

[0094] Herein, unless otherwise stated, the term "about" encompasses a 10% tolerance of any value or values ​​associated with the term.

[0095] "Weight %" herein may be abbreviated as "wt %" or "wt. %".

[0096] "Pharmaceutical composition" or "pharmaceutical formulation" refers to a composition suitable for pharmaceutical use in subjects including humans and mammals. The pharmaceutical composition comprises a pharmacologically effective amount of the composition used in the methods described herein, and further comprises a pharmaceutically acceptable carrier. The pharmaceutical composition encompasses a composition comprising an active ingredient and an inert ingredient (e.g., an excipient) constituting a carrier, as well as any product produced directly or indirectly by the combination, complex or aggregation of any two or more ingredients and / or by the dissociation of one or more ingredients, and / or by other types of reactions or interactions of one or more ingredients. Therefore, the pharmaceutical composition of the present invention encompasses any composition prepared by mixing the composition used in the methods described herein with a pharmaceutically acceptable carrier.

[0097] Unless defined alternatively, "pharmaceutically acceptable carrier" or simply "carrier" refers to any standard pharmaceutical carrier, buffer and excipient. A pharmaceutically acceptable carrier can be a saline solution, such as a phosphate buffered saline solution or a 5% dextrose solution in water. Additional examples of pharmaceutically acceptable carriers include, but are not limited to, emulsions, such as oil / water or water / oil emulsions, and various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 19th Edition. (Mack Publishing Co., Easton, 1995). Preferred pharmaceutical carriers depend on the intended mode of administration of the active agent. Example modes of administration include enteral (e.g., oral) or parenteral (e.g., subcutaneous, intramuscular, intravenous or intraperitoneal injection; or topical, transdermal or transmucosal administration).

[0098] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of an active agent that is toxicologically safe for systemic or local administration, such as a salt prepared from a pharmaceutically acceptable non-toxic base or acid (including an inorganic or organic base and an inorganic or organic acid). Pharmaceutically acceptable salts can be selected from the group consisting of alkali metals and alkaline earth metals, ammonium, aluminum, iron, amines, glucosamine, chlorides, sulfates, sulfonates, bisulfates, nitrates, citrates, tartrates, bitartrates, phosphates, carbonates, bicarbonates, malate, maleate, naphthalenesulfonate, fumarate, succinate, acetate, benzoate, terephthalate, palmitate, piperazine, pectinate, and S-methylmethionine salts, etc.

[0099] As used herein, the term "cosmetic composition" refers to a composition used in cosmetics. Cosmetics are products that are intended to be rubbed, poured, splashed or sprayed onto, introduced into, or otherwise applied to the human body for cleaning, beautification, attractiveness enhancement, or alteration of appearance. Typically, cosmetic compositions do not provide therapeutic effects and are not formulated as medicaments. However, in some cases, cosmetic compositions are incorporated into pharmaceutical products to provide cosmetic benefits (e.g., treatment of skin or hair conditions). The present disclosure also contemplates the use of a cosmetic product in animals other than humans.

[0100] As used herein, "cosmetically acceptable" indicates that the particular ingredient or excipient is generally regarded as safe and non-toxic at the level employed.

[0101] As used herein, the term "complex" is to be understood as a non-covalent physical interaction between two or more chemical entities.

[0102] As used herein, the terms "non-lamellar lyotropic liquid crystal phase" and "lyotropic liquid crystal (LLC) lipid carrier" refer to a self-assembled non-lamellar liquid crystal phase formed by at least one amphiphilic molecule to obtain a two-dimensional and / or three-dimensional mesophase structure capable of carrying an active agent. It is shown herein that dispersed non-lamellar lyotropic liquid crystal phase particles result in pH-responsive structural behavior. The term non-lamellar means that the lyotropic liquid crystal phase or lipid carrier or particle is not a liposome (or L α Liquid crystal phases are phases of matter that do not present a planar structure consisting of a lipid bilayer separated by water, in which case the polar head groups of the amphiphilic molecules associate and are in direct contact with the water, while the hydrophobic tails are away from the water. As described herein, a liquid crystal phase is a phase of matter that exhibits properties that are intermediate between those of a conventional liquid and those of a solid crystal. There are different types of liquid crystal phases that can be distinguished based on their different optical properties and other properties known in the art.

[0103] In an embodiment, the LLC lipid carrier of the present invention comprises only liquid crystals. That is, the LLC lipid carrier of the present invention does not comprise any solid lipid component. Therefore, the LLC lipid carrier of the present invention is not a solid lipid nanocarrier (SLN) or a nanostructured lipid carrier (NLC).

[0104] In an embodiment, the term "non-lamellar lyotropic liquid crystal phase" or "lyotropic liquid crystal phase" covers only cubic, hexagonal and sponge morphologies. Although the "sponge phase" or "sponge particles" (L3) are considered to have no long-range order and do not exhibit crystalline periodicity comparable to the anti-bicontinuous cubic phase (Q2), they are generally considered to be "molten" Q2 cubic phases and are therefore considered to be included as particles of the first aspect or the second aspect. Therefore, the short-range ordered sponge phase is explicitly considered to be within the scope of this term. In an embodiment, the term "non-lamellar lyotropic liquid crystal phase" or "lyotropic liquid crystal phase" can be used to include one or more phases selected from the group consisting of: hexagonal phases (positive phases and negative phases), cubic phases (positive discrete phases, negative discrete phases, negative bicontinuous phases - including original phases, gyroscopic phases and diamond phases - and negative discontinuous phases) and other "intermediate phases", including ribbon, network or non-cubic "sponge" bicontinuous phases.

[0105] Amino lipids

[0106] In a first aspect, the present disclosure provides a lyotropic liquid crystal (LLC) lipid vector comprising a structural lipid and an amino lipid, wherein the structural lipid is a lyotropic liquid crystal phase-forming lipid, the amino lipid has an amide linker, and the LLC lipid vector is suitable for undergoing a mesophase transition when exposed to a decrease in pH.

[0107] In an embodiment, the amino lipid has the structure of formula (I):

[0108] Cyc-LR

[0109] (I)

[0110] Wherein Cyc is a nitrogen heterocycle or a heteroaryl;

[0111] L is an amido linker; and

[0112] R is a C10 to C44 carbon chain.

[0113] As used herein, the term "amino lipid" refers to an amphiphilic lipid compound comprising a functional group including a nitrogen atom (preferably a head group comprising a nitrogen atom). In an embodiment, the lipid compound comprises an alkaline ionizable functional group, such as an aromatic ring containing an amine or nitrogen. In an embodiment, the amino lipid disclosed herein is an ionizable lipid and / or a cationic lipid. In an embodiment, the amino lipid provides pH sensitivity to the LLC lipid carrier disclosed herein. In any embodiment disclosed herein, the lipid may be referred to as an "amino lipid" based on the presence of at least one nitrogen atom.

[0114] In the embodiments disclosed herein, the amino lipids generally comprise a polar hydrophilic head group including a nitrogen heterocycle or a heteroaryl group and a non-polar hydrophobic tail group comprising a C10 to C44 carbon chain. In embodiments, the amino lipids in the LLC lipid carriers disclosed herein generally may comprise one or more cationic head groups and / or ionizable head groups.

[0115] In an embodiment, nitrogen heterocycle or heteroaryl is 5 yuan or 6 yuan nitrogen heterocyclic radical or heteroaryl, such as piperidinyl, piperazinyl, pyrrolinyl, pyrrolidinyl, pyrazolidinyl, morpholinyl, imidazolinyl, imidazolidinyl, pyrrolinyl, pyrazolinyl, thiazolinyl, thiomorpholinyl, tetrahydropyridinyl, dihydropyridinyl, tetrahydropyrimidinyl, pyrimidinyl, pyridinyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, oxadiazolyl, pyrazinyl, tetrazolyl, thiazolyl, isoxazolyl, isothiazolyl, isoxazolone, triazolyl, oxadiazolyl, thiadiazolyl or pyridazinyl.In some preferred embodiments, nitrogen heterocycle or heteroaryl is selected from pyridinyl, pyrimidinyl, piperidinyl, piperazinyl, morpholinyl and pyrazinyl.Preferably, nitrogen heterocycle or heteroaryl is pyridinyl or piperidinyl.

[0116] In an embodiment, the amino lipid in the LLC lipid vector disclosed herein can comprise a cationically ionizable amino head group bound (e.g., covalently bound) to a non-polar hydrophobic tail group comprising a C10 to C44 carbon chain via an amide linker.

[0117] As used herein, the term "amido linker" refers to a linker comprising an amide group (eg, an alkyl chain interrupted by amide groups).

[0118] In an embodiment, the amido linker comprises an optional carbon chain between the nitrogen heterocycle or heteroaryl and the amide group. In an embodiment, the optional carbon chain comprises 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms, or 1 to 2 carbon atoms, or 1 carbon atom; preferably 1 to 4 carbon atoms.

[0119] In embodiments, when present, the carbon chain is optionally interrupted by one or more heteroatoms. In embodiments, the heteroatom for each interruption can be independently selected from O, N and S.

[0120] In an embodiment, the non-polar hydrophobic tail group comprises a C10 to C44 carbon chain. The carbon chain can be a straight or branched alkyl, alkenyl or alkynyl chain. In certain embodiments, the tail group comprises a C10 to C36 alkyl, alkenyl or alkynyl chain, a C12 to C24 alkyl, alkenyl or alkynyl chain, preferably a C12 to C24 alkyl or alkenyl chain. In certain preferred embodiments, the tail group comprises a C12 to C18 alkyl, alkenyl or alkynyl chain, preferably a C12 to C18 alkyl or alkenyl chain, preferably a C12 to C18 alkenyl chain.

[0121] For example, the non-polar hydrophobic tail group may comprise oleyl, linoleoyl, linolenoyl, phytanoyl, and farnesoyl; preferably oleyl or linoleoyl; preferably oleyl.

[0122] In some embodiments, the LLC lipid vector disclosed herein comprises an amino lipid having the structure of Formula (Ib):

[0123]

[0124] Wherein Cyc is a nitrogen heterocycle or a heteroaryl;

[0125] R is a C12 to C24 alkyl or alkenyl chain; and

[0126] n is an integer of 1-6, preferably 1-4.

[0127] In an embodiment, the Cyc group is a 6-membered nitrogen heterocycle or a heteroaryl. In some embodiments, Cyc is selected from the group consisting of: pyridyl, pyrimidyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydropyridyl, dihydropyridyl, tetrahydropyrimidyl, pyrazinyl and pyridazinyl. Preferably, Cyc is selected from the group consisting of: pyridyl, pyrimidyl, piperidyl, piperazinyl, morpholinyl and pyrazinyl.

[0128] In any embodiment disclosed herein, the R group of the amino lipid can be selected from a hydrophobic chain that follows the design rules of the anti-bicontinuous cubic phase. The selection of the R group hydrophobic chain can be based on certain requirements understood in the art. For example, the hydrophobic chain can be selected from a hydrophobic chain that promotes a type II lyotropic liquid crystal phase at ambient temperature and physiological temperature. Parameters that can be suitable for selecting an appropriate hydrophobic chain include: 1. The temperature used should be higher than the chain melting temperature so that there is a molten chain; and 2. In a carbon chain consisting of at least 14 carbons, at least one cis-unsaturated bond should be present at least in the middle position along the skeleton; or 3. The carbon skeleton should contain at least 12 carbons, 3 of which are secondary carbons with methyl branches; and 4. The molecular weight of the hydrophobic chain should be at least> 200amu.

[0129] In an embodiment, the R group of the amino lipid is C12 to C24 alkenyl, preferably C14 to C22 alkenyl.

[0130] In embodiments, the R group is selected from the group consisting of: oleyl, linoleoyl, linolenoyl, phytanoyl, and farnesoyl. In some embodiments, the R group is oleyl. In some embodiments, R is:

[0131]

[0132] In an embodiment, the R group can be selected from a lipid tail known to form a cationic or ionizable cationic lipid with a polar head group. For example, the following lipids are known to form lipid nanoparticles (LNPs) for nucleic acid delivery. The lipid tails of these lipids and the lipid tails in this structural class can be suitable for use as R groups as defined herein:

[0133]

[0134] In an embodiment, the amino lipid in the LLC lipid carrier disclosed herein is selected from the group consisting of:

[0135]

[0136] wherein R is as defined above.

[0137] In some embodiments, the amino lipid is selected from the group consisting of:

[0138]

[0139] In another aspect, the present disclosure provides an amino lipid of formula (Ib):

[0140]

[0141] wherein Cyc is a 5- or 6-membered nitrogen heterocyclic radical or heteroaryl selected from the group consisting of piperidinyl, piperazinyl, pyrrolinyl, pyrrolidinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, pyrrolinyl, pyrazolinyl, thiazolinyl, thiomorpholinyl, tetrahydropyridinyl, dihydropyridinyl, tetrahydropyrimidinyl, pyrimidinyl, pyridinyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, oxadiazolyl, pyrazinyl, tetrazolyl, thiazolyl, isoxazolyl, isothiazolyl, isoxazolone, triazolyl, oxadiazolyl, thiadiazolyl, and pyridazinyl;

[0142] L is an amido linker;

[0143] R is a C10 to C44 carbon chain, optionally a C12 to C24 alkyl or alkenyl group, which is optionally interrupted by one or more heteroatoms; and

[0144] n is an integer of 1 to 6.

[0145] In an embodiment, Cyc is selected from the group consisting of pyridinyl, pyrimidinyl, piperidinyl, piperazinyl and pyrazinyl.

[0146] In an embodiment, R is selected from the group consisting of oleyl, linoleoyl, linolenoyl, phytanoyl and farnesoyl.

[0147] In an embodiment, the amino lipid is selected from the group consisting of:

[0148]

[0149] wherein R is as defined above.

[0150] In an embodiment, the amino lipid is selected from the group consisting of:

[0151]

[0152] Wherein R is oleyl.

[0153] The amino lipids described herein can be synthesized by any suitable method known to those skilled in the relevant art.

[0154] For example, amino lipids can be synthesized using an amide coupling reaction between a suitable carboxylic acid or derivative thereof carrying a hydrophobic tail group and an amine carrying a hydrophilic head group. Alternatively, amino lipids can be synthesized using an amide coupling reaction between a suitable carboxylic acid or derivative thereof carrying a hydrophilic head group and an amine carrying a hydrophobic tail group. The conditions of the amide coupling reaction are well known in the art.

[0155] Lyotropic Liquid Crystalline (LLC) Lipid Carriers

[0156] According to a first aspect, there is provided a lyotropic liquid crystal (LLC) lipid carrier comprising a structural lipid and an amino lipid, wherein the structural lipid is a lyotropic liquid crystal phase-forming lipid, the amino lipid has an amide linker, and the LLC lipid carrier is suitable for undergoing a mesophase transition when exposed to a decrease in pH.

[0157] Lyotropic liquid crystal (LLC) materials are generally composed of amphiphilic molecules that self-assemble into various mesophase structures in polar media (e.g., aqueous solutions or water). In general, the present disclosure relates to non-lamellar-formed mesophases, which can consist of a well-defined network of aqueous channels and lipid bilayer membranes, such as a bicontinuous cubic phase or an inverse hexagonal mesophase. As used herein, the terms "mesophase", "lyotropic phase", "liquid crystal phase", "lyotropic liquid crystal phase" or just "phase" are used interchangeably. Non-lamellar mesophases can be used as drug carriers in the form of bulk phases or fabricated colloidal nanocarriers (e.g., cubes and hexagons).

[0158] As used herein, the terms "amphiphile," "amphiphilicity," and "amphiphilic lipid" refer to compounds that contain both a hydrophilic portion and a hydrophobic portion. Typically, such compounds will have a hydrophilic head group and a hydrophobic tail. Suitable examples include fatty acids and a series of lipid molecules. In aqueous solution, amphiphiles spontaneously form structures that conceal the hydrophobic portion but allow the hydrophilic head group to hydrate.

[0159] As used herein, a "lyotropic liquid crystal" material refers to a material having fluid properties similar to those of a viscous liquid and a molecular order similar to that of a crystalline solid. The LLC lipid vectors disclosed herein may have some or all of the components of a lyotropic liquid crystal (LLC) phase. In embodiments, the LLC lipid vectors disclosed herein comprise at least 90% of the lyotropic liquid crystal (LLC) phase. In some embodiments, the LLC lipid vectors disclosed herein do not contain any solid crystalline components.

[0160] In the art, a lyotropic phase is characterized by the amount of curvature it exhibits. For example, where the amphiphilic monolayer bends toward the aqueous phase, the lyotropic phase is referred to as the reverse phase. Conversely, where the monolayer bends away from the aqueous phase, the lyotropic phase is referred to as the normal phase.

[0161] LLC lipid carriers disclosed herein can be in the form of LLC bulk phases. Bulk phase LLC materials are viscous gel-like materials. The present disclosure generally relates to self-assembled mesophases of non-lamellar bulks, such as inverse bicontinuous cubic (Q2) phases, inverse hexagonal (H2) phases, or inverse micellar cubic (I2) phases, which can be formed based on the complex interactions between the molecular shape of amphiphilic molecules, the total free energy of the lipid-water system, and environmental conditions.

[0162] The bulk phase LLC materials disclosed herein can be dispersed in aqueous solution to produce LLC nanoparticles, such as cubes and hexagons. Thus, in embodiments, the LLC lipid carriers disclosed herein are in the form of LLC nanoparticles.

[0163] Compared to prior art lipid carriers such as solid lipid nanoparticles (SLN) or nanostructured lipid carriers (NLC), the lipid carriers disclosed herein are lyotropic liquid crystal (LLC) lipid carriers in which the amphiphilic molecules retain fluid properties. Therefore, the lyotropic liquid crystal (LLC) lipid carriers disclosed herein advantageously can undergo a mesophase transition, such as when exposed to a pH change (e.g., a pH drop).

[0164] For example, the prior art SLNs described by Mhule et al. (Int. J. Pharm., 2018, 550, 149-159) are solid lipid nanoparticles that contain crystalline amino lipids as the only lipid component in addition to a surfactant and the drug vancomycin.

[0165] The LLC materials disclosed herein are typically formed under conditions of excess water, which makes them particularly suitable for drug delivery applications. For example, the LLC lipid carriers disclosed herein can comprise about 0.1 wt % to about 90 wt %, about 0.1 wt % to about 85 wt %, about 0.1 wt % to about 80 wt %, about 0.1 wt % to about 75 wt %, about 0.1 wt % to about 70 wt %, about 0.1 wt % to about 65 wt %, about 0.1 wt % to about 60 wt %, about 0.1 wt % to about 55 wt %, about 0.1 wt % to about 50 wt %, about 0.1 wt % to about 45 wt %, about 0.1 wt % to about 50 wt %, about 0.1 wt % to about 5 ... % to about 40 wt %, about 0.1 wt % to about 35 wt %, about 0.1 wt % to about 30 wt %, about 0.1 wt % to about 25 wt %, about 0.1 wt % to about 20 wt %, about 0.1 wt % to about 15 wt %, about 0.1 wt % to about 10 wt %, about 0.1 wt % to about 5 wt %, about 0.1 wt % to about 4 wt %, about 0.1 wt % to about 3 wt %, about 0.1 wt % to about 2 wt %, or about 0.1 wt % to about 1 wt %.

[0166] In embodiments, LLC lipid carriers of the present disclosure comprise water in an amount of at least 1 wt%, at least 5 wt%, at least 10 wt%, at least 15 wt%, at least 20 wt%, at least 25 wt%, at least 30 wt%, at least 35 wt%, at least 40 wt%, at least 45 wt%, at least 50 wt%, at least 55 wt%, at least 60 wt%, at least 65 wt%, at least 70 wt%, at least 75 wt%, at least 80 wt%, at least 85 wt%, at least 90 wt%, or at least 95 wt%. In embodiments, LLC lipid carriers of the present disclosure comprise water in an amount of at least 0.1 wt%, at least 0.5 wt%, at least 1 wt%, at least 2.5 wt%, at least 3 wt%, at least 3.5 wt%, at least 4 wt%, at least 4.5 wt%, or at least 5 wt%.

[0167] In embodiments, the LLC lipid carrier disclosed herein comprises water in an amount of about 1 wt % to about 10 wt %, about 5 wt % to about 15 wt %, about 10 wt % to about 20 wt %, about 15 wt % to about 25 wt %, about 20 wt % to about 30 wt %, about 25 wt % to about 35 wt %, about 30 wt % to about 40 wt %, about 35 wt % to about 45 wt %, about 40 wt % to about 50 wt %, about 45 wt % to about 55 wt %, about 50 wt % to about 60 wt %, about 55 wt % to about 65 wt %, about 60 wt % to about 70 wt %, about 65 wt % to about 75 wt %, or about 70 wt % to about 80 wt %. In some embodiments, LLC lipid carriers disclosed herein comprise water in an amount of about 10% to about 60% by weight, about 20% to about 60% by weight, about 30% to about 60% by weight, about 40% to about 60% by weight, or about 50% to about 60% by weight. In some embodiments, LLC lipid carriers disclosed herein comprise water in an amount of about 40% to about 50% by weight.

[0168] The LLC lipid carrier disclosed herein is formed by at least one structural lipid other than an amino lipid having an amide linker, the at least one structural lipid being a lyotropic liquid crystal phase forming lipid. In any embodiment herein, the at least one structural lipid is amphiphilic.

[0169] The at least one structured lipid may be selected from amphiphilic lipids known in the art to form lyotropic liquid crystal phases, specifically cubic and / or mesophase structures (e.g., cubic and / or hexagonal). In an embodiment, the structured lipid is capable of forming a cubic mesophase structure, such as a cube.

[0170] The selection of at least one structural lipid that is appropriate can be based on certain requirements understood in the art. For example, lipids can be selected from those lipids that adopt type II lyotropic liquid crystal phases at ambient temperature and physiological temperature. Parameters that can be suitable for selecting appropriate lipids include (i) about the hydrophobic component: 1. The temperature should be above the chain melting temperature so that there are molten chains; and 2. In a carbon chain consisting of at least 14 carbons, at least one cis unsaturated bond should be present at least in the middle position along the skeleton; or 3. The carbon skeleton should contain at least 12 carbons, 3 of which are secondary carbons with methyl branches; and 4. The molecular weight of the hydrophobic molecule should be at least> 200amu, and (ii) about the head group: 5. The head group should contain at least three functional groups (e.g., hydroxyl groups) with minimal hydrophilicity; 6. The head group should be able to form a head group-water hydrogen bond network; and 7. The head group area should be small relative to the space occupied by the hydrophobic molecule. As a guide, this is exemplified by the MO lipid used in the examples of the present disclosure, as the MO lipid: meets criteria 1, 2, and 4 for a hydrophobic molecule; and meets criteria 5, 6, and 7 for a head group. It will be appreciated that many other lipids are available that suitably meet these criteria, and that the lipids may be selected based on criteria of known or readily determined values.

[0171] Guidance for the selection of the at least one structural lipid may be found in one or more of the following publications, each of which is incorporated herein by reference in its entirety: (i) T. Kaasgaard and CJ Drummond "Ordered 2D and 3D Nanostructured Amphiphile Self-Assembly Materials Stable in Excess Solvent," Phys. Chem. Chem. Phys. 2006, 8, 4957-4975; (ii) C. Fong, T. Le and CJ Drummond "Lyotropic Liquid Crystal Engineering-Ordered Nanostructured Small Molecule Amphiphile Self-Assembly Materials by (iii) L. van't Hag, S.L. Gras, C.E. Conn, and C.J. Drummond. “Lyotropic liquid crystal engineering moving beyond binary compositional space–Ordered nanostructured amphiphile self-assembly materials by design.” Chem. Soc. Rev., 2012, 41, 1297–1322. DOI: 10.1039 / c1cs15148g. (iv) S. Sarkar, N. Tran, M.D. H. Rashid, T.C. Le, I. Yarovsky, C.E. Conn, and C.J.Drummond, “Toward cell membrane biomimetic lipidic cubic phases: a high-throughput exploration of lipid compositional space”, ACS Applied Biomaterials, 2019, 2, 182–195. DOI: 10.1021 / acsabm.8b00539.

[0172] Polyhydroxy (glycolipid) and polyether (polyethylene oxide) formation are two of the largest categories of the type II forming head groups of interest. Non-limiting examples of head group motifs include alcohols, fatty acids, monoacylglycerides, MAG, 2-MAG, glycerides, glyceryl ethers, ethylene oxide, amides, monoethanolamides, diethanolamides, serinolamides, methylpropylene glycol amides, ethylpropylene glycol amides, ureas, urea alcohols, biuret, biuretol, ureides, endogenous cannabinoids (cannabinoids, virodhamine, 2-glycerol, dopamine, 2-glyceryl ethers) and glycolipids. Examples include phospholipids, such as DMPC and DMPE.

[0173] In an embodiment, at least one structured lipid may be selected from the group consisting of ethylene oxide-based amphiphiles, monoacylglycerol-based amphiphiles, glycolipid-based amphiphiles, phosphatidylethanolamine-based amphiphiles and urea-based amphiphiles, and derivatives or analogs thereof.

[0174] The ethylene oxide amphiphilic molecules may include C12(EO)2, C12(EO)4, C12(EO)5 and C12(EO)6 as well as dialkyl ethylene oxide amphiphilic molecules. Monoacylglycerols may include monomyristolein, monoolein, monovaccenin and monoerucin. Amphiphilic molecules similar to monoacylglycerols may be suitable and include oleyl glyceride, phytane glyceride, glyceryl monooleyl ether, glyceryl phytane ether, phytantriol and monononadecenoin. Possibly suitable glycolipids with sugar moieties include monosubstituted glycolipids: β-Mal3(Phyt)2, β-Glc(Phyt), β-Xyl(Phyt), β-Glc-(TMO)2, β-Mal2(Phyt)2, and β-Glc(Phyt)2; and disubstituted unbranched glycolipids: 1,2-diacyl-(β-D-glucopyranosyl)-sn-glycerol; 1,2-dialkyl-(β-D-glucopyranosyl)-sn-glycerol; 1,3-diacyl-(β-D-glucopyranosyl)-sn-glycerol; 1,3-dialkyl-(β-D-glucopyranosyl)-sn-glycerol. Phosphatidylethanolamine amphiphiles may include dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE). Urea amphiphiles may include dodecyl urea (DU), octadecyl urea (ODU), oleyl urea (OU), oleyl biuret (OBU), linoleyl urea (LU), phytanyl urea (PU), hexahydrofarnesyl urea (HFU).

[0175] In an embodiment, at least one structural lipid can be selected from the group consisting of: 1-monoolein, 2-monoolein, citrem, oleyl lactate, oleamide, monoelaidin, linoleic acid, oleic acid, monopalmitolein, monolinolein, phytantriol, diolein, triolein, dioleoylglycerol, didodecyldimethylammonium bromide, dioctadecyl (dimethyl) ammonium bromide, ) ammonium chloride (DOAC / DODMAC) or dioctadecyl (dimethyl) ammonium bromide (DODAB), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-phosphatidylglycerol (DOPG), oleic acid, lyso-1-hydroxy-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-dihexyl-phosphocholine, vitamin E tocopherol, vitamin E (tocopherol) acetate, phytanyl monoethanolamide, farnesyl monoethanolamide, oleyl monoethanolamide, linoleyl monoethanolamide and linolenoyl monoethanolamide.

[0176] The single-chain amphiphilic lipid can be selected from the group consisting of: saturated fatty acids C7-C16, oleic acid, elaidic acid, linoleic acid, sodium oleate / gadolinium, oleamide, 1-glyceryl monooleyl ether (GME), GMO, 2-MO, oleyl lactate, monoglyceryl citrate, diglyceryl monooleate (DGMO), lyso(1-oleoyl)-phosphatidyl-choline, (Z)-octadec-9-enylferrocene, N-dodecyl-caprolactam (C12), vitamin K1, ubiquinone-10 (coenzyme Q10), vitamin E, vitamin E acetate, vitamin A palmitate, α-tocopheryl PEO1000 succinate (vitamin E TPGS), PEG2000-MO, PEG-PT, PEOx-stearate (x=40-100), polysorbate 80.

[0177] The amphiphilic lipid having multiple alkyl chains can be selected from the group consisting of: didodecyldimethylammonium bromide (DDAB); bis(rapeseed ethyl)dimethylammonium chloride (DEEDAC); dioctadecyl(dimethyl)ammonium chloride (DOAC / DODMAC) or dioctadecyl(dimethyl)ammonium bromide (DODAB); diolein; dioleoyl-glycerol (DOG), EDTA-bis-oleoyl; EDTA-bis-phytyl; 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); 1,2-dioleoyl-phosphatidic acid (DOPA); 1,2-dioleoyl-phosphatidylglycerol (DOPG); 1,2-distearoyl-phosphatidylglycerol (DSPG); 1,2-dioleoyl 1,2-dioleoyl-phosphatidylethanolamine (DOPE); 1,2-distearoyl-glycero-3-phosphoethanolamine (DSPE); 1,2-dioleoyl-phosphatidylcholine (DOPC); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS); 1,2-dipalmitoylphosphatidylserine (DPPS); DSPE-mPEG350, 750, 2000 (X=7, 16 or 45); DSPE-PEG2000, 3400, 5000; DMPE-mPEG550; (C18)2DTPA (Gd), cardiolipin, cyclodextrin derivative (βCD-nC10).

[0178] In any of the embodiments herein, the at least one structured lipid can be glyceryl monooleate and / or phytantriol.

[0179] In any of the embodiments herein, the lipid carriers of the present disclosure may comprise a combination of MO or phytantriol and one or more of cholesterol, DLPC, DSPC, DPPE, DPPS, DOPS, DPPC, DMPC, DMPS, and DLPS.

[0180] It is known that monoacylglycerols form inverted phases over large areas of their phase diagrams, with monoolein being the most prominent. The formation of inverted phases is favored by the kinks introduced by the cis double bonds. Longer acyl chains increase the volume of the hydrophobic chains and make monoolein more wedge-shaped and shift toward the Type 2 phase in the spectrum of the mesophase. If the double bond is closer to the end of the lipid, the double bond will weaken its effect and make it less wedge-shaped. It is expected that acyl chain extension will drive further mesophase formation toward the Type 2 phase, and on this basis, it is not surprising that the H2 phase becomes the dominant phase with this change.

[0181] In embodiments where the LLC lipid carrier comprises at least two structured amphiphilic lipids, then at least one structured amphiphilic lipid may be selected from monoolein and phytantriol.

[0182] In such embodiments, at least one structural lipid of monoolein and / or phytantriol can be combined with one or more of triolein, vitamin E and DOPE, either alone or in combination. If it is desired that there is a charge on the lipid carrier, these combinations themselves can be further combined with one or more cationic lipids, which are selected from the cationic lipids that are well known in the art and commercially available and include various quaternary ammonium cationic compounds.

[0183] Certain structural lipids of the at least one structural lipid may be selected specifically for their effect on the internal curvature of the final lipid particle (eg, DOPE).

[0184] Representative cationic lipids can be selected from the following non-limiting examples: 3-β[4N(1N 8-Biguanidinospermidine)-carbamoyl] cholesterol (BGSC); 3-β[(N,N-biguanidinoethyl-aminoethane)-carbamoyl] cholesterol (BGTC); N,N1,N2,N3-tetramethyltetrapalmitospermine (cellfectin); NtN'-butyl-N'-tetradecyl-3-tetradecyl-aminopropane-amidine (CLONfectin); dimethyldioctadecyl ammonium bromide (DDAB); 1,2-myristoylpropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE); 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propane(nitrotrifluoroacetate) (DOSPA); 1,3-dioleoyloxy-2-(6-carboxyspermyl)-propylamide (DOSPER); 4-(2,3-bis-palmitoyloxy-propyl)-propane N,N,N',N'-tetramethyl-N,N'-bis(2-hydroxyethyl)-2,3-dioleoyloxy-1,4-butane diammonium iodide (Tfx-50); N-1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA) or other N-(N,N-1-dialkoxy)-alkyl-N,N,N-trisubstituted ammonium surfactants; the trimethylammonium group is double-chained through a butanol spacer arm (DOT), 1,2-dioleoyl-3-(4'-trimethylammonio)butanol-sn-glycerol (DOBT) or cholesteryl (4'-trimethylamino)butyrate (ChOTB) linked to a cholesterol group (for ChOTB), DORI (DL-1,2-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium) or as WO DORIE (DL-1,2-O-dioleoyl-3-dimethylaminopropyl) or its analogs disclosed in 93 / 03709 (-β-hydroxyethylammonium) (DORIE); 1,2-dioleoyl-3-succinyl-sn-glycerocholine ester (DOSC); tetraoctylammonium bromide (TOAB) as a cationic phase transfer agent; cholesteryl hemisuccinate (ChOSC);Midon glycyl spermine (DOGS) and dipalmitoylphosphatidylethanolpentyl spermine (DPPES) or cationic lipids disclosed in U.S. Pat. No. 5,283,185, cholesteryl-3β-carboxy-amido-ethylenetrimethylammonium chloride, 1-dimethylamino-3-trimethylammonium-DL-2-propyl-cholesteryl formate iodide, cholesteryl-3-O-carboxyamidoethyleneamine, cholesteryl-3-β-oxysuccinamide-ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonium-DL-2-propyl-cholesteryl-3-β-oxysuccinamide iodide, 2-(2-trimethylammonium)-ethylmethylaminoethyl-cholesteryl-3-β-oxysuccinamide iodide, 3- β-N-(N',N'-dimethylaminoethane)carbamoylcholesterol (DC-chol) and 3-β-N-(polyethyleneimine)-carbamoylcholesterol; O,O-dimyristyl-N-lysyl-aspartic acid (DMKE); O,O-dimyristyl-N-lysyl-glutamate (DMKD); 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE); 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC); 1,2-dimyristyl-sn-glycero-3-ethylphosphocholine (DMEPC); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-dipal Toyl-sn-glycero-3-ethylphosphocholine (DPEPC); 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSEPC); 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); dioleoyldimethylaminopropane (DODAP); 1,2-palmitoyl-3-trimethylammonium propane (DPTAP); 1,2-distearoyl-3-trimethylammonium propane (DSTAP); 1,2-myristoyl-3-trimethylammonium propane (DMTAP); and sodium dodecyl sulfate (SDS).

[0185] In an embodiment, it is particularly preferred that the cationic lipid is DOTAP and / or DODAB and / or tetraoctylammonium bromide (TOAB).

[0186] In embodiments, the lipid carrier may comprise, in addition to the at least one structural lipid, a fatty acid, such as oleic acid.

[0187] In other embodiments, suitable amphipathic lipids include, but are not limited to, lipid compounds comprising a hydrophobic tail group selected from the group consisting of: oleyl, linoleoyl, linolenoyl, phytanoyl, farnesyl or an extended aliphatic hydrophobic tail. In certain embodiments, the structured lipid compound comprises an oleyl, linoleoyl or phytanoyl hydrophobic tail, preferably an oleyl tail.

[0188] Generally, any amphiphilic lipid typically used to form cubic phase nanoparticles can be used as the structural lipid.

[0189] In an embodiment, the one or more structured amphiphilic lipids can be selected from the group consisting of monoolein (also known as monoolein), monoglyceryl citrate, oleyl lactate, oleamide, mono-elaidin, linoleic acid, elaidic acid, monopalmitolein, monolinolein, phytantriol, diolein, triolein, dioleoylglycerol, didodecyldimethylammonium bromide, dioctadecyl(dimethyl)ammonium chloride (DOAC / DODMAC), or dioctadecyl(dimethyl)ammonium bromide (DOAC / DODMAC). ODAB), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-phosphatidylglycerol (DOPG), oleic acid, lyso-1-hydroxy-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-dihexyl-phosphocholine, vitamin E tocopherol, vitamin E (tocopherol) acetate, phytanyl monoethanolamide, farnesyl monoethanolamide, oleyl monoethanolamide, linoleyl monoethanolamide and linolenoyl monoethanolamide.

[0190] In a preferred embodiment, the structured amphipathic lipid is glyceryl monooleate.

[0191] In an embodiment, the LLC lipid carrier is a non-lamellar lyotropic liquid crystal phase, such as a self-assembled non-lamellar lyotropic liquid crystal phase. In an embodiment, the non-lamellar lyotropic liquid crystal phase is formed by the self-assembly of one or more amphiphilic lipids. It should be understood that suitable amphiphilic lipids will self-assemble in the presence of an aqueous solution (such as water or an aqueous buffer solution) to form a lyotropic liquid crystal structure exhibiting a non-lamellar mesophase. In an embodiment, the non-lamellar lyotropic liquid crystal phase is a mesophase having an inverse bicontinuous cubic (Q2) structure, an inverse hexagonal (H2) structure, or an inverse micellar cubic (I2) structure.

[0192] The bulk lyotropic liquid crystal phase can be dispersed to form nanoparticles. When dispersed in aqueous solution, these nanoparticles retain their internal nanostructure and are referred to as cubes, hexagons, and micellar cubes.

[0193] Thus, in some embodiments, LLC lipid carriers disclosed herein are in the form of nanoparticles, such as lipid nanoparticles (LNPs). Such nanoparticles may be referred to as lyotropic liquid crystal (LLC) lipid nanoparticles or lyotropic liquid crystal lipid nanoparticles (LCNPs). The terms "nanoparticles", "LLC lipid nanoparticles", "LNPs" and "LCNPs" are used interchangeably herein.

[0194] Nanoparticles can be formed by dispersing a bulk non-lamellar lyotropic liquid crystal phase into a colloidal system. For example, upon dispersion (e.g., in aqueous solution), nanoparticles such as cubes, hexagons, and micellar cubes are produced.

[0195] The LLC lipid carrier of the present invention may preferably comprise at least one stabilizer selected from stabilizers known in the art. Suitable stabilizers may help disperse the non-lamellar lyotropic liquid crystal phase, and / or may help maintain the nanostructure of the nanoparticles.

[0196] In embodiments, the stabilizer may be a poloxamer or a surfactant or a PEGylated lipid stabilizer or a modified version thereof.

[0197] In an embodiment, stabilizer is selected from PEG-PPO-PEG triblock copolymer and nonionic block copolymer surfactant and PEO copolymerized with charged part.Poloxamer 407 and Pluronic 127 can be suitable examples of stabilizer, and can be incorporated into any embodiment of the first aspect or second aspect as described herein.PEO copolymerized with (3-acrylamidopropyl) trimethylammonium chloride or similar charged part can also be suitable.The lipid stabilizer of PEGization is also suitable, including but not limited to PEG2000-MO, PEG-PT, DSPE-PEG (2000) amine, 18:0PEG2000 PE and DSPE-PEG (5000) amine.Many other such stabilizers are known in the art.

[0198] The use of stabilizers is preferred, and while the nature of the stabilizer may be selected based on the nature of the lipid, the choice of these components and an understanding of their compatibility may be based on information known in the art.

[0199] The many steric stabilizers reported so far can be divided into four groups: (i) amphiphilic block copolymers (i.e., Poloxamer TM), (ii) PEGylated lipids, (iii) tailored lipid-copolymers, and (iv) alternative steric stabilizers (e.g., bile salts, proteins). Ideally, the selected stabilizer will prevent aggregation of particles by providing an electrostatic or more commonly a steric barrier between particles in proximity. Stabilizers that can best function in lipid particles of the present disclosure share similar properties, including (i) being highly hydrophilic, having a high HLB (hydrophile-lipophile balance) value, due to an asymmetric amphiphilic polymer structure with a larger hydrophilic domain. Importantly, the hydrophilic portion of the molecule is not surrounded by hydrophobic regions. High HLB can be achieved by using longer PEG chains or multiple PEG chains; (ii) the presence of hydrogen bond acceptors and the absence of hydrogen bond donors; and (iii) electrical neutrality. One skilled in the art can select an appropriate stabilizer based on this. In addition, the following journal articles describe key aspects of stabilizers that may be suitable for use with the lipid carriers of the present disclosure and are hereby incorporated by reference in their entirety: (i) J. Y. Chong, X. Mulet, B. J. Boyd, and C. J. Drummond; "Steric Stabilizers for Cubic Phase Lyotropic Liquid Crystal Nanodispersions (Cubosomes)" in "Advances in Planar Lipid Bilayers and Liposomes", Vol. 21, Chapter 5, (2015) pp. 131-187, ISSN 1554-4516, Elsevier; (ii) J. Zhai, B. Fan, S. H. Thang, C. J. Drummond "Novelamphiphilic block copolymers for the formation of stimuli-responsive non-lamellar lipid nanoparticles" in "Advances in Planar Lipid Bilayers and Liposomes", Vol. 21, Chapter 5, (2015) pp. 131-187, ISSN 1554-4516, Elsevier; non-lamellar lipid nanoparticles)" "Molecules", 2021, 26, 3648-3664. DOI: 10.3390 / molecules26123648; (iii) J. Zhai, R. Suryadinata, B. Luan, N. Tran, TMHinton, J. Ratcliffe, X. Hao and CJDrummond, J. “Amphiphilic brush polymers produced by the RAFT polymerisation method stabilise and reduce the cell toxicity of lipid lyotropic liquid crystalline nanoparticles”, Faraday Discussions, 2016, 191, 545–563. DOI: 10.1039 / C6FD00039H; Faraday Discussion 191 on Nanoparticles with Morphological and Functional Anisotropy; (iv) J. Zhai, T. J. Hinton, L. J. Waddington, C. Fong, N. Tran, X. Mulet, C. J. Drummond and BW Muir. “Lipid-PEG Conjugates Sterically Stabilise and Reduce the Toxicity of Phytantriol-Based Lyotropic Liquid Crystalline Nanoparticles.” Langmuir, 2015, 31, 10871–10880. DOI: 10.1021 / acs.langmuir.5b02797. (v) JYT Hong, X. Mulet, D. Keddie, LJ Waddington, S. T. Mudie, B. J. Boyd and C. J. Drummond. “Novel Steric Stabilisers for Lyotropic Liquid Crystalline Nanoparticles: Pegylated Phytanyl Copolymers.” Copolymers)” Langmuir, 2015, 31, 2615-2629. DOI: 10.1021 / la501471z; (vi) JYT Hong, X. Mulet, A.Postma, DJ Keddie, LJ Waddington, BJ Boyd and CJ Drummond “Novel RAFT Amphiphile Brush Copolymer Steric Stabilisers for Cubosomes: Poly(octadecyl acrylate)-block-poly(polyethylene glycol methyl ether acrylate)” Soft Matter, 2014, 10, 6666-6676. DOI: 10.1039 / C4SM01064G; (vii) A. Tilley, CJ Drummond and BJ Boyd “Disposition and Association of the Steric Stabiliser Pluronic F127 in Lyotropic Liquid Crystalline Nanostructured Particle Dispersions” (viii) JYT Chong, X. Mulet, LJ Waddington, BJ Boyd and CJ Drummond “High Throughput Discovery of Novel Steric Stabilisers for Cubic Lyotropic Liquid Crystal Nanoparticle Dispersions” Langmuir, 2012, 28, 9223-9232. DOI: 10.1021 / la301874v; (ix) JYT Chong, X. Mulet, LJ Waddington, BJ Boyd and CJ Drummond “High Throughput Discovery of Novel Steric Stabilisers for Cubic Lyotropic Liquid Crystal Nanoparticle Dispersions” Langmuir, 2012, 28, 9223-9232. DOI: 10.1021 / la301874v;Drummond "Steric Stabilisation of Cubic Lyotropic Liquid Crystalline Nanoparticles: High Throughput Evaluation of Triblock Polyethylene Oxide–Polypropylene Oxide–Polyethylene Oxide Copolymers". Soft Matter, 2011, 7, 4768–4777. DOI: 10.1039 / c1sm05181d. .

[0200] A stabilizer may be present during the formation of the LLC lipid nanoparticles disclosed herein.

[0201] Certain preferred stabilizers include stabilizing polymers such as nonionic triblock copolymers. In certain embodiments, the stabilizer is Pluronic F127 (also known as Poloxamer 407) or Poloxamer 80; preferably Pluronic F127.

[0202] LLC lipid carriers disclosed herein can be prepared by mixing amino lipids and structured lipids disclosed herein. For example, the amino lipids and structured lipids can be mixed as solutions in an organic solvent, followed by evaporation of the solvent. Preferably, at least a stabilizer is also mixed with these lipid components.

[0203] A mixture of two or more structural lipids may be used as the structural lipid. Equally, two or more amino lipids may be used to form the lipid carrier of the present disclosure.

[0204] The amino lipids and structured lipids of different weight ratios can be used. These amino lipids and structured lipids can be used to determine the precise pH when the desired structural transformation occurs. The preferred weight ratio of amino lipids and structured lipids or total lipids in the lipid carrier can be simply determined by testing multiple ratios and determining which ratio is the best for the pH environment under consideration. This method is described in the experimental section. Therefore, in an embodiment, the weight ratio of amino lipids and structured lipids or total lipids in the lipid carrier can be the weight ratio that causes the mesophase transformation when the pH drops to the pH value determined first.

[0205] In an embodiment, the structural lipid can constitute the bulk of the lipid carrier, or can be used in an equal ratio with the amino lipid. In an embodiment, the ratio of the structural lipid to the lipid having an amide linker (such as an amino lipid) is 95:5wt%, 90:10wt%, 85:15wt%, 80:20wt%, 75:25wt%, 70:30wt%, 60:40wt%, 50:50wt%, 40:60wt%, 30:70wt%, 20:80wt%, 10:90wt%, 5:95wt%; preferably 95:5wt %, 90:10wt%, 85:15wt%, 80:20wt%, 75:25wt%, 70:30wt%, 60:40wt%, 50:50wt%, 40:60wt%, 30:70wt%; preferably 95:5wt%, 90:10wt%, 85:15wt%, 80:20wt%, 75:25wt%, 70:30wt%, 60:40wt%, 50:50wt%, 40:60wt%, 30:70wt%; preferably 95:5wt%, 90:10wt%, 85:15wt%, 80:20wt%, 75:25wt%, 70:30wt%, 60:40wt%, 50:50wt%. In an embodiment, the corresponding weight fraction of the amino lipid is 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95; preferably 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4 and 0.5, 0.6, 0.7; preferably 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4 and 0.5.

[0206] In embodiments, the amino lipid having an amide linker is present in a weight fraction of 0.1 to 0.7 relative to the total amount of lipids in the lipid carrier.

[0207] In embodiments, the amino lipid with an amide linker is present in a weight fraction of between 0.1 and 0.6, or between 0.2 and 0.6, or between 0.1 and 0.55, or between 0.2 and 0.55 relative to the total amount of lipids in the lipid carrier.

[0208] In certain preferred embodiments, the weight fraction of the amino lipid is between 0.2 and 0.6, preferably between 0.3 and 0.5, preferably between 0.4 and 0.5.

[0209] In any embodiment described herein, amino lipids can be present in a suitable wt% relative to the total lipid content of LLC lipid carriers to provide an intermediate phase transition when exposed to a pH drop. For example, amino lipids can account for about 5wt% to about 50wt% of the total lipid content of LLC lipid carriers disclosed herein. LLC lipid carriers can include amino lipids in an amount greater than about 5%, greater than about 10%, greater than about 15%, greater than about 20%, greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75% or greater than about 80% of the total lipid content of LLC lipid carriers. In embodiments, the amino lipid may comprise about 0.5 wt % to about 50 wt % of the total lipid content of the LLC lipid carrier disclosed herein, for example, about 0.5 wt % to about 40 wt %, about 1 wt % to about 35 wt %, about 1.5 wt % to about 30 wt %, about 2 wt % to about 25 wt %, about 2.5 wt % to about 20 wt %, about 3 wt % to about 15 wt %, about 3.5 wt % to about 10 wt %, or about 4 wt % to 9 wt % of the total lipid content of the LLC lipid carrier disclosed herein. In one embodiment, the amino lipid comprises about 40 wt % of the total lipid content of the LLC lipid carrier disclosed herein.

[0210] In order to prepare LLC lipid nanoparticles disclosed herein, a mixture of amino lipids and structural lipids can be dispersed in an aqueous solution. For example, the aqueous solution can be water, a buffer solution or another aqueous solution. The aqueous solution can also contain a stabilizer. Suitable stabilizers include the above-mentioned stabilizers. In an embodiment, the stabilizer can be a poloxamer or a surfactant or a PEGylated lipid stabilizer or a modified version thereof. Preferred stabilizers include but are not limited to Pluronic F127 or Poloxamer 80.

[0211] The LLC lipid nanoparticles of the present disclosure may comprise about 0.1 wt % to about 90 wt %, about 0.1 wt % to about 85 wt %, about 0.1 wt % to about 80 wt %, about 0.1 wt % to about 75 wt %, about 0.1 wt % to about 70 wt %, about 0.1 wt % to about 65 wt %, about 0.1 wt % to about 60 wt %, about 0.1 wt % to about 55 wt %, about 0.1 wt % to about 50 wt %, about 0.1 wt % to about 45 wt %, about 0.1 wt % to about 50 wt %, about 0.1 wt % to about 5 ... % to about 40 wt %, about 0.1 wt % to about 35 wt %, about 0.1 wt % to about 30 wt %, about 0.1 wt % to about 25 wt %, about 0.1 wt % to about 20 wt %, about 0.1 wt % to about 15 wt %, about 0.1 wt % to about 10 wt %, about 0.1 wt % to about 5 wt %, about 0.1 wt % to about 4 wt %, about 0.1 wt % to about 3 wt %, about 0.1 wt % to about 2 wt %, or about 0.1 wt % to about 1 wt %.

[0212] In embodiments, LLC lipid nanoparticles disclosed herein comprise at least 1 wt%, at least 5 wt%, at least 10 wt%, at least 15 wt%, at least 20 wt%, at least 25 wt%, at least 30 wt%, at least 35 wt%, at least 40 wt%, at least 45 wt%, at least 50 wt%, at least 55 wt%, at least 60 wt%, at least 65 wt%, at least 70 wt%, at least 75 wt%, at least 80 wt%, at least 85 wt%, at least 90 wt% or at least 95 wt% of water. In embodiments, LLC lipid nanoparticles disclosed herein comprise at least 0.1 wt%, at least 0.5 wt%, at least 1 wt%, at least 2.5 wt%, at least 3 wt%, at least 3.5 wt%, at least 4 wt%, at least 4.5 wt% or at least 5 wt% of water.

[0213] In embodiments, LLC lipid nanoparticles disclosed herein comprise water in an amount of about 1 wt % to about 10 wt %, about 5 wt % to about 15 wt %, about 10 wt % to about 20 wt %, about 15 wt % to about 25 wt %, about 20 wt % to about 30 wt %, about 25 wt % to about 35 wt %, about 30 wt % to about 40 wt %, about 35 wt % to about 45 wt %, about 40 wt % to about 50 wt %, about 45 wt % to about 55 wt %, about 50 wt % to about 60 wt %, about 55 wt % to about 65 wt %, about 60 wt % to about 70 wt %, about 65 wt % to about 75 wt %, or about 70 wt % to about 80 wt %. In some embodiments, LLC lipid nanoparticles disclosed herein comprise water in an amount of about 10 wt % to about 60 wt %, about 20 wt % to about 60 wt %, about 30 wt % to about 60 wt %, about 40 wt % to about 60 wt %, or about 50 wt % to about 60 wt %. In some embodiments, LLC lipid nanoparticles disclosed herein comprise water in an amount of about 40 wt % to about 50 wt %.

[0214] LLC lipid nanoparticles disclosed herein can be provided as nanoparticle dispersions in polar media (such as aqueous media). Therefore, in a second aspect, a lyotropic liquid crystal (LLC) lipid nanoparticle composition is provided, which is a dispersion of LLC lipid carriers disclosed herein in polar media, such as aqueous dispersions of LLC lipid carriers disclosed herein. The term "polar medium" should be understood to mean polar media including but not limited to water, glycerol, propylene glycol, propylene carbonate, methanol, ethanol, glycogen, etc. and solutions based on these liquids and mixtures thereof. For example, the polar medium can be water or an aqueous buffer. Generally, LLC lipid nanoparticle compositions disclosed herein can include solvents or dispersion media, and the dispersion media include but are not limited to water, ethanol, polyols (such as, glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (such as, triglycerides, vegetable oils and injectable organic esters, such as ethyl oleate) and combinations thereof. In some cases, it will be preferred to include isotonic agents, such as sugars, sodium chloride or combinations thereof.

[0215] In some embodiments, LLC lipid nanoparticle compositions disclosed herein will be formed by dispersing LLC lipid carriers disclosed herein in pharmaceutically or cosmetically acceptable carriers or diluents, including but not limited to water or any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and such similar materials and combinations thereof as will be known to those skilled in the art (Remington's, 1990). Unless any conventional pharmaceutically or cosmetically acceptable carrier is incompatible with the active ingredient to some extent, its use in therapeutic compositions, pharmaceutical compositions, and cosmetic compositions is contemplated. The compositions used in the present disclosure may contain different types of carriers, depending on whether the carrier is administered in solid form, liquid form, or aerosol form and whether it needs to be sterile for routes of administration such as injection.

[0216] In embodiments, the amount of polar medium (e.g., water) in the LLC lipid nanoparticle compositions disclosed herein is at least about 10 wt%, at least about 15 wt%, at least about 20 wt%, at least about 25 wt%, at least about 30 wt%, at least about 35 wt%, at least about 40 wt%, at least about 45 wt%, at least about 50 wt%, at least about 55 wt%, at least about 60 wt%, at least about 65 wt%, at least about 70 wt%, at least about 75 wt%, at least about 80 wt%, at least about 85 wt%, at least about 90 wt%, or at least about 95 wt%. In some embodiments, the amount of polar medium (e.g., water) in the LLC lipid nanoparticle compositions disclosed herein is about 80% to about 99% by weight, such as about 80%, about 82%, about 84%, about 86%, about 88%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%. In some embodiments, the amount of polar medium (e.g., water) in the LLC lipid nanoparticle compositions disclosed herein is about 90% by weight.

[0217] The nanostructure of LLC lipid carriers can be determined by any technique known to those skilled in the relevant art. For example, small angle X-ray scattering (SAXS) can be used to determine the structure.

[0218] In embodiments, the mesophase transition of the LLC lipid vector upon exposure to a decrease in pH is a transition caused by a decrease in pH to below pH 10, below pH 9, below pH 8, or below pH 7. In embodiments, the LLC lipid vector disclosed herein can undergo a mesophase transition of pH 4-pH 7, pH 5-7, or pH 5-6.5, e.g., pH 5.5-6.

[0219] In embodiments, the mesophase transition upon exposure to a decrease in pH is a transition caused by a decrease in pH of 1 to 4 pH units.

[0220] In embodiments, the mesophase transition upon exposure to a decrease in pH is a transition to a mesophase having an interfacial curvature lower than the interfacial curvature of the mesophase occupied by the lipid vector prior to the decrease in pH.

[0221] In embodiments, the mesophase transition upon exposure to a decrease in pH is a transition to a mesophase corresponding to a lower CPP value.

[0222] In embodiments, the mesophase transition upon exposure to a drop in pH is a transition from one mesophase to another, the transition following the general mesophase transition sequence of L2→H2→Q2→L3. In embodiments, the phase transition is H2→Q2.

[0223] In an embodiment, the nanostructure of the non-lamellar lyotropic liquid crystal phase (LLC lipid carrier) is hexagonal (H2) or cubic (Q2). In an embodiment, the non-lamellar lyotropic liquid crystal phase is hexagonal at a pH above about pH 10, about pH 9, about pH 8, or about pH 7. In an embodiment, the non-lamellar lyotropic liquid crystal phase is hexagonal at a pH of about pH 7 or higher (such as about pH 7.4 or higher, or about pH 7.5 or higher). In an embodiment, the non-lamellar lyotropic liquid crystal phase is cubic at a pH below about pH 7.5 (such as about pH 7.4 or lower).

[0224] In preferred embodiments, the non-lamellar lyotropic liquid crystal phase transitions from a hexagonal phase at a pH above about pH 8 to a cubic phase at a pH below about pH 7.5. In certain embodiments, the non-lamellar lyotropic liquid crystal phase transitions from a hexagonal phase at a pH above about pH 7.5 to a cubic phase at a pH below about pH 7. In certain embodiments, the non-lamellar lyotropic liquid crystal phase transitions from a hexagonal phase at a pH above about pH 6.5 to a cubic phase at a pH below about pH 6.5. In preferred embodiments, the non-lamellar lyotropic liquid crystal phase undergoes a transition from a predominantly hexagonal phase to a predominantly cubic phase at a pH below about pH 7. In some embodiments, the transition is complete within 0.5 pH.

[0225] In a preferred embodiment, the non-lamellar lyotropic liquid crystal phase of the lipid carrier undergoes a transition from a predominantly hexagonal phase at pH 7 to a predominantly cubic phase at pH 5.5-6.0. In other words, the lipid carrier undergoes a transition to a mesophase with lower interfacial curvature; and / or a transition to a mesophase corresponding to a lower CPP (critical packing parameter) value; and / or a transition from one mesophase to another, the transition being a transition in the general order of L2→H2→Q2→L3, but not necessarily a transition through each of these mesophases.

[0226] Advantageously, the pH range of 5.5-6.0 is a pathologically relevant pH range (the range is reported to be found in tumors, infection sites of certain bacteria and fungi, and subcellular (intracellular) organelles), and is significantly lower than the pH of healthy tissue (about pH 7). The present disclosure is predicted, at least in part, by the recognition that the amino lipids disclosed herein can be used to manipulate the structural behavior of lipid carriers to provide carrier particles that can undergo phase changes in response to pH, such as a pH between 5 and 7. For example, a non-lamellar lyotropic liquid crystal phase can undergo a phase change to an intermediate phase with a lower interfacial curvature in response to a decrease in pH from an ambient pH of about 7 to 7.5 to a reduced pH below 7, such as a pH between 5 and 7 (e.g., between 5.5 and 6.0). Since the reduced pH range can be found in tumors, infection sites of certain bacteria and fungi, and subcellular (intracellular) organelles, it is believed that the LLC lipid carriers disclosed herein undergo phase transitions at such sites. For example, the LLC lipid nanoparticles disclosed herein can undergo a phase transition from a hexagonal phase at pH 7 to a cubic phase at pH 5.5-6.0.

[0227] LLC lipid carrier containing active agent

[0228] In embodiments, the LLC lipid carrier disclosed herein comprises an active agent. In embodiments, the active agent is encapsulated in the LLC lipid carrier.

[0229] In the context of the present disclosure, encapsulating or including an active agent in an LLC lipid carrier means that when the encapsulated active agent is placed in a biological system, most agents are not released immediately, but rather the release is slow and is promoted by diffusion of the agent into the lipid carrier, or the release is promoted by phase transition of the LLC lipid carrier.

[0230] In embodiments, the active agent is a pharmaceutical or cosmetic active agent.In embodiments, the active agent is selected from the group consisting of: a peptide, a protein, an enzyme, a small molecule drug, and a nucleic acid.

[0231] For example, suitable active agents include radionuclides, imaging agents, polymers, antibiotics, antifungals, metal-containing nanoparticles, anti-inflammatory agents, anti-cancer agents, cardiovascular agents, anxiolytic agents, hormones, growth factors, steroid agents, gene expression regulators, knockdown agents, siRNA, RNAi agents, DNA, mRNA, dicer substrates, miRNA, shRNA, antisense oligonucleotides, aptamers, and microbial-derived toxins.

[0232] In an embodiment, the active agent is a small molecule drug. In a preferred embodiment, the active agent is an anticancer chemotherapy drug. Non-limiting examples of anticancer chemotherapy drugs include temozolomide, dacarbazine, carmustine, lomustine, paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine, etoposide, teniposide, topotecan, irinotecan, The invention relates to an anti-cancer drug, for example, otecan, doxorubicin, daunomycin, epirubicin, idarubicin, methotrexate, cytarabine, gemcitabine, capecitabine, cisplatin, carboplatin, cyclophosphamide, oxaliplatin, or a mixture thereof, but is not limited thereto.

[0233] In some embodiments, the anti-cancer chemotherapy drug is a topoisomerase I inhibitor, such as camptothecin, irinotecan, and SN-38, preferably SN-38.

[0234] In embodiments, the active agent is an antibiotic.

[0235] In embodiments, the active agent is a fungicide.

[0236] In embodiments, the active agent is an oligonucleotide or a nucleic acid.

[0237] In embodiments, the active agent is a cosmetic active agent.

[0238] As described above, the LLC lipid carriers disclosed herein can undergo an intermediate phase transition in response to a predefined pH (e.g., a pH drop) such as a pH below 10, a pH below 9, a pH below 8, a pH below 7.5, a pH below 7; preferably pH 4-pH 7, pH 5-7, or pH 5-6.5 (e.g., pH 5.5-6).

[0239] In embodiments, LLC lipid carriers disclosed herein undergo a transition to an intermediate phase with a lower interfacial curvature in response to a decrease in pH. For example, a decrease in pH may result in a phase transition of LLC lipid carriers disclosed herein to a phase with a lower critical packing parameter (CPP), such as a transition from a dominant hexagonal phase (e.g., H2) to a dominant cubic phase (e.g., Q2). For example, the phase transition may be L2→H2→Q2→L3 (i.e., CPP high to low). In embodiments, the phase transition is H2→Q2.

[0240] In embodiments, LLC lipid carriers disclosed herein undergo a transition to a mesophase with lower interfacial curvature at about pH 10, about pH 9, about pH 8, about pH 7, or about pH 6. In embodiments, LLC lipid carriers disclosed herein undergo a transition to a mesophase with lower interfacial curvature at pH below 10, pH below 9, pH below 8, or pH below 7 (e.g., pH 4.0-pH 7.0, pH 5-pH 7, or pH 5.5-pH 6.5). In embodiments, LLC lipid carriers disclosed herein undergo a transition to a predominantly cubic phase at pH below 10, pH below 9, pH below 8, or pH below 7 (e.g., pH 4-pH 7, pH 5-pH 7, or pH 5.5-pH 6.5). In embodiments, LLC lipid carriers disclosed herein undergo a transition from a predominantly hexagonal phase to a predominantly cubic phase at pH below 10, pH below 9, pH below 8, or pH below 7 (e.g., pH 4-pH 7, pH 5-pH 7, or pH 5.5-pH 6.5). The transition can be from a predominantly hexagonal phase at about pH 7 or higher (e.g., pH 7.4 or higher) to a predominantly cubic phase at a pH below 7 (e.g., pH 4.0-pH 7.0, pH 5-pH 7, or pH 5.5-pH 6.5). In some embodiments, LLC lipid carriers disclosed herein undergo an intermediate phase transition from a hexagonal phase at about pH 7 or higher (e.g., pH 7.4 or higher) to a cubic phase at about pH 4.0-pH 7.0, pH 5-pH 7, or pH 5.5-pH 6.5. In certain embodiments, the mesophase transition is from an inverse hexagonal phase at about pH 7 or higher (e.g., pH 7.4 or higher) to a bicontinuous cubic phase at about pH 4.0-pH 7.0, pH 5-pH 7, or pH 5.5-pH 6.5.

[0241] Previous studies have shown that hexagonal structured hosts will release encapsulated guests at a much slower rate than corresponding cubic structured hosts. Consistent with previous studies, but not wishing to be bound by theory, it is believed that the LLC lipid carrier disclosed herein releases the encapsulated active agent when it is converted from a hexagonal phase structure to a cubic phase structure at low pH. Therefore, when the lipid carrier is in an environment with an ambient pH (e.g., physiological of about 7-7.5), the encapsulated agent will have a slow release profile, and when the lipid carrier is in an environment with a reduced pH (e.g., a pH lower than 7 as found, such as a pH between 7 and 5), the encapsulated agent will have a faster release profile. This reduced pH range can be found in environments including tumor sites, infection sites of certain bacteria and fungi, and subcellular organelles.

[0242] Compositions of LLC lipid carriers containing active agents

[0243] In a third aspect, a cosmetic composition is provided, comprising the LLC lipid carrier according to the first aspect and a cosmetically active agent and a cosmetically acceptable carrier, diluent and / or excipient.

[0244] In a fourth aspect, a pharmaceutical composition is provided, comprising the LLC lipid carrier according to the first aspect and a pharmaceutically active agent and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0245] Suitably, the cosmetically or pharmaceutically acceptable carrier, diluent and / or excipient may be or include one or more of a diluent, a solvent, a pH buffer, a binder, a filler, an emulsifier, a disintegrant, a polymer, a lubricant, an oil, a fat, a wax, a coating, a viscosity modifier, a glidant, and the like.

[0246] The diluent may include one or more of microcrystalline cellulose, lactose, mannitol, calcium phosphate, calcium sulfate, kaolin, dry starch, powdered sugar, etc. The binder may include one or more of povidone, starch, stearic acid, gum, hydroxypropyl methylcellulose, etc. The disintegrant may include one or more of starch, cross-linked sodium carboxymethyl cellulose, cross-linked polyvidone, sodium starch glycolate, etc. The solvent may include one or more of ethanol, methanol, isopropanol, chloroform, acetone, methyl ethyl ketone, methylene chloride, water, etc. The lubricant may include one or more of magnesium stearate, zinc stearate, calcium stearate, stearic acid, sodium stearyl fumarate, hydrogenated vegetable oil, behenic acid glyceride, etc. The glidant may be one or more of colloidal silicon dioxide, talc, or corn starch, etc. The buffer may include phosphate buffer, borate buffer, and carbonate buffer, but is not limited thereto. The filler may include one or more gels, including gelatin, starch, and synthetic polymer gel, but is not limited thereto. The coating may include one or more of a film former, a solvent, a plasticizer, etc. Suitable film formers may be one or more of hydroxypropyl methylcellulose, methyl hydroxyethyl cellulose, ethyl cellulose, hydroxypropyl cellulose, povidone, sodium carboxymethyl cellulose, polyethylene glycol, acrylates, etc. Suitable solvents may be one or more of water, ethanol, methanol, isopropanol, chloroform, acetone, methyl ethyl ketone, methylene chloride, etc. Plasticizers may be one or more of propylene glycol, castor oil, glycerol, polyethylene glycol, polysorbate, etc.

[0247] Regarding pharmaceutically acceptable carriers, diluents and / or excipients, reference is made to Handbook of Excipients, 6th edition, ed. Rowe, Sheskey and Quinn (Pharmaceutical Press), which provides non-limiting examples of excipients that can be used in accordance with the present disclosure.

[0248] The cosmetic compositions disclosed herein may further comprise nutrients, such as carnitine, iron in ferrous form, iron salts or elemental iron, iodine, folic acid, vitamins or trace nutrients.

[0249] The cosmetic compositions may also contain coloring or flavoring agents.

[0250] It will be appreciated that the choice of cosmetically or pharmaceutically acceptable carriers, diluents and / or excipients will depend, at least in part, on the mode of administration of the formulation. By way of example only, the composition may be in the form of a tablet, capsule, caplet, powder, injectable liquid, suppository, sustained release formulation, osmotic pump formulation, or any other form that is effective and safe for administration.

[0251] In an embodiment, the cosmetic composition or pharmaceutical composition is a liquid dispersion of an LLC lipid carrier comprising a cosmetic active agent or a pharmaceutical active agent, respectively. The liquid dispersion can be an aqueous dispersion. The liquid dispersion can be encapsulated in a standard capsule known for delivering a liquid formulation. The liquid dispersion can be formulated for delivery by injection, or for topical application or for subcutaneous administration.

[0252] Methods for delivering active agents using LLC lipid carriers

[0253] In a fifth aspect, a method for delivering an active agent to a biological system is provided, the method comprising the step of administering to the biological system the LLC lipid carrier according to the first aspect, the LLC lipid nanoparticle composition according to the second aspect, or the composition according to the third aspect or the fourth aspect.

[0254] In a sixth aspect, a method for controlled release of an active agent is provided, the method comprising the following steps: forming an LLC lipid carrier according to the first aspect, an LLC lipid nanoparticle composition according to the second aspect, or a composition according to the third aspect or the fourth aspect, and applying the lipid carrier or the composition to a biological system comprising a target area with a predefined pH, thereby achieving preferential release of the active agent at the target area.

[0255] As discussed, the loaded LLC lipids disclosed herein can provide a desired release profile for the host active agent due to the active agent being trapped within the complex internal architecture of the non-lamellar lyotropic liquid crystal phase at ambient pH, such as at physiological pH between about 7.4 (e.g., between 7.35 and 7.45).

[0256] The target region can be any region to which the active agent is desired to be delivered. The target region can be within a biological sample or tissue or fluid of a mammalian subject. For example, the target region can be a tumor, a tissue infected with a bacterial infection, or a tissue infected with a fungal infection. The target region can be a population of cells in a subject or cell culture. For example, the target region can be a subcellular organelle.

[0257] LLC lipid carriers containing active agents for therapeutic and diagnostic applications

[0258] In a seventh aspect, a method for treating or preventing a disease, disorder or condition in a mammal is provided, the method comprising administering to the mammal a therapeutically effective amount of the LLC lipid carrier according to the first aspect, the LLC lipid nanoparticle composition according to the second aspect, or the composition according to the third aspect or the fourth aspect.

[0259] In an eighth aspect, there is provided the LLC lipid carrier according to the first aspect, the LLC lipid nanoparticle composition according to the second aspect, or the composition according to the third aspect or the fourth aspect for use in treating or preventing a disease, disorder or condition.

[0260] In the ninth aspect, provided is a use of the LLC lipid carrier according to the first aspect, the LLC lipid nanoparticle composition according to the second aspect, or the composition according to the third aspect or the fourth aspect in the preparation of a medicament for treating a disease, disorder or condition.

[0261] In the tenth aspect, a method for diagnosing a disease, disorder or condition in a mammal is provided, the method comprising the following steps: administering the LLC lipid carrier according to the first aspect, the LLC lipid nanoparticle composition according to the second aspect, or the composition according to the third aspect or the fourth aspect to the mammal or a biological sample obtained from the mammal to facilitate diagnosis of the disease, disorder or condition in the mammal, wherein the active agent is a labeled active agent.

[0262] As generally used herein, the terms "administering" or "administration" and the like describe the introduction of the relevant particles or compositions into a mammal, such as by a specific route or vehicle. The routes of administration can include topical, parenteral and enteral, including oral, buccal, sublingual, nasal, anal, gastrointestinal, subcutaneous, intramuscular and intradermal routes of administration, but are not limited thereto. The compounds, compositions and methods of the present invention can be used for both delivery to a specific site of administration or systemic delivery.

[0263] "Treat", "treatment" or "treating" means administering the relevant particles or compositions to a subject to at least improve, reduce or inhibit existing signs or symptoms of a disease, disorder or condition experienced by the subject to the extent that the disease, disorder or condition is improved according to clinically acceptable standard medical conditions. For example, "treating a bacterial infection or a fungal infection" means reducing the infection, or eradicating the infection, or alleviating the symptoms of the infection in the patient, wherein the improvement and relief are evaluated using clinically acceptable standardized tests and / or empirical tests (including swab sample tests, etc.).

[0264] "Prevent," "prevention," or "preventing" means the prophylactic administration of the relevant particles or compositions to a subject who does not exhibit signs or symptoms of a disease, disorder, or condition but who is expected or predicted to exhibit such signs or symptoms in the absence of prevention. Prophylactic treatment may at least alleviate or partially ameliorate the expected symptoms or signs.

[0265] As used herein, "effective amount" or "therapeutically effective amount" refers to the amount of the relevant particles or compositions administered that is sufficient to prevent the occurrence of symptoms of the condition being treated, or to stop the worsening of symptoms, or to treat and alleviate symptoms, or at least reduce the severity of symptoms. The effective amount will vary with the patient's age, sex, weight, etc. in a manner that is understood by those skilled in the art. The appropriate dose or dosage regimen can be determined by routine experimentation or based on the current treatment regimen for delivering the active agent via the particles of the first aspect or the second aspect.

[0266] As used herein, the term "subject" or "individual" or "patient" may refer to any subject for which therapy is desired, specifically a vertebrate subject, and even more specifically a mammalian subject. Suitable vertebrates include, but are not limited to, primates, avian species, livestock animals (e.g., sheep, cows, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs), and captive wild animals (e.g., foxes, deer, wild dogs). Preferred subjects are those who need to treat a disease, disorder, or condition as described herein. However, it should be understood that the aforementioned terms do not mean that symptoms must exist. In one embodiment, the subject is a person who is treated for a bacterial infection or a fungal infection (specifically, a gram-negative bacterial infection).

[0267] As used herein, the terms "co-therapy" and "combination therapy" shall mean the treatment of a subject in need thereof by any suitable means, simultaneously, sequentially, separately or in a single pharmaceutical formulation or in combination, with one or more particles or compositions as described herein and one or more agents for treating a disease, disorder or condition. When administered in separate dosage forms, the number of doses administered per day for each compound may be the same or different. The relevant particles or compositions and one or more active agents for treating a disease, disorder or condition may be administered by the same or different routes of administration.

[0268] As discussed above, the LLC lipid carrier according to the first aspect may comprise an active agent, which may be pharmaceutically active. The lipid carrier may comprise two or more different pharmaceutically active agents, for example, where the agents are effective against the same or different targets. The agents may have synergistic activity. For example, in the treatment of multi-strain bacterial infections, or in targeting tumor cells expressing two or more different molecular targets, it may be advantageous to deliver different agents.

[0269] The LLC lipid carriers (e.g., LLC lipid nanoparticles) disclosed herein are particularly suitable for controlled delivery of active agents, which may be pharmaceutically active or cosmetically active. Thus, the present disclosure also provides methods for treating, preventing or diagnosing a disease, disorder or condition, the method comprising administering to a subject an LLC lipid carrier (e.g., LLC lipid nanoparticle) disclosed herein, an LLC lipid nanoparticle composition disclosed herein, or a cosmetic or pharmaceutical composition disclosed herein.

[0270] As discussed above, the LLC lipid carriers disclosed herein can undergo a phase change in response to a pH decrease from a physiological pH of about 7.4 (e.g., 7.35 to 7.45) to a pH between 7 and 5. This reduced pH range can be found in environments including tumors, infection sites of certain bacteria and fungi, and subcellular organelles, and can trigger a phase change to the disclosed non-lamellar lyotropic liquid crystal phase. The phase change, in turn, can promote the release of the encapsulated active agent.

[0271] Thus, in some embodiments, a disease, disorder or condition suitable for treatment, prevention or diagnosis by the methods disclosed herein is cancer or a bacterial infection or a fungal infection.

[0272] In some embodiments, the disease, disorder or condition suitable for treatment, prevention or diagnosis by the methods disclosed herein is a tumor or other malignancy. As used herein, cancer, tumor and malignancy refer to a disease, disorder or condition characterized by aberrant or abnormal cell proliferation, differentiation and / or migration, or cells or tissues associated with the disease, disorder or condition, which are usually accompanied by an aberrant or abnormal molecular phenotype, and the aberrant or abnormal molecular phenotype includes one or more gene mutations or other genetic changes associated with tumorigenesis, expression of tumor markers, loss of expression or activity of tumor suppressors and / or aberrant or abnormal cell surface marker expression. In general embodiments, cancer, tumor and malignancy may include sarcomas, lymphomas, leukemias, solid tumors, blastomas, gliomas, carcinomas, melanomas and metastatic cancers, but are not limited thereto. A more comprehensive list of cancers, tumors and malignancies can be found on the National Cancer Institutes website.

[0273] Found at http: / / www.cancer.gov / cancertopics / types / alphalist.

[0274] In some embodiments, the disease, disorder or condition is a disease, disorder or condition of bacterial infection. In one embodiment, the disease, disorder or condition is caused by or associated with a pathogen. The pathogen can be a bacterium or fungus that can infect a mammal.

[0275] In embodiments where the infection amenable to treatment, prevention, or diagnosis by the methods disclosed herein is a Gram-negative infection, the bacteria can be selected from the group consisting of Enterobacteriaceae, Pseudomonas, Vibrio, Campylobacter, Legionella, Neisseria, Hemophilus, and Bartonella.

[0276] In an embodiment, the Gram-negative bacteria may be selected from the group consisting of E. coli, Pseudomonas aeruginosa, Klebsiella, Acinetobacter baumannii, Neisseria gonorrhoeae, and Enterobacteriaceae.

[0277] In embodiments where the infection suitable for treatment, prevention, or diagnosis by the methods disclosed herein is a Gram-positive infection, the bacteria can be selected from the group consisting of Staphylococci, Streptococci, Pneumococci, Enterococci, Bacilli, Clostridia, Corynebacterium, Listeria, and Actinomyces.

[0278] Non-limiting examples of pathogenic bacteria include Staphylococcus aureus, Helicobacter pylori, Bacillus anthracis, Bordetella pertussis, Corynebacterium diphtheriae, Clostridium tetani, Clostridium botulinum, Streptococcus pneumoniae, Streptococcus pyogenes, Listeria monocytogenes, Hemophilus influenzae, Pasteurella multocida, Shigella dysenteriae, Mycobacterium tuberculosis, Mycobacterium leprae, leprae, Mycoplasma pneumoniae, Mycoplasma hominis, Neisseria meningitidis, Neisseria gonorrhoeae, Rickettsia rickettsii, Legionella pneumophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Propionibacterium acnes, Treponema pallidum, Chlamydia trachomatis, Vibrio cholerae, Salmonella typhimurium, Salmonella typhi, Borrelia burgdorferi, and Yersinia pestis, but are not limited thereto.

[0279] Non-limiting examples of fungi include Candida species and Aspergillus species, but are not limited thereto. In an embodiment, the fungi associated with or causing a disease, disorder or condition are selected from the group consisting of: C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. dubliniensis, C. krusei, C. lusitaniae, C. Auris and A. fumigatus.

[0280] As discussed above, the phase change of LLC lipid carrier disclosed herein and the relevant release of the encapsulated active agent can occur in subcellular (intracellular) organelles. Generally, the delivery of therapeutic nucleic acid depends on the delivery of nucleic acid to the cell in which its mode of action is exerted. Therefore, any condition suitable for therapy by oligonucleotides or nucleic acids can be treated by LLC lipid carriers comprising active agents disclosed herein. Suitable oligonucleotides or nucleic acids can be selected from gene expression regulators, knockdown agents, small interfering RNA (siRNA), RNA interference (RNAi), mRNA, DNA, antisense oligonucleotides, functional nucleic acids such as ribozymes, aptamers and mirror image isomers (spiegelmer), dicer substrates, microRNA (miRNA) and short hairpin RNA (shRNA).

[0281] In the case where the active agent is a labeled active agent, the lipid carrier according to the second aspect comprising the active agent or the composition according to the third aspect or the fourth aspect comprising the active agent can be used to detect or diagnose the diseases, disorders or conditions discussed above.

[0282] Typically, detection or diagnosis will be performed in a mammal or cell population.

[0283] In embodiments, the labeled active agent is a contrast agent.

[0284] The following numbered embodiments are disclosed herein.

[0285] Example 1: A lipid compound of formula (I):

[0286] Cyc-LR

[0287] (I)

[0288] Wherein Cyc is a nitrogen heterocycle or a heteroaryl;

[0289] L is an amido linker; and

[0290] R is a C10 to C44 carbon chain.

[0291] Embodiment 2: The lipid compound according to embodiment 1, wherein Cyc is selected from a 5-membered or 6-membered nitrogen heterocyclic group or a heteroaryl group.

[0292] Embodiment 3: The lipid compound according to embodiment 1 or embodiment 2, wherein Cyc is selected from the group consisting of piperidinyl, piperazinyl, pyrrolinyl, pyrrolidinyl, pyrazolidinyl, morpholinyl, imidazolinyl, imidazolidinyl, pyrrolinyl, pyrazolinyl, thiazolinyl, thiomorpholinyl, tetrahydropyridinyl, dihydropyridinyl, tetrahydropyrimidinyl, pyrimidinyl, pyridinyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, oxadiazolyl, pyrazinyl, tetrazolyl, thiazolyl, isoxazolyl, isothiazolyl, isoxazolone, triazolyl, oxadiazolyl, thiadiazolyl and pyridazinyl;

[0293] Embodiment 4: The lipid compound according to any one of the preceding embodiments, wherein L comprises an optional carbon chain located between Cyc and the amide group.

[0294] Embodiment 5: A lipid compound according to embodiment 4, wherein the carbon chain between Cyc and the amide group, when present, is a C1 to C6 chain.

[0295] Embodiment 6: The lipid compound according to any one of the preceding embodiments, wherein R is a C10 to C36 alkyl, alkenyl or alkynyl chain.

[0296] Embodiment 7: The lipid compound according to any one of the preceding embodiments, wherein R is a C12 to C24 alkyl or alkenyl chain.

[0297] Embodiment 8: The lipid compound according to any one of the preceding embodiments, wherein R is a C12 to C18 alkenyl chain.

[0298] Embodiment 9: The lipid compound according to any one of the preceding embodiments, wherein R is optionally interrupted by one or more heteroatoms.

[0299] Embodiment 10: The lipid compound according to any one of the preceding embodiments, wherein the amino lipid compound of formula (I) is a compound of formula (Ib):

[0300]

[0301] Wherein Cyc is a nitrogen heterocycle or a heteroaryl;

[0302] R is a C12 to C24 alkyl or alkenyl chain; and

[0303] n is an integer of 1 to 6.

[0304] Embodiment 11: The lipid compound according to embodiment 10, wherein Cyc is a 6-membered nitrogen heterocycle or a heteroaryl group.

[0305] Embodiment 12: The lipid compound according to embodiment 11, wherein Cyc is selected from the group consisting of pyridyl, pyrimidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydropyridinyl, dihydropyridinyl, tetrahydropyrimidinyl, pyrazinyl and pyridazinyl.

[0306] Embodiment 13: The lipid compound according to embodiment 12, wherein Cyc is selected from the group consisting of pyridyl, pyrimidinyl, piperidinyl, piperazinyl, morpholinyl and pyrazinyl.

[0307] Embodiment 14: The lipid compound according to any one of embodiments 10 to 13, wherein n is an integer from 1 to 4.

[0308] Embodiment 15: The lipid compound according to any one of embodiments 10 to 14, wherein R is a C12 to C24 alkenyl group.

[0309] Embodiment 16: The lipid compound according to embodiment 14, wherein R is C14 to C22 alkenyl.

[0310] Embodiment 17: The lipid compound according to any one of the preceding embodiments, wherein R is selected from the group consisting of oleyl, linoleoyl, linolenoyl, phytanoyl and farnesoyl.

[0311] Embodiment 18: The lipid compound according to any one of embodiments 10 to 17, wherein R is optionally interrupted by one or more heteroatoms.

[0312] Embodiment 19: The lipid compound according to any one of the preceding embodiments, wherein the lipid compound is selected from the group consisting of:

[0313]

[0314] wherein R is as defined in any one of the preceding embodiments.

[0315] Embodiment 20: The lipid compound according to embodiment 19, wherein the lipid compound is selected from the group consisting of:

[0316]

[0317] Embodiment 21: The lipid compound according to any one of the preceding embodiments, wherein R is:

[0318]

[0319] Example 22: A lipid carrier comprising a lipid having an amido linker, the lipid carrier being adapted to undergo a mesophase transition when exposed to a decrease in pH.

[0320] Embodiment 23: The lipid carrier according to embodiment 22, further comprising a structured lipid compound, wherein the structured lipid compound is amphiphilic.

[0321] Embodiment 24: The lipid carrier according to embodiment 23, wherein the structured lipid compound comprises a hydrophobic tail group, and the hydrophobic tail group is selected from the group consisting of: oleyl, linoleoyl, linolenoyl, phytanoyl, farnesoyl or an extended aliphatic hydrophobic.

[0322] Embodiment 25: The lipid carrier according to embodiment 24, wherein the hydrophobic tail group is selected from oleyl, linoleoyl and phytanoyl.

[0323] Embodiment 26: The lipid carrier of Embodiment 24 or Embodiment 25, wherein the hydrophobic tail group is oleyl.

[0324] Embodiment 27: The lipid carrier according to any one of embodiments 23 to 26, wherein the structured lipid compound is glyceryl monooleate (glyceryl monooleate).

[0325] Embodiment 28: The lipid carrier according to any one of Embodiments 22 to 27, wherein the lipid carrier is a non-lamellar lyotropic liquid crystalline phase lipid carrier.

[0326] Embodiment 29: The lipid carrier according to Embodiment 28, wherein the non-lamellar lyotropic liquid crystal phase lipid carrier is a self-assembled non-lamellar lyotropic liquid crystal phase lipid carrier.

[0327] Embodiment 30: The lipid carrier according to Embodiment 28 or Embodiment 29, wherein the non-lamellar lyotropic liquid crystal phase lipid carrier is in the form of nanoparticles.

[0328] Embodiment 31: The lipid carrier according to any one of Embodiments 28 to 30, wherein the non-lamellar lyotropic liquid crystal phase lipid carrier further comprises a stabilizer.

[0329] Embodiment 32: The lipid carrier of Embodiment 31, wherein the stabilizer is a stabilizing polymer.

[0330] Embodiment 33: The lipid carrier of Embodiment 32, wherein the stabilizing polymer is a nonionic triblock copolymer.

[0331] Embodiment 34: The lipid carrier according to Embodiment 32 or Embodiment 33, wherein the stabilizing polymer is Pluronic F127 (Poloxamer 407) or Poloxamer 80.

[0332] Embodiment 35: The lipid carrier according to any one of embodiments 22 to 34, wherein the lipid having an amido linker is present in a suitable wt % of the total lipid content of the lipid carrier to provide an intermediate phase transition at a selected pH.

[0333] Embodiment 36: The lipid carrier according to any one of embodiments 22 to 35, wherein the lipid having an amido linker accounts for about 5% to about 50 wt% of the total lipid content of the lipid carrier.

[0334] Embodiment 37: The lipid carrier according to any one of embodiments 22 to 36, wherein the lipid having an amido linker is a lipid compound according to any one of embodiments 1 to 21.

[0335] Embodiment 38: The lipid carrier according to any one of Embodiments 28 to 37, wherein the non-lamellar lyotropic liquid crystal phase is a hexagonal or cubic non-lamellar lyotropic liquid crystal phase.

[0336] Embodiment 39: The lipid carrier of Embodiment 38, wherein the non-lamellar lyotropic liquid crystal phase is a hexagonal non-lamellar lyotropic liquid crystal phase at a pH above about pH 8.

[0337] Embodiment 40: The lipid carrier of Embodiment 38 or Embodiment 39, wherein the non-lamellar lyotropic liquid crystal phase is a cubic non-lamellar lyotropic liquid crystal phase at a pH below about pH 7.5.

[0338] Embodiment 41: A lipid carrier according to Embodiment 38 to Embodiment 40, wherein the non-lamellar lyotropic liquid crystal phase is a hexagonal non-lamellar lyotropic liquid crystal phase at a pH above about pH 8, and is a cubic non-lamellar lyotropic liquid crystal phase at a pH below about pH 7.5.

[0339] Embodiment 42: A lipid carrier according to Embodiment 38 or Embodiment 39, wherein the non-lamellar lyotropic liquid crystal phase is a hexagonal non-lamellar lyotropic liquid crystal phase at a pH above about pH 7.5 and a cubic non-lamellar lyotropic liquid crystal phase at a pH below about pH 7.

[0340] Embodiment 43: A lipid carrier according to Embodiment 38 or Embodiment 39, wherein the non-lamellar lyotropic liquid crystal phase is a hexagonal non-lamellar lyotropic liquid crystal phase at a pH above about pH 7, and is a cubic non-lamellar lyotropic liquid crystal phase at a pH below about pH 6.5.

[0341] Embodiment 44: A lipid carrier according to Embodiment 35 to Embodiment 37, wherein the non-lamellar lyotropic liquid crystal phase is a hexagonal non-lamellar lyotropic liquid crystal phase at a pH above about pH 6.5, and is a cubic non-lamellar lyotropic liquid crystal phase at a pH of about pH 6.5 or below.

[0342] Embodiment 45: The lipid carrier according to any one of Embodiments 22 to 44, further comprising an active agent.

[0343] Embodiment 46: The lipid carrier of Embodiment 45, wherein the active agent is pharmaceutically active or cosmetically active.

[0344] Embodiment 47: The lipid carrier of Embodiment 45 or Embodiment 46, wherein the active agent is selected from the group consisting of peptides, proteins, enzymes, small molecule drugs and nucleic acids.

[0345] Embodiment 48: According to any one of embodiments 45 to 47, the active agent is selected from the group consisting of: radionuclides, imaging agents, polymers, antibiotics, fungicides, metal-containing nanoparticles, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones, growth factors, steroid agents, gene expression regulators, knockdown agents, siRNA, RNAi agents, mRNA, DNA, dicer substrates, miRNA, shRNA, antisense oligonucleotides, aptamers and microbial toxins.

[0346] Embodiment 49: The lipid carrier according to any one of embodiments 45 to 48, wherein the active agent is a topoisomerase I inhibitor.

[0347] Embodiment 50: The lipid carrier according to embodiment 49, wherein the topoisomerase I inhibitor is selected from camptothecin, irinotecan and SN-38.

[0348] Embodiment 51: A cosmetic composition comprising the lipid carrier according to any one of embodiments 22 to 46 and a cosmetically acceptable carrier, diluent and / or excipient.

[0349] Embodiment 52: A pharmaceutical composition comprising the lipid carrier according to any one of Embodiments 22 to 50 and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0350] Embodiment 53: The cosmetic composition according to Embodiment 51 or the pharmaceutical composition according to Embodiment 52, wherein the composition is a composition for injection.

[0351] Embodiment 54: The cosmetic composition according to Embodiment 51 or the pharmaceutical composition according to Embodiment 52, wherein the composition is a composition for topical administration.

[0352] Embodiment 55: The cosmetic composition according to Embodiment 51 or the pharmaceutical composition according to Embodiment 52, wherein the composition is a composition for subcutaneous administration.

[0353] Embodiment 56: A method of delivering an active agent to a biological system, the method comprising the step of administering to the biological system the lipid carrier of any one of Embodiments 45 to 50 or the composition of any one of Embodiments 51 to 55.

[0354] Embodiment 57: The method of Embodiment 56, wherein delivery of the active agent into the biological system is regulated by the response of the lipid carrier to a predefined pH range.

[0355] Embodiment 58: The method of embodiment 57, wherein the response comprises a phase structure transition of the lipid carrier.

[0356] Embodiment 59: The method according to embodiment 58, wherein the phase structure transition is a transition from a hexagonal phase structure to a cubic phase structure of the lipid carrier.

[0357] Embodiment 60: The method of Embodiment 59, wherein the conversion occurs at a pH below about pH 8.

[0358] Embodiment 61. The method of Embodiment 59 or Embodiment 60, wherein the conversion occurs at a pH of about pH 7.5 or below.

[0359] Embodiment 62: The method of any one of Embodiments 59 to 61, wherein the conversion occurs at a pH of about pH 7 or below.

[0360] Embodiment 63: The method of any one of Embodiments 59 to 62, wherein the conversion occurs at a pH of about pH 6.5 or below.

[0361] Embodiment 64: The method of any one of Embodiments 56 to 63, wherein the response provides for preferential release of the active agent in the biological system at the predefined pH range.

[0362] Embodiment 65: A method for controlled release of an active agent, the method comprising the steps of forming a lipid carrier according to any one of Embodiments 45 to 50 or a composition according to any one of Embodiments 51 to 55, and applying the lipid carrier or the composition to a biological system comprising a target area with a predefined pH, thereby achieving preferential release of the active agent at the target area.

[0363] Embodiment 66: The method according to Embodiment 65, wherein the lipid carrier undergoes a mesophase structural transition in response to the predefined pH.

[0364] Embodiment 67: The method according to Embodiment 66, wherein the phase structure transition is a transition from a hexagonal phase structure to a cubic phase structure of the lipid carrier.

[0365] Embodiment 68: A method of treating or preventing a disease, disorder or condition in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the lipid carrier of any one of Embodiments 45 to 50 or the composition of any one of Embodiments 51 to 55.

[0366] Embodiment 69: The lipid carrier according to any one of Embodiments 45 to 50 or the composition according to any one of Embodiments 51 to 55 for use in treating or preventing a disease, disorder or condition.

[0367] Embodiment 70: Use of the lipid carrier of any one of Embodiments 45 to 50 or the composition of any one of Embodiments 51 to 55 in the preparation of a medicament for treating a disease, disorder or condition.

[0368] Embodiment 71: A method for diagnosing a disease, disorder or condition in a mammal, the method comprising the following steps: administering a lipid carrier according to any one of Embodiments 45 to 50 or a composition according to any one of Embodiments 51 to 55 to the mammal or a biological sample obtained from the mammal to facilitate diagnosis of the disease, disorder or condition in the mammal, wherein the active agent is a labeled active agent.

[0369] Embodiment 72: The method of embodiment 68 or embodiment 71, the lipid carrier for use or the pharmaceutical composition of embodiment 69, or the use of embodiment 70, wherein the disease, disorder or condition is cancer or a bacterial infection or a fungal infection.

[0370] Embodiment 73: The lipid carrier according to any preceding embodiment, wherein the lipid carrier itself has a water content of between about 20 wt% and about 60 wt%.

[0371] The following experimental section describes in more detail the characterization of certain compounds of the present invention and their efficacy. The purpose is to illustrate certain specific embodiments of the compounds of the present invention and their efficacy, but not to limit the present invention in any way.

[0372] Examples

[0373] The present disclosure will now be further described with reference to the following non-limiting examples and with reference to the accompanying drawings.

[0374] Material

[0375] 4-(Aminomethyl)pyridine, methylpyridinamine, 4-(2-aminoethyl)morpholine, 1-(2-aminoethyl)piperidine, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), hydroxybenzotriazole (HOBt), dimethylaminopyridine (DMAP), dichloromethane (DCM), n-hexane, ethyl acetate, sodium sulfate, sodium chloride, deuterated chloroform, deuterated dimethyl sulfoxide, deuterium oxide, and Pluronic F-127 were purchased from Sigma-Aldrich. Milli-Q water (18.2 18.2 MΩ·cm) was used for all aqueous formulations. Monoolein (MO) and oleic acid (OA) were obtained with purity >99% from Nu-chek-Prep, Inc (Elysian, MN, USA). SN-38 was purchased from Adooq bioscience.

[0376] Example 1: Synthesis, purification and analysis of amino lipids

[0377] The amino lipids disclosed herein comprise a hydrophilic head group comprising a nitrogen heterocycle or a heteroaryl group, conjugated to a hydrophobic carbon chain tail by an amide linker. In the following examples, a C18-oleyl tail was used, and the C18-oleyl tail was kept constant for all synthetic lipids, but the head group and length of the amide linker were varied.

[0378] Amino lipids were synthesized by amide coupling reaction between oleic acid (OA) and the amine of the head group. EDC (3.9mmol, 1.1 equivalent) was added to the cooled OA solution (3.54mmol, 1.0 equivalent) in DCM. The solution was stirred for 30 minutes at 0-5°C, and amine (3.54mmol, 1.0 equivalent) and DMAP (0.7mmol, 0.2 equivalent) were added to the cooled reaction mass. The reaction temperature was slowly raised to room temperature (RT), and stirred at room temperature for 24 hours. The solvent was removed using a rotary evaporator, and a fast silica gel column was used to purify the crude material obtained. The synthesized amino lipids were subjected to nuclear magnetic resonance (NMR) imaging ( 1 H) Analysis. Bruker 300 Ultrashield TM NMR spectrometer acquisition 1 H NMR spectrum.

[0379] result:

[0380] The physical state of the amino lipid synthesized is semisolid at room temperature. Generally speaking, the productive rate of reaction is in the scope of 60-75%, and purity is 85-95%. The structure and purity of the amino lipid synthesized are confirmed by NMR (data not shown). The exemplary structure synthesized by the method is shown in Figure 2 middle.

[0381] The following amino lipids were used or mentioned in the examples below:

[0382]

[0383] Example 2: Preparation of LLC Nanoparticles Containing Amino Lipids

[0384] LLC lipid carriers disclosed herein can be in bulk phase, or can be dispersed in the form of nanoparticles comprising amino lipids and structural amphipathic lipids disclosed herein. The structural amphipathic lipids can constitute the bulk of the nanoparticles, depending on the amount of amino lipids used. In the following examples, monoolein (MO) is used as the structural amphipathic lipid.

[0385] LLC nanoparticles composed of amino lipids and MO were formulated using a high-throughput method as reported in Tran et al. 2016, which is incorporated herein by reference. The amount of amino lipid added to the formulation was determined by R MO Indicates that the R MO is defined as the wt / wt ratio of amino lipid to the total amount of lipid in the system and may be referred to herein as R ALBriefly, MO (20 mg / 1 mL ethanol) and amino lipids (20 mg / 1 mL ethanol) were mixed at increasing amino lipid ratios of 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 60:40, 50:50 to obtain R values ​​of 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, and 0.5. MO Value. 1 mL of lipid solution was added to each well of a 96-well deep well block (Greiner Bio-One, Interpath Inc., Australia). The organic solvent was then removed using a centrifugal evaporator (GeneVac, NSW, Australia) to obtain a dried lipid mixture.

[0386] In these examples, Pluronic F-127 was used to stabilize the nanoparticles. Therefore, 1 mL of F127 (20 mg in 1 mL of DI water) was added to the dried lipid mixture. The resulting mixture was sonicated by a high-throughput multi-probe sonicator (QSonica) to obtain an opaque dispersion.

[0387] Nanoparticles can also be prepared using mixtures of two or more amino lipids to fine tune the pH responsiveness of the nanoparticles.

[0388] result:

[0389] Various amounts of amino lipids from 5 wt% to 50 wt% were added to MO and dispersed with Pluronic F-127. The amount of Pluronic F-127 was kept at 10 wt% of the total amount of lipids in the system. These formulations dispersed well with no visible precipitation or phase separation.

[0390] Example 3: Particle size and polydispersity index of LLC lipid nanoparticles

[0391] The average hydrodynamic diameter and polydispersity index (PDI) of lipid nanoparticles were measured by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS (Malvern Instruments, UK). All measurements were performed at 25°C (n=3) by loading the lipid formulations (10 μL) into a clear 96-well half-area polystyrene plate and diluting with 190 μL milli-Q water (Zhai et al. 2015).

[0392] result

[0393] The average particle size and polydispersity index (PDI) of the above nanoparticle formulations were investigated. The obtained values ​​are presented in Tables 1 and 2, respectively.

[0394] Table 1

[0395]

[0396] Table 2

[0397]

[0398] Tables 1 and 2 show the particle size and polydispersity index (PDI) data of nanoformulations prepared by adding amino lipids to MO dispersed with the stabilizer Pluronic F-127. The amount of amino lipid added to the formulation is determined by R MO Indicates that the R MO is defined as the wt / wt ratio of amino lipids to total lipids and may be referred to herein as R AL Measurements were averaged from triplicates and results are reported as mean + / - standard deviation.

[0399] In general, the hydrodynamic diameter of the nanoparticles ranges from 137nm to 300nm, and the PDI ranges from 0.15 to 0.40 (Tables 1 and 2). In order to evaluate the stability of the nanoparticles at room temperature, the particle size of the nanoparticles was also measured when they were left standing at room temperature for 30 days after preparation. No significant differences in size or physical appearance were observed, which indicates that the nanoparticles are stable at room temperature for at least 30 days (data not shown).

[0400] Example 4: Phase behavior of LLC lipid nanoparticles

[0401] High throughput formulation and Synchrotron small angle X-ray scattering (SAXS) were used to determine the effects of amino lipid concentration and pH on the nanoparticle mesophase.

[0402] Nanoparticle preparations (50 μL) were loaded into transparent 96-well half-area polystyrene plates (Greiner Biotech, Interpath, VIC, Australia), and 50 μL of a suitable buffer solution (citrate buffer) of a specified pH was added to the plates. The phase behavior of the nanoparticles was characterized at the SAXS / WAXS beamline at the Australian Synchrotron. The instrument used The X-ray wavelength is 11.0 keV and the typical flux is about 10 photons / second. The sample-to-detector distance is chosen to be 1.6 m, which provides The q range (scattering vector q = 4πsin(θ / 2) / λ, where θ is the scattering angle and λ is the wavelength) of the samples was obtained at 25°C and 37°C following the procedure reported in the literature. The plate was placed on a holder perpendicular to the X-ray beam. The sample holder was moved by a motor controlled by the in-house developed ScatterBrain software for automated SAXS screening. The sample temperature was controlled by a circulating water bath and maintained at 25°C or 37°C. Two-dimensional X-ray diffraction images were recorded using ScatterBrain on a Decris-Pilatus 1-M detector. The scattering images were integrated into a one-dimensional plot of intensity versus q for phase identification. The intermediate phases of the nanoparticles were assigned by matching the relative positions of the scattering peaks with the known patterns of bicontinuous cubic, hexagonal, lamellar, and micellar cubic phases. The lattice parameters of the nanoparticles were calculated using the IDL-based software AXcess as described (Tran et al. 2018; Tran et al. 2016).

[0403] result

[0404] As detailed in Example 2 above, the synthesized amino lipids were prepared in 8 different compositions (R MO =0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5) were doped into MO and dispersed with Pluronic F-127 to prepare the lipid nanoparticles described herein. The mesophase structure of the drug-free formulation was determined by SAXS over ten different pH values ​​(pH range of 2.5 to 10). Figure 3 The SAXS partial phase diagram of these nanoparticles at 25°C is shown. The shape of the amphiphilic molecule can be characterized by the critical packing parameter (CPP). CPP = V / al, where V is the effective volume of the hydrophobic tail of the amphiphilic molecule, a is the effective cross-sectional area of ​​the head group, and l is the effective length of the hydrocarbon chain. The lamellar phase (L α ). For CPP values ​​greater than 1, as the negative interface curvature increases, inverse phases (such as Q2 phase and H2 phase) are formed. The Q2 phase consists of lipid bilayers arranged in a periodic 3D structure by twisting the bilayer into the shape of an infinitely periodic minimal surface. The H2 is a closed extended micellar columnar structure in which there is no direct contact between the water inside the H2 phase and the water outside.

[0405] High-throughput SAXS experiments demonstrated that both amino lipid composition and pH affect the internal mesophase of the nanoparticles, as discussed below.

[0406] (a) Effect of adding amino lipids to MO nanoparticles at neutral pH

[0407] In the case of MO, the head group size is relatively small, leading to the formation of an inverse bicontinuous cubic phase with negative surface Gaussian curvature in an aqueous environment. Previous studies have shown that MO nanoparticles stabilized with Pluronic F-127 exhibit a pristine cubic phase (Im3m space group) at room temperature (Tran et al. 2015; Sarkar et al. 2018). At neutral pH, the addition of increasing amounts of amino lipids to MO nanoparticles resulted in changes in the mesophase structure in the order of bicontinuous cubic phase → hexagonal phase → inverse micellar phase (Q2 → H2 → L2) for lipid 10, lipid 11, and lipid 13. The sequential changes can be reasonably explained by changes in the effective molecular critical packing parameter (CPP). At neutral pH, the addition of lipid 10, lipid 11, and lipid 13 to MO can further increase the effective CPP, thereby inducing a larger curvature of the lipid membrane. MO has a 2-OH (hydroxyl) group as part of its head group, and the hydroxyl group can also participate in hydrogen bonding with neighboring water molecules, making the effective head group area of ​​MO larger than that of lipids 10, lipids 11 and lipids 13.

[0408] For nanoparticles doped with lipid 10, the MO = 0.4 at pH 7, the transition from Q2 to H2 occurs. For the nanoparticles doped with lipid 11, at R MO When =0.4, L2 phase was observed.

[0409] The lipids 10 and 11 prepared herein can be compared to comparator lipids, namely lipids 1 and lipid 2, which have ester rather than amide linkers (Rajesh et al. 2021, the contents of which are incorporated herein by reference). The amino lipids disclosed herein contain an amide group in the linker, while the comparator lipids disclosed are ester-containing amino lipids. Interestingly, for MO nanoparticles doped with lipid 10, at R MO Much higher R MO At neutral pH, the transition from Q2 to H2 occurs. For the amide-containing amino lipids of the present invention, at much higher R MO The fact that the transition from Q2 to H2 occurs at pH 7 is most likely due to the hydrophilicity of the amide bond. The partition coefficient P is defined as the concentration ratio of a compound between the organic phase and the aqueous phase, and the logP value can be used as a measure of lipophilicity. The predicted logP values ​​of the comparator lipid 1 and the currently disclosed lipid 10 are 6.95 and 6.12, respectively (Chemdraw software), which indicates that the comparator amino lipid, i.e., lipid 1, is more lipophilic.

[0410] For lipid 12, the opposite trend was observed, such that with increasing amounts of lipid 12, the change was from bicontinuous cubic mesophase → (mixed cubic + sponge) mesophase → sponge (Q2 → Q2 + L3 → L3) mesophase. The L3 phase is usually found between the L a The L3 phase is in a narrow region between the Q2 phase and the L3 phase, and represents a "molten" cubic phase structure without long-range order. Due to the absence of a periodic structure, the SAXS diffraction pattern of the L3 phase contains only a single broad scattering peak. It was observed that the sponge phase coexists with the Q2 phase, and adding more lipid 12 produces a single sponge phase. This difference is attributed to the chemical structure of lipid 12, i.e., the amide linker attached to the hydrophilic morpholine makes the effective head group area larger than the effective head group area of ​​MO. Adding additives that are more polar than MO or have a head group size larger than the head group size of MO can further reduce the effective CPP, thereby inducing a smaller curvature of the lipid membrane.

[0411] (b) Effect of pH on the mesophase structure of amino lipid-containing MO nanoparticles

[0412] For each system, the pH was varied from pH 2 to pH 8, and synchrotron radiation center SAXS was used to determine the effect of pH on the mesophase structure of MO nanoparticles doped with the four different amino lipids studied. The SAXS results of the nanoparticles stabilized by F-127 are presented in Figure 3 In. MO nanoparticles stabilized by F-127 exhibited a pristine cubic phase independent of the pH between pH 2.5 and pH 8 (data not shown). In general, as the pH decreases, the intermediate phase of the amino lipid-doped MO nanoparticles transforms into a structure with a lower interface curvature. This observation confirms that the ionization state of the amino lipid plays a crucial role in the pH responsiveness of MO nanoparticles. The head group containing the tertiary amine portion obtains protons at low pH and obtains a positive charge generated by protonation. This combined with the electrostatic repulsion of the ionized head group will produce a larger effective head group size and a smaller CPP. At higher pH, according to the apparent pKa of the amino lipid, the head group of the amino lipid remains unionized and therefore has a small effective head group (larger CPP). Therefore, in the self-assembled lipid microenvironment, the reduction in pH leads to a transition from a high CPP to a low CPP, or in other words, a phase transition sequence of L2→H2→Q2 that reduces the interface curvature, such as Figure 4 As summarized. Figure 4 As presented, at pH-7, the observed phase transition was Q2→H2→L2 upon addition of lipids 10, 11, or lipid 13, and a phase transition of Q2→Q2+L3→L3 was observed for nanoparticles doped with lipid 12. Changing the pH value resulted in a decrease in the interfacial curvature, and the transition trend followed the order of L2→H2→Q2.

[0413] The ionizable head group part of amino lipid resides at lipid-water interface. In the above nanoparticle formulations prepared in each of the type and amount of amino lipids in different cases, each of the above nanoparticle formulations prepared is unique, and therefore can have different apparent pKa due to pH and ionic strength being changed. This apparent pKa is mainly affected by the surface charge density at the interface solvation medium effect and the weak base interface. The surface charge density at the interface will be managed by head group ionization degree (pH effect) and ionic strength (electrostatic shielding effect) (Drummond et al., 1989).

[0414] The pH range in which the phase transition occurs depends on the molecular structure of the amino lipid and its doping level in the MO nanoparticles. For lipid 10, in the concentration range R MO The expected phase transition from H2 at a higher pH of 7.5 to Q2 at pH 5.5 was observed at pH = 0.4-0.5, and a further decrease in pH to 4.0 resulted in liposome formation. MO = 0.4-0.5, a transition from L2 to Q2 was observed at pH 3.0. For lipid 12, lowering the pH produced a mixed L3 and weekly ordered Q2, and further lowering the pH became L α For lipid 13, in R MO = 0.2-0.3, the expected H2→Q2 transition was observed at pH 7.0, and the system appears to be very sensitive, with the change from H2 to Q2 observed within a 0.5 pH unit difference. It should be noted that for the system with lipid 13, there are large regions in the phase diagram where the intermediate phase cannot be determined (denoted as N / D). For these samples, the scattering peaks are too weak or not found, thus hindering phase identification.

[0415] The results show that the ionizable amino lipids studied promote the formation of H2 phases in MO nanoparticles at neutral pH. However, at acidic pH, amino lipids are gradually protonated, and the electrostatic repulsion at the lipid-water interface among the protonated head groups reduces the surface curvature. It is believed that the result is that the reduction in pH triggers phase transitions, thereby converting hexagons into cubes under acidic conditions. In exemplary four amino lipid-MO nanoparticle systems, two of them (i.e. lipid 10 and lipid 13) show pH-dependent H2→Q2 phase transitions at pH (pH 5.0-pH 6.5) (this is the pathologically relevant range of solid tumors).

[0416] (c) Effect of temperature on the mesophase structure of amino lipid-containing MO nanoparticles

[0417] The lyotropic liquid crystal phase is temperature dependent, and therefore to evaluate the fate of the nanoparticles at body temperature, SAXS experiments were also performed at 37°C to study and compare the phase behavior of the nanoparticles at 25°C and 37°C. For this purpose, R MO 0.4 of the formulation with lipid 10, which exhibits H2 at pH 7.5 and transforms to Q2 at lower pH. The one-dimensional SAXS spectra of the nanoparticles at pH 4.0 to pH 7.5 are shown in Figure 5 From the SAXS spectra, it is evident that temperature has a slight effect on the mesophase structure of the amino lipid-doped nanoparticles. The phase transition from H2 to Q2 is 0.5 pH units higher at 37 °C than at 25 °C. However, both systems are eligible for loading with active agents using drug SN-38 and were further investigated for drug loading, encapsulation efficiency (EE), and drug release studies.

[0418] Example 5: Preparation of LLC lipid nanoparticles containing active agents

[0419] LLC lipid carriers disclosed herein (such as LLC lipid nanoparticles disclosed herein) can contain active agents, such as chemotherapeutic drugs, cosmetically active molecules or nucleic acids. In the following examples, the potent anticancer drug SN-38 was used as an active agent.

[0420] The drug-containing formulation was prepared by first preparing a dried lipid mixture of MO and amino lipids described in Example 2 above. Solid SN-38 was added to the dried lipid mixture (20 mg). The resulting mixture was heated at 60 ° C overnight. The resulting mixture was mixed for 2 minutes using a vortex mixer, and then an aqueous solution of Pluronic F-127 (2 mg / ml) was added. The subsequent mixture was probe-ultrasonicated in pulse mode for 5 minutes (Ranneh et al., 2016).

[0421] result

[0422] In this example, 1 wt%, 2 wt%, 5 wt% or 10 wt% of SN-38, which accounts for the total amount of lipid mixture in the formulation, is added to a mixture containing lipid 10 (R MO =0.4) to prepare four formulations with different amounts of SN-38.

[0423] Visual inspection of the resulting formulations indicated that the formulations dispersed well, however some precipitation was observed, particularly at 5 wt% and 10 wt%.

[0424] Example 6: Particle Size and Polydispersity Index of LLC Lipid Nanoparticles Containing Active Agents

[0425] The physicochemical properties of SN-38-containing lipid nanoparticles prepared as described in Example 5 above were studied.

[0426] result

[0427] The particle size and polydispersity index (PDI) of each of the four lipid nanoparticle formulations as described in Example 3 above were studied and the results are shown in Figure 6 middle.

[0428] The particle size of the drug-loaded formulations ranged from 244-274 nm with a PDI of 0.19-0.26. In general, the particle size increased slightly gradually as more SN-38 was added to the system. Carsado et al. reported that the lipid / drug molar ratio strongly affected the size and amount of SN-38 within the vesicles and observed a significant increase in liposome size for their novel microfluidic liposome formulations for delivery of SN-38 (Carsado et al., 2018). However, in formulations with lipid 10, the average particle size increased by about 10-15%, and these were suitable for delivery of SN-38.

[0429] Example 7: Phase Behavior of LLC Lipid Nanoparticles Containing Active Agents

[0430] The mesophase structure of each of the SN-38-containing lipid nanoparticle formulations prepared as described in Example 5 was determined over a range of pH.

[0431] result

[0432] SAXS experiments were performed as described in Example 4 above to study the internal mesophase structure in response to a pH ranging from 2.5 to 8 at 25°C (data not shown) and 37°C. Figure 7 A partial phase diagram at 37°C is presented in and compared with that of a formulation without drug.

[0433] SAXS experiments were performed with drug loaded formulations to observe the effect of drug loading on the behavior of the intermediate phase and to compare the phase behavior with that of formulations without drug. Interestingly, it was observed that all drug loaded formulations behaved slightly differently than the control without drug. For formulations with 1 wt%, 2 wt%, and 5 wt%, the phase transition of H2→Q2 occurred at pH 5.0. For the drug loaded formulations with a 10 wt% ratio, a coexistence phase window of (X+H2) was observed at pH 5.5, where X was not an identifiable phase and was likely the weakly scattering Q2 phase. At pH 5.0, a complete transition to the Q2 phase occurred. The slightly higher pH of the H2→Q2 transition can be explained by studies conducted by Casado et al. on the interaction between the SN-38 drug and a model biomembrane, and reported that SN-38 was inserted into the hydrophobic core of the bilayer due to its highly hydrophobic nature and the drug was localized in the outer hydrophobic region of the bilayer (Carsado et al., 2018). Shifting the H2→Q2 phase transition pH upward by 0.5 pH units would be an additional advantage, and the results are encouraging because SN-38 can be inserted into the hydrophobic core of the mixed lipid bilayer without disrupting its structure or affecting the stability of the nanoparticles.

[0434] SAXS results showed that drug loading did not change the mesophase structure at neutral pH, and drug loading shifted the phase transition (H2→Q2) pH to 0.5 units higher than that of the control without drug.

[0435] Example 8: Drug Loading and Drug Encapsulation Efficiency of LLC Lipid Nanoparticles Containing Active Agents

[0436] The drug loading (DL) and encapsulation efficiency (EE) of each of the four formulations of Example 5 were determined using an HPLC method.

[0437] The drug-containing nanoparticles were centrifuged at 1000 × g for 25 min at 25 °C to remove any unencapsulated drug aggregates. The supernatant was collected and vortexed at 2500 rpm for 10 min to homogenize the distribution.

[0438] For the SN-38 drug content assay, a UV variable detector with a wavelength of 265 nm and an Agilent Zorbax SB-C18 column (4.6 mm×250 mm, 5 μm) were used. The mobile phase consisted of a 50:50 (v / v) mixture of 25 mM NaH2PO4 (pH=3.1) buffer and acetonitrile. The mobile phase pH was maintained at 3.8 to ensure that the analyte SN-38 was in a closed lactone ring form during the assay. The samples for HPLC were prepared by diluting the nanoparticles 100-fold with HPLC solvent (10 μL of formulation and 990 μL of a 50 / 50 mixture of buffer and acetonitrile). During the assay, aliquots of 20 μL of the sample were injected into the HPLC system in duplicate at a flow rate of 1 ml / min. The SN-38 content in the formulation was quantitatively determined using a standard curve prepared as outlined in a previously published journal article (Zhang et al. 2004; Escoriaza et al. 2000).

[0439] The encapsulation efficiency (EE%) was calculated using the following equation,

[0440]

[0441] Wa is the weight of SN-38 after centrifugation, and Wi is the weight before centrifugation.

[0442] Formulations were prepared by adding 1 wt%, 2 wt%, 5 wt% and 10 wt% of SN-38 to the total amount of lipid mixture in the formulation. The unencapsulated free drug was removed from the formulation using centrifugation and the EE% was determined using HPLC as outlined above. When 1 wt% of the drug was added to the formulation, the average EE% was found to be 82%, indicating that most of the added drug was encapsulated in the bilayer and only a very small amount of free unencapsulated drug was present. For the drug loaded with OAPy-4, it was observed that the higher the amount of drug to lipid ratio, the more the EE% decreased. Each drug has specific requirements for efficient and stable encapsulation into lipid nanoparticles.

[0443] result

[0444] The DL and EE% of the SN-38 loaded formulations are shown in Tables 3 and Figure 8 For the data in Table 3 representing the drug loading and EE% of samples prepared by adding 1 wt%, 2 wt%, 5 wt%, and 10 wt% of SN-38 based on the total amount of the lipid mixture (MO+OAPy-4) in the formulation and dispersing with F-127, the experiments were performed in triplicate on three independently prepared SN-38-loaded formulations, and all values ​​are presented as mean ± SD (n = 3).

[0445] Table 3

[0446]

[0447] A high loading of 844 μg / ml SN-38 was achieved, which is about 100 times the solubility of SN-38 in water. A previous study by Ranneh et al. loaded SN-38 in phytantriol-based cubes with surfactants to enhance solubility and achieved high loading formulations of SN-38 with 90-120 μg / ml loading using cationic surfactants (Ranneh et al., 2016). Compared to the study conducted by Ranneh, the loading of the formulation of the present invention with ionizable amino lipids was about 8 times.

[0448] The dynamic properties of the bilayer may greatly hinder or facilitate the incorporation of hydrophobic molecules such as SN-38. Although carriers with rigid bilayers have significant advantages such as low permeability and stability in vivo, they have many problems with drug loading limitations. In contrast, fluid bilayers more easily accommodate hydrophobic molecules within their hydrophobic cores. For drugs loaded with lipid 10, it was observed that the higher the amount of drug to lipid ratio, the lower the EE%. Each drug has specific requirements for efficient and stable encapsulation into nanoparticles. Many factors contribute to the success of the final formulation, including lipid composition and the manufacturing process that determines the size, surface charge, and bilayer fluidity (Bala et al., 2013). It was observed that the loading was pH-dependent, and the loading of nanoparticles prepared with PBS buffer was better than that prepared with DI water. The higher loading using PBS may be because SN-38 undergoes a structural change from a closed lactone ring to an open carboxylate form as pH increases, and the two forms are in equilibrium at pH 6.7. The open carboxylate form is water-soluble and is therefore incorporated into the water channel with bilayers and higher drug loadings. The efficiency of drug loading will depend on its encapsulation in the aqueous core, its incorporation into the bilayer or its partitioning between these two phases.

[0449] In summary, the results demonstrate that the novel LLC lipid nanoparticles disclosed herein enable high drug loading of the drug SN-38 to be achieved.

[0450] Example 9: Drug Release Study

[0451] The in vitro release of SN-38 from nanoparticles was evaluated using dynamic dialysis (Zambito, Y. International Journal of Pharmaceutics 2012, 434(1), 28-34). The dispersion was placed in a dialysis tube (Pur-A-Lyzer TMThe drug release rate was determined by exhaustive dialyzing in 1% 1% iodine-free Maxi dialysis tubing (MW cut-off 3500).

[0452] After free drug removal, the nanoparticles with the highest loading of SN-38 (1 mL of formulation) were placed inside the dialysis tubing. The dialysis tubing was then immersed in 1000 mL of buffer maintained at 37 ± 1.0 °C and stirred at 400 rpm. The separation of free drug from the drug-loaded formulation was analyzed over a period of 420 minutes. Release experiments were performed in triplicate on three independently prepared SN-38-loaded formulations. For physiological pH, the experiments were performed by dialyzing the formulations against 1000 mL of phosphate buffer (pH 7.0). Lower pH experiments were performed by dialyzing the formulations against 1000 mL of phosphate buffer adjusted to the desired pH. The remaining SN-38 in the dialysis tubing was quantitatively measured by HPLC. At predetermined time intervals (60 minutes, 120 minutes, 240 minutes, and 420 minutes), 10 μL samples were withdrawn from the dialysis tubing and diluted with 990 μL of HPLC solvent. Protocols for predicting release kinetics obtained by withdrawing samples from the inside of a dialysis bag are described in the literature.

[0453] result

[0454] Calibration experiments of drug diffusion through the dialysis membrane in the absence of lipid nanocarrier drug delivery vehicles were performed to evaluate the effect of the dialysis membrane on release kinetics. More than 90% of SN-38 was released from a suspension of pure drug within 60-90 minutes (data not shown here), and therefore any membrane effects were negligible with respect to drug release kinetics.

[0455] The release profiles of SN-38 from SN-38-lipid 10 nanoparticles at different pH values ​​are shown in Fig. 9 The SN-38-lipid 10 formulation showed a burst release (about 37%) in the first 60 minutes of the study for the pH studied (7.0 and 5.0), and sustained release after the first hour. In the dialysis setting, sink conditions were maintained at 10-20 times the volume required for a saturated drug solution so that free drug could quickly equilibrate across the dialysis membrane. Since the solubility of SN-38 in water is 7–11 μg / mL, 1000 mL of sink solution was used for 1 mL of formulation. This 1000-fold dilution increased the diffusion-controlled drug release rate by several orders of magnitude, resulting in a significant burst release of SN-38.

[0456] After the first hour of the study, the drug release rate from the sample at pH = 5.0 was faster than that of the sample at pH = 7.0. This difference in sustained release behavior is consistent with the presence of different intermediate phases (reverse hexagonal phase or cubic phase) under different pH conditions. The cubic phase, which is open to the external aqueous phase and has two interpenetrating water channels, provides a larger surface area and more accessible pathways for drug release. On the other hand, the hexagonal phase with a closed columnar micellar structure hinders drug release. Compared to the channels of smaller diameter hexagonal stacking, the drug release from the bicontinuous cubic phase network of large open channels is released faster.

[0457] Previous in vitro and in vivo studies have shown that changing the release rate is one of the possible ways to increase the distribution of drugs into tumors and improve the anti-tumor effect of drugs. The release results of SN-38-lipid 10 nanoparticles revealed that the drug release rate was slower in the simulated normal cell physiological environment (pH = 7.4) and faster in the simulated tumor microenvironment and lysosomal environment (pH = 5.0). The slower release rate at pH 7.4 is potentially beneficial because it may reduce drug losses in the circulation and increase the bioavailability of the drug at the tumor site. The faster release rate at pH = 5.0 is beneficial because it allows the drug to accumulate in the pathological environment, and the cytotoxicity of SN-38 to healthy cells is reduced and the efficacy is improved.

[0458] The results showed that the novel LLC lipid nanoparticles disclosed herein enable the controlled release of the drug SN-38 in response to pH value.

[0459] Example 10: In vivo toxicity study of LLC lipid nanoparticles

[0460] The LLC lipid nanoparticles disclosed herein were evaluated for in vivo toxicity in mice.

[0461] Nanoparticles were prepared using the evaporation and sonication method described above using amino lipids OAPi-1 and OAPy-4 and monoolein (MO) as the structural lipid:

[0462]

[0463] Structures of OAPi-1 (N-(2-(piperidin-1-yl)ethyl)oleamide) and OAPy-4 (N-(pyridin-4-ylmethyl)oleamide).

[0464] Briefly, lipids were dissolved in ethanol at appropriate ratios. The ratios of MO:OAPi-1 were 75:25 and 85:15 (wt:wt). The ratio of MO:OAPy-4 was 60:40. The solvent was evaporated in a vacuum oven at 40°C overnight. An aqueous solution containing Pluronic F-127 was added to the dried lipid mixture. The ratio of F-127 to lipid was kept constant at 1:10 (wt / wt). The solution was sonicated using a probe sonicator to produce stable nanoparticles.

[0465] Six-week-old Balb / c nude mice were injected intraperitoneally with the formulated nanoparticles at indicated doses (50 mg / kg, 100 mg / kg and 200 mg / kg). The mice were monitored for any side effects over a 24-hour period.

[0466]

[0467] *Note: The concentrations here refer to the lipid concentration in the formulation.

[0468] The results showed that at the concentration tested, the nanoparticles showed no side effects after 24 hours. This experiment shows that the nanoparticles do not cause acute toxicity to mice. The experiment also showed that the maximum tolerated dose is higher than 200mg / kg.

[0469] The results indicate that the novel LLC lipid nanoparticles disclosed herein are suitable for clinical applications as pharmaceutical or cosmetic agent carriers.

[0470] Those skilled in the art will appreciate that many changes and / or modifications may be made to the above embodiments without departing from the general broad scope of the present disclosure. Therefore, the present embodiments should be considered in all aspects as illustrative and not restrictive.

[0471] References

[0472] Bala, V.; Rao, S.; Boyd, BJ; Prestidge, CA, Prodrug and nanomedicine approaches for the delivery of thecamptothecin analogue SN38. J Control Release, 2013, 172(1), 48-61.

[0473] Casadó, A.; Sagristá, ML; Mora, M., A novel microfluidic liposomal formulation for the delivery of the SN-38camptothecin: characterisation and in vitro assessment of its cytotoxic effect on two tumor cell lines. Int J Nanomedicine, 2018, 13, 5301-5320.

[0474] Drummond, CJ; Grieser, TF; Healy, TW, Acid-Base Equilibria in Aqueous Micellar Solutions. Journal of the Chemical Society, Faraday Transactions 1. 1989, 85(3), 521-535.

[0475] Escoriaza, J.; Castellanos, AAC; Calvo, E.; Giraldez, J. Simple and rapid determination of Irinotecan and its metabolite SN-38 in plasma by high-performance liquid-chromatography: application to clinical pharmacokinetic studies. Journal of Chromatography B, 2000, 740 (2000) 159–168.

[0476] Rajesh, S.; Zhai J.; Drummond, C. J., Tran, N. Synthetic ionizable aminolipids induce a pH dependent inverse hexagonal to bicontinuous cubiclyotropic liquid crystalline phase transition in monoolein nanoparticles. Journal of Colloid and Interface Science, 2021, Vol. 589, 85-95.

[0477] Ranneh, AH; Iwao, Y.; Noguchi, S.; Oka, T.; Itai, S. The use of surfactants to enhance the solubility and stability of the water-insoluble anticancer drug SN38 into liquid crystalline phase nanoparticles. Int J Pharm, 2016, 515(1-2), 501-505.

[0478] Sarkar, S.; Tran, N.; Rashid, MH; Le, TC; Yarovsky, I.; Conn, CE; Drummond, CJ. Toward Cell Membrane Biomimetic Lipidic Cubic Phases: A High-Throughput Exploration of Lipid Compositional Space. ACS Applied BioMaterials, 2018, 2(1), 182-195.

[0479] Tran, N.; Hawley, A. M.; Zhai, J.; Muir, B. W.; Fong, C.; Drummond, C. J.; Mulet, X., High-Throughput Screening of Saturated Fatty Acid Influence on Nanostructure of Lyotropic Liquid Crystalline Lipid Nanoparticles, Langmuir, 2016, 32(18), 4509-4520.

[0480] Tran, N.; Hocquet, M.; Eon, B.; Parveen Sangwan; Ratcliffe, J.; Hinton, TM; White, J.; Ozcelik, B.; Reynolds, NP; Muir, BW, Non-Lamellar Lyotropic Liquid Crystalline Nanoparticles Enhance the Antibacterial Effects of Rifampicin against Staphylococcus Aureus Journal of Colloid and Interface Science, 2018, 519(1), 107-118.

[0481] Tran, N.; Mulet, X.; Hawley, A. M.; Hinton, T. M.; Mudie, S. T.; Muir, B. W.; Giakoumatos, E. C.; Waddington, L. J.; Kirbyb, N. M.; Drummond, C. J. Nanostructure and cytotoxicity of self-assembled monoolein–capric acid lyotropic liquid crystalline nanoparticles. Royal Society of Chemistry Advances, 2015, 5(34), 26785-26795.

[0482] Zhai, J.; Hinton, TM; Waddington, LJ; Fong, C.; Tran, N.; Mulet, X.; Drummond, CJ; Muir, BW. Lipid-PEG conjugates sterically stabilise and reduce the toxicity of phytantriol-based lyotropic liquid crystalline nanoparticles. Langmuir, 2015, 31(39), 10871-10880.

[0483] Zhang, JA; Xuan, T.; Parmar, M.; Ma, L.; Ugwu, S.; Ali, S.; Ahmad, I., Development and characterisation of a novel liposome-based formulation of SN-38. International Journal of Pharmacy, 2004, 270(1-2), 93-107.

Claims

1. A lyotropic liquid crystal (LLC) lipid carrier comprising a structural lipid and an amino lipid, wherein the structural lipid is a lyotropic liquid crystal phase-forming lipid, the amino lipid has an amide linker, and the LLC lipid carrier is suitable for undergoing a mesophase transition when exposed to a decrease in pH.

2. The LLC lipid carrier of claim 1, wherein the structural lipid is capable of forming a cubic mesophase structure.

3. The LLC lipid carrier according to claim 2, wherein the structured lipid comprises a hydrophobic tail group selected from the group consisting of oleyl, linoleoyl, linolenoyl, phytanoyl, farnesoyl or an extended aliphatic hydrophobic, optionally selected from oleyl, linoleoyl and phytanoyl.

4. The LLC lipid carrier according to any one of claims 1 to 3, wherein the structural lipid is glyceryl monooleate (glyceryl monooleate).

5. The LLC lipid carrier according to any one of claims 1 to 4, wherein the amino lipid is present in a suitable wt% of the total lipid content of the LLC lipid carrier to provide the mesophase transition when exposed to a drop in pH.

6. The LLC lipid carrier according to any one of claims 1 to 5, wherein the amino lipid accounts for about 5% to about 50 wt% of the total lipid content of the LLC lipid carrier.

7. The LLC lipid carrier according to any one of claims 1 to claim 6, wherein the amino lipid has the structure of formula (I): Cyc-LR (I)， Wherein Cyc is a nitrogen heterocycle or a heteroaryl; L is an amido linker; and R is a C10 to C44 carbon chain.

8. The LLC lipid carrier according to any one of claims 1 to claim 7, wherein the amino lipid has the structure of formula (Ib): wherein Cyc is a 5-membered or 6-membered nitrogen heterocyclic group or heteroaryl group, the nitrogen heterocyclic group or heteroaryl group is optionally selected from the group consisting of pyridyl, pyrimidinyl, piperidinyl, piperazinyl, morpholinyl and pyrazinyl; L is an amido linker; R is a C10 to C44 carbon chain, optionally a C12 to C24 alkyl or alkenyl group, which is optionally interrupted by one or more heteroatoms; and n is an integer of 1 to 6.

9. The LLC lipid carrier of claim 7 or claim 8, wherein R is selected from the group consisting of oleyl, linoleoyl, linolenoyl, phytanoyl and farnesoyl.

10. The LLC lipid carrier according to any one of claims 1 to claim 9, wherein the amino lipid is selected from the group consisting of: wherein R is as defined in any one of claims 10 to 12.

11. The LLC lipid carrier according to any one of claims 1 to claim 10, wherein the LLC lipid carrier further comprises a stabilizer, optionally wherein the stabilizer is a stabilizing polymer, optionally a non-ionic triblock copolymer.

12. The LLC lipid carrier of claim 11, wherein the stabilizer is present in an amount of about 10 wt% of the total lipid content of the LLC lipid carrier.

13. The LLC lipid carrier of claim 12 or claim 13, wherein the stabilizer is Pluronic F127 (Poloxamer 407) or Poloxamer 80.

14. The LLC lipid carrier according to any one of claims 1 to claim 13, wherein the LLC lipid carrier is in the form of lyotropic liquid crystal (LLC) nanoparticles.

15. The LLC lipid carrier of any one of claims 1 to 14, wherein the mesophase transition upon exposure to a drop in pH is a transition from a hexagonal phase to a cubic phase.

16. The LLC lipid carrier of claim 15, wherein the LLC lipid carrier comprises a hexagonal lyotropic liquid crystal phase structure at a pH of about pH 7 or higher, optionally about pH 7.4 or higher.

17. The LLC lipid carrier of claim 15 or claim 16, wherein the LLC lipid carrier comprises a cubic lyotropic liquid crystal phase structure at a pH of about pH 4.0-pH 7.

0.

18. The LLC lipid carrier according to any one of claims 15 to 17, wherein the LLC lipid carrier comprises a hexagonal lyotropic liquid crystal phase structure at a pH of about pH 7 or higher, optionally about pH 7.4 or higher, and comprises a cubic lyotropic liquid crystal phase structure at a pH of about pH 4.0-pH 7.

0.

19. The LLC lipid carrier according to any one of claims 1 to claim 18, further comprising an active agent, optionally wherein the active agent is pharmaceutically active or cosmetically active.

20. The LLC lipid carrier of claim 19, wherein the active agent is selected from the group consisting of peptides, proteins, enzymes, small molecule drugs, and nucleic acids, optionally wherein the active agent is selected from the group consisting of radionuclides, imaging agents, polymers, antibiotics, fungicides, metal-containing nanoparticles, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, antianxiety agents, hormones, growth factors, steroid agents, gene expression regulators, knockdown agents, siRNA, RNAi agents, mRNA, DNA, dicer substrates, miRNA, shRNA, antisense oligonucleotides, aptamers, and microbial-derived toxins.

21. The LLC lipid carrier of claim 19 or claim 20, wherein the active agent is a topoisomerase I inhibitor, optionally selected from camptothecin, irinotecan and SN-38.

22. The LLC lipid carrier of any one of claims 1 to 21, wherein the water content of the LLC lipid carrier is between about 20% to about 60% by weight.

23. A lyotropic liquid crystal (LLC) lipid nanoparticle composition, which is a dispersion of the LLC lipid carrier according to any one of claims 19 to claim 22 in a polar medium.

24. A cosmetic composition comprising the LLC lipid carrier according to any one of claims 19 to 22 or the LLC lipid nanoparticle composition according to claim 23 and a cosmetically acceptable carrier, diluent and / or excipient.

25. A pharmaceutical composition comprising the LLC lipid carrier according to any one of claims 19 to 22 or the LLC lipid nanoparticle composition according to claim 23 and a pharmaceutically acceptable carrier, diluent and / or excipient.

26. The cosmetic composition according to claim 24 or the pharmaceutical composition according to claim 25, wherein the composition is formulated as a composition for injection, topical administration or subcutaneous administration.

27. A method of delivering an active agent to a biological system, the method comprising the step of administering to the biological system an LLC lipid carrier according to any one of claims 19 to claim 22, an LLC lipid nanoparticle composition according to claim 23, or a composition according to any one of claims 24 to claim 26.

28. The method of claim 27, wherein delivery of the active agent into the biological system is regulated by the response of the LLC lipid carrier to a predefined pH range.

29. The method of claim 28, wherein the response comprises a mesophase structure transition of the LLC lipid carrier, optionally wherein the phase transition is a transition from a hexagonal phase structure to a cubic phase structure of the LLC lipid carrier.

30. The method of claim 29, wherein the mesophase structural transition occurs at a pH of about pH 7.0-pH 4.0, optionally about pH 7.0-pH 5.

5.

31. The method of any one of claims 27 to claim 30, wherein the response provides for preferential release of the active agent in the biological system at the predefined pH range.

32. A method for controlled release of an active agent, the method comprising the steps of forming an LLC lipid carrier according to any one of claims 19 to claim 22, an LLC lipid nanoparticle composition according to claim 23, or a composition according to any one of claims 24 to claim 26, and applying the LLC lipid carrier, the LLC lipid nanoparticle composition, or the composition to a biological system comprising a target area with a predefined pH, thereby achieving preferential release of the active agent at the target area.

33. The method of claim 32, wherein the LLC lipid carrier undergoes a mesophase structural transition in response to the predefined pH.

34. The method of claim 33, wherein the mesophase structure transition is a transition from a hexagonal phase structure to a cubic phase structure of the LLC lipid carrier.

35. A method of treating or preventing a disease, disorder or condition in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the LLC lipid carrier according to any one of claims 19 to claim 22, the LLC lipid nanoparticle composition according to claim 23, or the composition according to any one of claims 24 to claim 26.

36. A method of diagnosing a disease, disorder or condition in a mammal, the method comprising the steps of: The LLC lipid carrier according to any one of claims 19 to claim 22, the LLC lipid nanoparticle composition according to claim 23, or the composition according to any one of claims 24 to claim 26 is administered to the mammal or a biological sample obtained from the mammal to facilitate diagnosis of the disease, disorder or condition in the mammal, wherein the active agent is a labeled active agent.

37. The method of claim 35 or claim 36, wherein the disease, disorder or condition is cancer or a bacterial infection or a fungal infection.

38. An amino lipid of formula (Ib): wherein Cyc is a 5- or 6-membered nitrogen heterocyclic radical or heteroaryl selected from the group consisting of piperidinyl, piperazinyl, pyrrolinyl, pyrrolidinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, pyrrolinyl, pyrazolinyl, thiazolinyl, thiomorpholinyl, tetrahydropyridinyl, dihydropyridinyl, tetrahydropyrimidinyl, pyrimidinyl, pyridinyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, oxadiazolyl, pyrazinyl, tetrazolyl, thiazolyl, isoxazolyl, isothiazolyl, isoxazolone, triazolyl, oxadiazolyl, thiadiazolyl, and pyridazinyl; L is an amido linker; R is a C10 to C44 carbon chain, optionally a C12 to C24 alkyl or alkenyl group, which is optionally interrupted by one or more heteroatoms; and n is an integer of 1 to 6.

39. The amino lipid of claim 38, wherein Cyc is selected from the group consisting of pyridyl, pyrimidyl, piperidinyl, piperazinyl and pyrazinyl.

40. The amino lipid of claim 38 or claim 39, wherein R is selected from the group consisting of oleyl, linoleoyl, linolenoyl, phytanoyl and farnesoyl.

41. The amino lipid according to any one of claims 38 to claim 40, wherein the amino lipid is selected from the group consisting of: wherein R is as defined in any one of claims 40 to 42.

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