Lyotropic liquid crystal phase particles

Abstract

The present disclosure provides non-lamellar lyotropic liquid crystalline phase particles useful for delivering active agents to treat Gram-negative bacterial and / or fungal infections. The non-lamellar lyotropic liquid crystalline phase particles have been shown to be beneficial in delivering active agents through fusion events at the Gram-negative bacterial outer membrane or fungal outer layer.

Description

[Technical Field]

[0001] The present invention relates to the field of medical treatment and disease diagnosis. More specifically, the present invention relates to non-lamellar lyotropic liquid crystalline phase particles that are carrier particles for active agents and use thereof. [Background technology]

[0002] Any reference to background art in this specification should not be construed as an admission that such art constitutes common general knowledge in Australia or elsewhere.

[0003] The increasing prevalence of antibiotic-resistant bacteria is perceived as one of the major health risks to humanity. Resistance is primarily caused by the overuse of antibiotics, which promotes resistant genetic mutations. Financial and health complications due to resistant bacteria are predicted to increase in the future. New avenues of antimicrobial treatment are currently under extensive investigation, but the development of new antibiotics remains slow. The outer cell membrane of Gram-negative bacteria, including notorious species such as Escherichia coli and Pseudomonas aeruginosa, provides an additional permeability barrier, making them particularly resilient to traditional small-molecule antibiotics. Gram-negative bacteria resistant to last-resort antibiotics have already been reported. For this reason, the World Health Organization (WHO) has prioritized the particularly urgent need for new treatments for Gram-negative bacteria.

[0004] Due to the continuing low discovery rate for new antibiotics, the development of technologies to complement existing treatments may be important. Delivery technologies and carrier-controlled internalization may provide more effective treatments to effectively combat bacteria. In particular, nanomaterials may act as carriers for single or multiple therapeutic compounds and may potentially act to weaken bacterial membrane barriers, amplifying antibacterial outcomes. However, the interactions between nanomaterials and bacteria remain poorly understood. 1 .

[0005] In contrast to mammalian cells, which typically internalize nanomaterials via the endocytic pathway 2、3 Bacteria rely on the permeability of their cell walls to transport materials. The inherent complexity of cell walls is a major cause of difficulties in developing effective antibiotics, and therefore carriers suitable for mammalian cells are rarely also suitable for delivery to bacterial cells. Lyotropic liquid crystalline interlayer carriers, such as cubic phase lipid nanocarriers (cubosomes), have shown some promise in the delivery of therapeutic agents to mammalian cell lines by means other than endocytosis. 4、5、6、7 The mechanism of delivery, and consequently the reliability and extent of delivery of any encapsulated active, remains unresolved in the existing literature.

[0006] Similarly, resistant fungal infections are increasing in prevalence and threatening current health practices. Once in the bloodstream, these infections have a mortality rate of approximately 25%. The estimated medical costs are $3 billion annually. Antifungal drugs are often highly cytotoxic and poorly water-soluble. As with bacteria, fungal outer cell wall materials, such as chitin, can provide a significant diffusion barrier to antimicrobial agents.

[0007] There is a need to provide additional carriers for the delivery of antimicrobial agents, including antibacterial and antifungal agents, as well as other biologically active agents. There is a further need for the development of new lyotropic liquid crystalline phase particles as carriers of such active agents for more effective treatment of microbial infections, among other medical uses. Summary of the Invention

[0008] According to a first aspect of the present invention, there is provided a non-lamellar lyotropic liquid crystalline phase particle comprising one or more fusogenic amphiphilic lipids, and a particle encapsulating an active agent.

[0009] In a second aspect, there is provided a pharmaceutical composition comprising the non-lamellar lyotropic liquid crystalline phase particles of the first aspect, a pharmaceutically acceptable carrier, diluent and / or excipient.

[0010] In a third aspect, there is provided a method for controlled release of an active agent, comprising forming the non-lamellar lyotropic liquid crystalline phase particles of the first aspect and administering the non-lamellar lyotropic liquid crystalline phase particles to a target area.

[0011] In a fourth aspect, there is provided a method of forming the non-lamellar lyotropic liquid crystalline phase particles of the first aspect, the method comprising: (i) providing one or more fusogenic amphiphilic lipids; and (ii) exposing the one or more fusogenic amphiphilic lipids to a solution in the presence of an active agent.

[0012] In a fifth aspect, there is provided a method of treating or preventing a disease, disorder or condition, comprising administering to a subject in need thereof a therapeutically effective amount of non-lamellar lyotropic liquid crystalline phase particles of the first aspect, or a pharmaceutical composition of the second aspect, thereby treating or preventing the disease, disorder or condition.

[0013] In a sixth aspect, there is provided the non-lamellar lyotropic liquid crystalline phase particles of the first aspect, or the pharmaceutical composition of the second aspect, for use in the treatment or prevention of a disease, disorder or condition.

[0014] In a seventh aspect there is provided the use of non-lamellar lyotropic liquid crystalline phase particles of the first aspect in the manufacture of a medicament for the treatment of a disease, disorder or condition.

[0015] In an eighth aspect, there is provided a method of delivering an active agent to a biological target, the method comprising administering non-lamellar lyotropic liquid crystalline phase particles of the first aspect.

[0016] In a ninth aspect, there is provided a method of diagnosing a disease, disorder or condition in a mammal, comprising the step of administering non-lamellar lyotropic liquid crystalline phase particles of the first aspect or a composition of the second aspect, wherein the active agent in the non-lamellar lyotropic liquid crystalline phase particles of the first aspect is an active agent labeled to a biological target in or obtained from the mammal so as to facilitate diagnosis of the disease, disorder or condition in the mammal.

[0017] The various features and embodiments of the invention referred to in individual sections above apply mutatis mutandis to other sections, as appropriate. Thus, features specified in one section may be combined with features specified in other sections, as appropriate.

[0018] Further features and advantages of the present invention will become apparent from the following detailed description.

[0019] In order that the present invention may be readily understood and put into practice, preferred embodiments will now be described by way of example with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0020] [Figure 1] A series of fluorescence images of bacteria in the absence of fluorescently tagged cubosome particles. Left and right images were recorded at λex = 405 nm and λex = 647 nm, respectively. Scale bar = 2 μm. [Figure 2]A series of images and graphic representations showing cubosome uptake by B. cereus. (a) TIRF snapshots showing in situ binding of a single cubosome to the B. cereus surface. Scale bar = 5 μm. (b) Mean square displacement of the binding cubosome. (c) Snapshots highlighting the continuous attachment of MO cubosomes to the B. cereus surface over approximately 4 hours. (d) Long-term transfer of fluorescence from the particle to B. cereus over 24 hours. Scale bar = 20 μm. (e) Extracted fluorescence intensity over time for an individual cubosome in contact with the B. cereus surface. Data are shown on a logarithmic scale in the insert, with an indicating slope of -1 / 2. (f) Extracted intensity over time for B. cereus. [Figure 3] A series of images and graphic representations showing cubosome uptake by Staphylococcus aureus. (a and b) TIRF snapshots highlighting the sequential binding of MO-DOTAP cubosomes to the surface of S. aureus. Scale bar = 2 μm. (c) Extracted fluorescence intensity over time for individual cubosomes in contact with the S. aureus surface. Data are shown in the inserts on a log-log scale, with indicated slopes of -1 / 3 and -1 / 2 shown as solid and dashed lines, respectively. (d) Fluorescence intensity over time for clusters of S. aureus, peptidoglycan extract, and peptidoglycan extract treated with lipoteichoic acid. (e) TIRF snapshots highlighting the sequential binding of MO cubosomes to the surface of S. aureus peptidoglycan extract. Scale bar = 5 μm. (f) SEM micrographs of S. aureus peptidoglycan extract in the absence and upon treatment with cubosomes. Scale bar = 1 μm. (g) SEM of fixed S. aureus after treatment with cubosomes. Scale bar = 300 nm. (h) A series of TIRF snapshots showing the lack of cubosome binding to the surface of S. aureus peptidoglycan and lipoteichoic acid extract. Scale bar = 5 μm. [Figure 4]A series of images showing the variability of cubosome uptake by Escherichia coli. (a) TIRF snapshots highlighting the sequential binding of MO-cubosomes to the surface of Escherichia coli. Scale bar = 2 μm. (b) Sequential binding and spreading of MO-DOTAP cubosomes to the surface of Escherichia coli. Scale bar = 2 μm. (c and d) Snapshots at λex = 647 nm highlighting the apparent bursts of fluorescence across E. coli for MO and MO-DOTAP, respectively. Scale bar = 2 μm. (e) Snapshots of the rapid interaction of two MO-DOTAP cubosomes. The first frame shows fluorescence from λex = 405 nm, and the following four frames are at λex = 647 nm. Two cubosomes are observed landing at 10 and 20 seconds. In the following frames, the cubosomes are no longer present, but an intensity gradient is visible around the bacteria. (f-m) SEM micrographs of Escherichia coli treated with MO-cubosomes. Scale bar = 400 nm. [Figure 5] A series of images and graphic representations showing cubosome uptake by Escherichia coli. (a and b) Peak intensity profiles across an individual E. coli cell for MO and MO-DOTAP, respectively. (c) Intensity across the entire Escherichia coli cell. The left axis indicates total area intensity. The right axis indicates peak intensity. (d) Peak intensity over time for an individual MO-DOTAP cubosome in contact with Escherichia coli. The insert contains data replotted on a logarithmic scale. The steep and shallow dashed lines indicate slopes of -1 and -1 / 6, respectively. (e) A single representative plot of an individual cubosome on a logarithmic scale. (f) Corresponding TIRF snapshot of the individual cubosome plotted in (e). Scale bar = 2 μm. [Figure 6]Graphic representation showing the incident of rapid uptake. (a) Peak intensity over time of an individual cubosome in contact with Escherichia coli. The arrow indicates the time point when fluorescence spread rapidly throughout the bacterium. (b) Data plotted in seconds over time on a log scale. The steep and shallow dashed lines indicate slopes of -1 and -1 / 6, respectively. [Figure 7] (a) A series of images and a graphical representation showing time-lapse TIRF time-lapse showing fusion of cubosomes (bright dots) with SLBs. Scale bar = 1.5 μm. (b) Cubosome uptake into E. coli shows two distinct regimes. Inset images are 10 s apart and show the spreading of nanocarriers (NCs). (c) Uptake into S. aureus shows one distinct regime. (c and d) TIRF time-lapse showing binding and internalization of NCs (bright dots) into E. coli and S. aureus, respectively. Scale bar = 2 μm. (e) Internalization of NCs (red) into C. albicans. [Figure 8] Formulations of cubosomes containing varying degrees of novobiocin against Pseudomonas aeruginosa are shown. Free antibiotic is shown as a plain black line. Concentration on the x-axis is the drug concentration in solution. Drug concentration is consistent across formulations. Increasing Novo from 1 to 3 to 5% molar reflects an increase in drug loading per particle, although fewer particles are present as the concentration increases. [Figure 9] Formulations of cubosomes containing varying degrees of novobiocin against Escherichia coli are shown. Free antibiotic is shown as a plain black line. Concentration on the x-axis is the drug concentration in solution. Drug concentration is consistent across formulations. Increasing Novo from 1 to 3 to 5% molar reflects an increase in drug loading per particle, although fewer particles are present as the concentration increases. [Figure 10]Figure 1 shows the inhibition of Pseudomonas aeruginosa by novobiocin formulations in the presence of varying serum environments. The x-axis is the concentration of the protein environment, and the antibiotic concentration is fixed at 20 μg / ml for each formulation. MO-TAP-3Novo was evaluated with HSA, BSA, and FBS. The equivalent free antibiotic loading is indicated by the black squares, circles, and triangles, respectively. The dashed line shows the performance of MO-TAP-3Novo in the absence of serum proteins. [Figure 11] Figure 1 shows the formulation of cubosomes containing varying levels of piperacillin against Pseudomonas aeruginosa, with the free antibiotic shown as a solid black line. [Figure 12] Figure 1 shows the formulation of cubosomes containing varying levels of piperacillin against Escherichia coli. Free antibiotic is shown as a solid black line. [Figure 13] Figure 1 shows formulations of cubosomes containing varying levels of meropenem against Pseudomonas aeruginosa. Free antibiotic is shown as a solid black line. [Figure 14] Figure 1 shows formulations of cubosomes containing varying levels of meropenem against Escherichia coli. Free antibiotic is shown as a plain black line. [Figure 15] Figure 1 shows the formulation of cubosomes containing varying levels of clarithromycin against Escherichia coli. Free antibiotic is shown as a solid black line. [Figure 16] Photographs showing that clarithromycin formulations are prone to sedimentation / poor dispersion. [Figure 17] Figure 1 shows the formulation of cubosomes containing varying levels of gentamicin against Pseudomonas aeruginosa, with the free antibiotic shown as a solid black line. [Figure 18] Formulations of cubosomes containing varying levels of gentamicin against Escherichia coli are shown, with the free antibiotic shown as a solid black line. [Figure 19] 1 shows the formulation of cubosomes containing dicloxacillin and tazobactam against Escherichia coli. [Figure 20] 1 shows the formulation of cubosomes containing benzylpenicillin against Escherichia coli. [Figure 21] 1 shows the formulation of cubosomes containing various strengths of rifampicin against Escherichia coli. [Figure 22] CFU counts for various doses of rifampicin, free in solution and encapsulated in MO-1TAP-3Fus (hash column), are shown. Minimum inhibitory concentrations (90% mortality) are indicated by asterisks. For rifampicin, the lowest concentration tested (0.05 μg / ml) already resulted in a 50% reduction in CFU compared to the free drug. Then, at 0.5 μg / ml, counts were dramatically reduced by approximately 7-fold. The respective MICs, again annotated by * on the plot, were 3-5 μg / ml and 1 μg / ml for free and encapsulated Rif, respectively, indicating at least a 3-fold reduction in MIC. The free MIC values ​​are in reasonable agreement with those reported for Escherichia coli O157:H7 (approximately 4 μg / ml). [Figure 23] Formulations of cubosomes containing 1% rifampicin with various degrees of DOPE (increased curvature) from 0 to 10 to 20% against Escherichia coli are shown. [Figure 24] As for Figure 23 after the delay test, formulations of cubosomes containing 1% rifampicin with various degrees of DOPE (increased curvature) against Escherichia coli are shown. [Figure 25] Formulations of cubosomes containing 1% DOTAP (positive charge) with varying degrees of DOPE (increased curvature) of 10 or 20 against Escherichia coli are shown. [Figure 26]For Escherichia coli, formulations of cubosomes containing 1% rifampicin 70% MO, 30% DOPE (mol%) or 1% rifampicin 60% MO, 40% DOPE (mol%) (increasing curvature with increasing DOPE) are shown, thus presenting a hexagonal phase. [Figure 27] A second data set for the formulations shown in Figure 26 against Escherichia coli. [Figure 28] We show that against Escherichia coli, formulations of cubosomes containing 1% rifampicin, 1% DOTAP (positively charged), 70% MO and 30% DOPE or 1% rifampicin, 1% DOTAP (positively charged), 60% MO and 40% DOPE (increasing curvature with increasing DOPE), thus presenting a hexagonal phase to E. coli. [Figure 29] A second data set for the formulations shown in Figure 28 against Escherichia coli. [Figure 30] Figure 1 shows CFU counts for various doses of fusidic acid free in solution and encapsulated in MO-1DOTAP-3Fus (hashed columns) against Escherichia coli. The minimum inhibitory concentration (90% death) is indicated by an asterisk. [Figure 31] Figure 1 shows the survival rates of Pseudomonas aeruginosa and Escherichia coli for various concentrations of lipid nanoparticles without encapsulated active agent as a control. [Figure 32] MIC determination results of rifampicin formulations and DOTAP (positive charge) incorporation into Mycobacterium smegmatis are shown. [Figure 33] Figure 1 shows cell death and incorporation of DOTAP (positive charge) achieved by rifampicin formulations against Mycobacterium smegmatis. [Figure 34]MIC determination results of rifampicin formulations and incorporation of DOTAP (positive charge) against Mycobacterium tuberculosis H37Ra are shown. [Figure 35] Figure 1 shows cell death and incorporation of DOTAP (positive charge) achieved by rifampicin formulations against Mycobacterium tuberculosis H37Ra. [Figure 36] Figure 1 shows a formulation of cubosomes containing 1% filipin, 0 / 1% DOTAP (positively charged), and 0 / 10% cholesterol against Candida albicans. [Figure 37] Figure 1 shows a formulation of cubosomes containing 1% amphotericin B, 0 / 1% DOTAP (positively charged), 0 / 10% cholesterol against Candida albicans. [Figure 38] Confocal and SEM images of fluconazole alone, lipid particles without fluconazole (control nanoparticles), and fluconazole-loaded lipid nanoparticles are shown. a) Confocal image at pH-5.0, b) SEM image at pH-5.0, c) Confocal image at pH-7.0, d) SEM image at pH-7.0. DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention is based, at least in part, on the realization that certain parameters and components of non-lamellar lyotropic liquid crystalline phase particles, such as component-induced lipid properties, internal or average spontaneous curvature, charge, drug loading, and microrheology, can be manipulated to provide carrier particles capable of one or more of: (i) improved encapsulation of active agents; (ii) improved derivatization behavior with microorganisms; (iii) optimal release profile of active agents; (iv) reduction in steric and / or electrostatic barriers in contact with biological membranes; (v) protection of active agents from damage or binding that would otherwise occur and inactivate or reduce the activity of the active agents; (vi) ability to deliver active agents that would otherwise be unable to cross bacterial membranes; and (vii) improved efficacy of active agents compared to delivery of free active agents.

[0022] The formation of non-lamellar lyotropic liquid crystalline phase particles that can be tailored to improve the delivery of specific active agents and / or to improve delivery to specific biological targets will enable a wide range of uses within medical applications including detection, targeted therapy, imaging, etc.

[0023] Specifically, it is shown herein that non-lamellar lyotropic liquid crystalline phase carrier particles can be designed that are capable of delivering payloads to fungi, mycobacteria, and Gram-negative bacteria. The particle designs herein may be particularly, but not exclusively, suitable for the delivery of antibacterial and antifungal agents by fusion of the particles with bacterial or fungal membranes of Gram-negative bacteria.

[0024] The delivery of active agents to fungi, specifically Gram-negative bacteria, is known to be difficult. As is well known, in Gram-positive species, the bacterial membrane consists of a thick outer layer of peptidoglycan and an inner plasma phospholipid membrane. Conversely, Gram-negative species exhibit a relatively thin peptidoglycan layer sandwiched between two phospholipid membranes, with lipopolysaccharides characteristic of the outer membrane. The outer membrane present in Gram-negative species makes them particularly resilient to antibacterial compounds, presenting a unique challenge for the delivery of antibacterial agents.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0026] In this patent specification, the terms "comprises," "comprising," "includes," "including," or similar terms are intended to imply a non-exclusive inclusion, such that a method or composition that includes a list of elements does not include only those elements, but may well include other elements that are not listed.

[0027] "Consisting of" means including, but not limited to, everything that follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that other elements may not be present.

[0028] "Consisting essentially of" means including any elements listed after this phrase, limited to other elements that do not interfere with or contribute to the activity or function set forth in this disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or essential, but that other elements are optional and may or may not be present depending on whether they affect the activity or function of the listed elements.

[0029] The term "non-lamellar lyotropic liquid crystalline phase carrier particles," as used herein, refers to self-assembled particles comprising a liquid crystalline non-lamellar structure formed from at least one amphiphilic substance to provide a two-dimensional and / or three-dimensional mesophase structure capable of carrying an active agent. Non-lamellar lyotropic liquid crystalline phase carrier particles have been shown herein to provide excellent integration with and delivery of active agents across biological membranes. The terms "lipid carrier," "non-lamellar lyotropic liquid crystalline phase carrier particles," "non-lamellar LLC particles," "particles," and "nanoparticles" are used interchangeably herein.

[0030] In embodiments, the term "non-lamellar lyotropic liquid crystalline phase carrier particles" can be used to include cubic, hexagonal, and sponge morphologies. A "sponge phase" or "sponge particle" (L3) does not have long-range order and is a reverse bicontinuous cubic phase (Q II ), but they are often referred to as "melt" Q II These are considered cubic phases and are therefore considered to be included as particles of the first aspect. Thus, short-range-ordered sponge phases are explicitly considered to be within the scope of this term. In embodiments, the term "non-lamellar lyotropic liquid crystalline phase carrier particles" may be used to include one or more phases selected from the group consisting of cubic phases (normal and inverse), cubic phases (normal discrete, inverse discrete, inverse bicontinuous - including simple, gyroid, and diamond - and inverse discontinuous), and other "mesophases" including ribbon, mesh, or non-cubic "sponge" bicontinuous. Preferably, this term is used for cubic phases and / or hexagonal phases.

[0031] The terms "amphiphile," "amphiphilic," and "amphiphilic lipid," as used herein, refer to compounds containing both hydrophilic and hydrophobic portions and may be used in lipid, fusogenic, or otherwise, in forming the LLC particles described herein. Typically, such compounds will have a hydrophilic head group and a hydrophobic tail. Suitable examples include regions of fatty acids and lipid molecules.

[0032] The term "fusogenic," as referred to herein, refers to a compound, usually a lipid, that, as part of a non-lamellar lyotropic liquid crystalline phase carrier particle, will promote or enhance fusion of the particle with a biological membrane, such as a bacterial cell membrane, thereby facilitating delivery of an active agent.

[0033] The term "pharmaceutically acceptable salts," as used herein, refers to salts of active agents that are toxicologically safe for systemic or topical administration, such as salts prepared from pharmaceutically acceptable non-toxic bases or acids, including inorganic or organic bases and inorganic or organic acids. Pharmaceutically acceptable salts may be selected from the group including alkali and alkaline earth, ammonium, aluminum, iron, amine, glucosamine, chloride, sulfate, sulfonate, bisulfate, nitrate, citrate, tartrate, bitarate, phosphate, carbonate, bicarbonate, malate, maleate, napsylate, fumarate, succinate, acetate, benzoate, terephthalate, palmoate, piperazine, pectinate, and S-methylmethionine salts, etc.

[0034] According to a first aspect of the present invention, there is provided a lipid carrier, which may be a non-lamellar lyotropic liquid crystalline phase particle comprising one or more fusogenic amphiphilic lipids, and a particle encapsulating an active agent.

[0035] In embodiments, the lipid carriers, which may be non-lamellar lyotropic liquid crystalline phase particles, are formed by the self-assembly of one or more fusogenic amphiphilic lipids. It will be appreciated that suitable amphiphilic lipids self-assemble in the presence of an aqueous solution, such as water or an aqueous buffer, to form lyotropic liquid crystal (LLC) structures that exhibit a non-lamellar mesophase.

[0036] In embodiments, the non-lamellar lyotropic liquid crystalline phase particles comprise at least two fusogenic amphiphilic lipids, optionally at least three or at least four, or at least five, or at least six, or at least seven, eight, nine, or ten or more fusogenic amphiphilic lipids.

[0037] In embodiments, the non-lamellar lyotropic liquid crystalline phase particles may consist of, or consist essentially of, one, two, three, or four fusogenic amphiphilic lipids.

[0038] In embodiments, the non-lamellar lyotropic liquid crystalline phase particles may consist of, or consist essentially of, one, two, three, or four fusogenic amphiphilic lipids, fusogenic or non-fusogenic.

[0039] In embodiments, non-lamellar lyotropic liquid crystal phase particles are formed by the self-assembly of one or more fusogenic amphiphilic lipids in the presence of an active agent.It will be understood that there are multiple ways that active agents can be bound to, incorporated into, or encapsulated in particles, and the final approach will depend on the nature of the active agent and the way the particles deliver it.For example, in certain embodiments, it may be appropriate to focus on binding the active agent to the majority of the particle's surface.However, typically, particles are formed in the presence of an active agent, so that the active agent is incorporated into the internal channels and folds of lipid particles, in addition to any additional surface-bound active agent.

[0040] In embodiments, the majority of the active agent is located within the internal channels of the particles of the first aspect. Preferably, upon delivery to the target area, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the active agent associated with the particles of the first aspect is located within the internal channels of the particles of the first aspect.

[0041] In embodiments, the non-lamellar lyotropic liquid crystalline phase particles may be colloidal particles, which are colloidal particles having a particle size of less than 10 micrometers.

[0042] In an embodiment, the particle size of the particles of the first aspect can be about 10 micrometers to about 40 nanometers. Preferably, the particle size is about 5 micrometers to about 50 nanometers, more preferably about 1 micrometer to about 50 nanometers, even more preferably about 800 nanometers to about 50 nanometers, even more preferably about 600 nanometers to about 50 nanometers, even more preferably about 500 nanometers to about 50 nanometers or about 400 nanometers to about 50 nanometers, or about 5 micrometers to about 100 nanometers, more preferably about 1 micrometer to about 100 nanometers, even more preferably about 800 nanometers to about 100 nanometers, even more preferably about 600 nanometers to about 100 nanometers, even more preferably about 500 nanometers to about 100 nanometers or about 400 nanometers to about 100 nanometers. Thus, the particles of the first aspect can function as nanocarriers for active agents within the above particle size range embodiments.

[0043] In embodiments, the non-lamellar lyotropic liquid crystalline phase particles optionally have a bulk phase selected from the group consisting of a cubic phase, a hexagonal phase, and a sponge phase, including each of the normal and reverse / inverse phases.

[0044] Non-lamellar LLC particle matrices offer a range of advantages over their lamellar analogs, such as liposomes. Their lipid composition can make them fusogenic with bacterial outer membranes, and due to their high internal surface area and amphiphilic nature, non-lamellar LLC particles, such as cubosomes, have the ability to encapsulate and release an array of active agents. Non-lamellar LLC particle matrices can also protect the structural integrity of the encapsulated active agents from enzymatic degradation.

[0045] In embodiments, the non-lamellar lyotropic liquid crystalline phase particles are those selected from the group consisting of hexagonal (normal and inverse), cubic (normal discrete, inverse discrete, inverse - including simple, gyroid, and diamond - and inverse discontinuous), and other "mesophases" including ribbon, mesh, or non-cubic "sponge" bicontinuous.

[0046] In embodiments, the non-lamellar lyotropic liquid crystalline phase particles are selected from the group consisting of cubosome particles, hexosome particles, and sponge particles.

[0047] In embodiments, the non-lamellar lyotropic liquid crystalline phase particles of the first aspect may be cubosomes or hexosomes.

[0048] Preferably, the cubosomes are bicontinuous cubic phase (V1) or reverse bicontinuous cubic phase (V2) cubosomes. Reverse bicontinuous cubic phase (V2) cubosomes are particularly preferred. Those skilled in the art will understand that V2 is a generic term for various cubic phases. V2 is a cubosome of V II or Q II It is also called Q II Inside, Q II D (Pn3m), Q II P (Im3m), Q II G (Ia3d) exists.

[0049] Inverse (reverse) phase particles may be preferred because they provide a complex series of internal channels that can accommodate the active agent and allow for a better controlled release profile under certain circumstances.

[0050] The cubic phase structure within cubosomes provides a lipid bilayer motif repeatedly wrapped around a triply periodic minimal surface. The increased surface curvature of the lipid membrane within these particles of the first embodiment may aid in promoting bilayer fusion upon contact with other self-assembly systems containing lipid membranes, such as bacterial membranes. Therefore, high curvature values ​​are preferred in the lipid carriers of the present disclosure. Due to their high internal surface area and amphiphilic nature, cubosomes have the ability to encapsulate and release a wide variety of currently available active agents, including antimicrobial agents such as small molecules, proteins, antimicrobial peptides, and other biocidal components.

[0051] For fusogenic lipids used to form the particles of the first embodiment, the lipid's critical packing parameter (CPP) can be used to rationalize the mean and Gaussian curvatures characteristic of the formed particles, thus indicating the nature of the mesophase that is formed or being formed, and allowing consideration of the suitability of the resulting non-lamellar LLC particles as active agent carriers. The CPP is related to the mean and Gaussian curvatures via the following equation:

number

[0052] The same approach can be applied to two or more amphiphilic lipids. In the case of multiple amphiphiles, amphiphiles with an intrinsic CPP less than 1 can be included in a composition in which the CPP of a secondary amphiphile is greater than 1, so that the average CPP is greater than 1. Secondary additives that can also contribute to increasing curvature to achieve a CPP > 1 include small hydrophobic molecules, polymers that interact with the amphiphilic head groups, strong kosmotropes, and siRNA and DNA. Conversely, curvature can be reduced by including amphiphiles with CPP < 1, high molecular weight PEG, strong chaotropes, charged head groups, and solvents with a LogP between -1.5 and 0.

[0053] LLC particles can therefore be classified based on their interfacial curvature, which can be calculated by approaches known in the art. Generally speaking, the curvature of reverse lyotropic phases increases in the order lamellar < bicontinuous cubic < hexagonal < micellar cubic.

[0054] In embodiments, one or more fusogenic amphiphilic lipids have a critical packing parameter (CPP) of about 1.0 or greater.

[0055] In embodiments, one or more fusogenic amphiphilic lipids have a CPP of about 1.0 to about 3.0, preferably about 1.0 to about 2.5, more preferably about 1.0 to about 2.0, even more preferably about 1.0 to about 1.75, and even more preferably about 1.0 to about 1.5.

[0056] When multiple lipids are incorporated into the particle of the first embodiment, it will be understood that all references above to the CPP value of each lipid refer to the average CPP value.That is, when LLC particle comprises two or more amphiphilic substances (lipids), the average CPP can be defined as the molar average of all the CPP values ​​of the component amphiphilic lipids.The average CPP value can be selected from those provided above.

[0057] In an embodiment, the non-lamellar lyotropic liquid crystalline phase particles have an average CPP value of about 1.0 to about 3.0, preferably about 1.0 to about 2.5, more preferably about 1.0 to about 2.0, even more preferably about 1.0 to about 1.75, and even more preferably about 1.0 to about 1.5.

[0058] CPP is next:v / a0l c where l c is the effective length of the amphiphilic (lipid) chain, a is the effective surfactant headgroup area (determined by the balance between interchain attractive and headgroup repulsive interactions), and v is the average volume occupied by the amphiphilic molecule.

[0059] Without wishing to be bound by theory, the inventors hypothesize that an average CPP value of about 1.0 to about 3.0, optimally about 1.0 to about 1.5, provides a particle curvature that allows for both entrapment and subsequent release of the active agent. The CPP value may also allow for prediction of the likelihood of the particle fusing with a biological membrane.

[0060] Similarly, the natural splay value corresponds to the natural curvature of non-lamellar LLC particles. When two lipids have corresponding natural splay energies, fusion between them becomes more energetically favorable. Therefore, particles of the first embodiment having the following splay value are more likely to undergo the desired fusion event with biological membranes, such as bacterial membranes.

[0061] The choice of fusogenic lipid will obviously affect the spray and can be determined based on the choice of hydrophobic substance to enhance chain spray, including, for example, using myristyl, pentadecenyl, oleyl, elaidyl, linoleyl, linolenyl, arachindonyl, docosenyl and / or isoprenoid-type hydrophobic substances, such as 3,7,11-trimethyl-dodecyl, 5,9,13-trimethyltetradecanyl, 3,7,11,15-tetramethyl-hexadecyl, 5,9,13,17-tetramethyloctadecyl. Non-limiting examples of such lipids include ME, MP, MM, MV, MO, ML, and MR, as known in the art.

[0062] The energy cost per surface area for spraying is approximated by the following formula:

number

number

number

number

[0063] In an embodiment, the non-lamellar lyotropic liquid crystalline phase particles have a particle size of −0.05 nm -1 Internal curvature-induced splay of less than

number

[0064] In an embodiment, the non-lamellar lyotropic liquid crystalline phase particles have a particle size of -0.10 nm -1 Internal curvature-induced splay of less than

number

[0065] In an embodiment, the non-lamellar lyotropic liquid crystalline phase particles have a particle size of −0.15 nm -1 Internal curvature-induced splay of less than

number

[0066] In an embodiment, the non-lamellar lyotropic liquid crystalline phase particles have a particle size of −0.20 nm -1 Internal curvature-induced splay of less than

number

[0067] In an embodiment, the non-lamellar lyotropic liquid crystalline phase particles have a particle size of −0.25 nm -1 Internal curvature-induced splay of less than

number

[0068] In embodiments, the non-lamellar lyotropic liquid crystalline phase particles have a particle size of about -0.05 nm -1 ~about -0.95nm -1 , or about -0.05 nm -1 ~about -0.85nm -1 , or about -0.05 nm -1 ~about -0.75nm -1 , or about -0.05 nm -1 ~about -0.65nm -1 , or about -0.05 nm -1 ~about -0.55nm-1 , or about -0.05 nm -1 ~about -0.40nm -1 , or about -0.10 nm -1 ~about -0.95nm -1 , or about -0.10 nm -1 ~about -0.85nm -1 , or about -0.10 nm -1 ~about -0.75nm -1 , or about -0.10 nm -1 ~about -0.65nm -1 , or about -0.10 nm -1 ~about -0.55nm -1 , or about -0.10 nm -1 ~about -0.40nm -1 , or about -0.15 nm -1 ~about -0.75nm -1 , or about -0.15 nm -1 ~about -0.65nm -1 , or about -0.15 nm -1 ~about -0.55nm -1 , or about -0.15 nm -1 ~about -0.40nm -1 , or about -0.20 nm -1 ~about -0.75nm -1 , or about -0.20 nm -1 ~about -0.65nm -1 , or about -0.20 nm -1 ~about -0.55nm -1 , or about -0.25 nm -1 ~about -0.75nm -1 , or about -0.25 nm -1 ~about -0.65nm -1 , or about -0.25 nm -1 ~about -0.55nm -1 Internal curvature-guided spray

number

[0069] Furthermore, one skilled in the art, in light of this disclosure, can use the following formula to ascertain the appropriate level of curvature to provide the benefits described herein:

number

[0070] In an embodiment, the lattice parameter of the non-lamellar lyotropic liquid crystal phase particles is from about 20 to about 684 Å, or from about 20 to about 500 Å, or from about 20 to about 400 Å, or from about 20 to about 200 Å, or from about 20 to about 190 Å, or from about 20 to about 180 Å, or from about 20 to about 170 Å, or from about 20 to about 160 Å, or from about 20 to about 150 Å, or from about 40 to about 684 Å, or from about 40 to about 500 Å, or from about 40 to about 400 Å, or about 40 to about 200 Å, or about 40 to about 190 Å, or about 40 to about 180 Å, or about 40 to about 170 Å, or about 40 to about 160 Å, or about 40 to about 150 Å, or about 60 to about 684 Å, or about 60 to about 500 Å, or about 60 to about 400 Å, or about 60 to about 200 Å, or about 60 to about 190 Å, or about 60 to about 180 Å, or about 60 to about 170 Å, or about 60 to about 160 Å, or about 60 to about 150 Å, or about 80 to about 684 Å, or about 80 to about 500 Å, or about 80 to about 400 Å, or about 80 to about 200 Å, or about 80 to about 190 Å, or about 80 to about 180 Å, or about 80 to about 170 Å, or about 80 to about 160 Å, or about 80 to about 150 Å, or about 100 to about 684 Å, or about 100 to about 500 Å, or about 100 to about 400 Å, or about 100 to about 200 Å, or about 100 to about 19 0 Å, or about 100 to about 180 Å, or about 100 to about 170 Å, or about 100 to about 160 Å, or about 100 to about 150 Å, or about 120 to about 684 Å, or about 120 to about 500 Å, or about 120 to about 400 Å, or about 120 to about 200 Å, or about 120 to about 190 Å, or about 120 to about 180 Å, or about 120 to about 170 Å, or about 120 to about 160 Å, or about 120 to about 150 Å.

[0071] Lattice parameters can range up to 684 Å, for example, with sponge particles typically having larger lattice parameters than cubosomes or hexosomes. More commonly, more expanded phases may have values ​​between 200 and 400 Å. Expanded lattice parameters have certain design rules, including that the head groups may contain electrostatic charges, which can be negatively charged (e.g., PG and PS phospholipids) or positively charged (e.g., DOTAP and DOMA). The head groups may contain hydrating agents with multiple hydroxyl groups (e.g., DGMO and OG). The hydrophobic regions may contain cholesterol or other stiffening agents to stabilize the membrane and / or amphiphiles (e.g., PC and PE phospholipids) to promote reduced membrane curvature. Lipid-PEG polymers (e.g., DOPE-PEG and MO-PEG) can be used in combination with charged lipids to expand water channels. Additionally, block copolymers (e.g., Pluronic F127, F108, and Polysorbate 80) can be used as stabilizers when nanoparticle dispersions are needed, although they may not have a direct effect on the swelling of the water channels.

[0072] In embodiments, at least one of the one or more lipids displays or is modified to display a charge.

[0073] In embodiments, the non-lamellar lyotropic liquid crystalline phase particles may further comprise non-amphiphilic and / or non-fusogenic charged compounds.

[0074] As shown in the results, the presence of charged species has been found to have beneficial effects in terms of promoting contact between the particles of the first embodiment and biological membranes. This is believed to be due to the positive charge on the non-lamellar LLC particles, which helps overcome any steric or electrostatic barriers that might otherwise separate the particles and biological membranes. The number of contact events is thereby increased, which can lead to a correspondingly greater number of fusion and delivery events. This is particularly important for bacterial membranes, such as Gram-negative bacterial membranes, which exhibit a net negative charge.

[0075] Charge can be generated by incorporating one or more charged species into the non-lamellar LLC particles. This can be achieved by including charged amphiphiles or surfactants. It will be understood that a wide range of cationic lipids, surfactants, and related compounds known in the art may be suitable.

[0076] In embodiments, the non-lamellar LLC particles may include a cationic lipid.

[0077] In embodiments, a cationic lipid may be a lipid that includes a nitrogen-containing head group that may bear a positive charge.

[0078] Positively charged species, such as the head groups of amphiphilic or surfactant molecules, are also hypothesized to be important in generating the initial perturbation of the biological membrane desired to fuse the non-lamellar LLC particles of the first embodiment, which may include membrane bending, tilting, expansion, etc., which may then be the initiating event to fusion.

[0079] In embodiments, the cationic lipid may be incorporated into the non-lamellar LLC particles in an amount of less than 0.1 to 20 mol%, or less than 0.1 to 10 mol%, or less than 0.1 to 5 mol%, or less than 0.1 to 4 mol%, or less than 0.1 to 3 mol%, or less than 0.1 to 2 mol%, or less than 0.5 to 20 mol%, or less than 0.5 to 10 mol%, or less than 0.5 to 5 mol%, or less than 0.5 to 4 mol%, or less than 0.5 to 3 mol%, or less than 0.5 to 2 mol%.

[0080] In embodiments, the lipid carriers, which may be non-lamellar lyotropic liquid crystalline phase particles, may have a zeta potential that is greater than 0 mV.

[0081] In embodiments, the non-lamellar lyotropic liquid crystalline phase particles may have a zeta potential of greater than about 0 mV to about 60 mV, greater than about 0 mV to about 50 mV, greater than about 0 mV to about 40 mV, about 1 mV to about 60 mV, about 1 mV to about 50 mV, or about 1 mV to about 40 mV.

[0082] The inventors further hypothesize that designing and controlling the viscosity of the particles of the first embodiment is an important parameter to consider so as to achieve proper fusion of the particles of the first embodiment with biological membranes, such as those of Gram-negative bacteria, mycobacteria, or fungi. Specifically, it is important to ensure that the particles of the first embodiment have sufficient viscosity to achieve sufficient adhesion upon contact with the biological membrane and not detach. This will also ensure that the nanostructure is maintained under most flow conditions.

[0083] In embodiments, the non-lamellar lyotropic liquid crystalline phase particles have a viscosity of about 10 Pas at 0°C to 40°C. -1 ~approximately 1×10^6Pas -1 The viscosity may be

[0084] In embodiments, the non-lamellar lyotropic liquid crystalline phase particles are -1 At shear rates below about 5 cST to about 30,000 cST, or about 10 cST to about 20,000 cST.

[0085] One or more fusogenic amphiphilic lipids can be selected from those known in the art, particularly for forming cubosomes and hexosomes. The selection of one or more suitable fusogenic amphiphilic lipids can be based on certain requirements understood in the art. For example, lipids can be selected from those that adopt a type II lyotropic liquid crystalline phase at ambient and physiological temperatures. Parameters that may be relevant for selecting a suitable lipid include (i) for the hydrophobic component: 1. The temperature must be above the chain melting temperature so that molten chains exist, and (ii) for the head group: 1. At least one cis-unsaturated bond in the carbon chain of at least 14 carbons must be present, at least in the middle position along the backbone, or (iii). The carbon backbone must contain at least 12 carbons, three of which are secondary carbons with methyl branches, and (iv) for the head group: 5. The molecular weight of the hydrophobic substance must be at least 200 amu, and (ii) for the head group: 5. The head group must contain at least three functional groups with minimal hydrophilicity (e.g., hydroxyl), 6. The head group must be capable of forming a head group-water hydrogen bond network, and 7. The head group area must be small compared to the hydrophobic footprint. As a guide, this includes the MO lipids used in the examples of this disclosure because they meet criteria 1, 2, and 4 for hydrophobic materials and criteria 5, 6, and 7 for head groups. It will be understood that many other lipids that adequately meet these criteria are available and can be selected based on these criteria being known or readily ascertained.

[0086] Guidance may be found in one or more of the following publications, each of which is incorporated herein by reference in its entirety: US Pat. (i)T.Kaasgaard and CJDrummond“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 CJDrummond“Lyotropic Liquid Crystal Engineering-Ordered Nanostructured Small Molecule Amphiphile Self-Assembly Materials by Design”Chem.Soc.Rev.,2012,41,1297-1322 DOI:10.1039 / c1cs15148g, (iii)L.van't Hag,SLGras,CEConn and CJDrummond“Lyotropic liquid crystal engineering moving beyond binary compositional space-Ordered nanostructured amphiphile self-assembly materials by design”Chem.Soc.Rev.,2017,46,2705-2731.DOI:10.1039 / c6cs00663a, and (iv) S. Sarkar, N. Tran, Md H. Rashid, TCLe, I. Yarovsky, CEConn and CJ Drummond “Toward cell membrane biomimetic lipidic cubic phases: a high-through explorationput of lipid compositional space”ACS Applied Biomaterials,2019,2,182-195.DOI:10.1021 / acsabm.8b00539.

[0087] Polyhydroxyls (glycolipids) and polyethers (polyethylene oxide) form two of the largest categories of Type II headgroups. Non-limiting examples of headgroup motifs include alcohols, fatty acids, monoacylglycerides, MAG, 2-MAG, glycerates, glyceryl ethers, ethylene oxide, amides, monoethanolamides, diethanolamides, serinolamides, methylpropanediolamides, ethylpropanediolamides, ureas, urea alcohols, biurets, biuret alcohols, urea derivatives, endocannabinoids (anandamide, virodamine, 2-glycerol, dopamine, 2-glycerol ether), and glycolipids. Examples include phospholipids such as DMPC and DMPE.

[0088] In embodiments, the one or more fusogenic amphiphilic lipids may be selected from the group consisting of ethylene oxide amphiphiles, monoacylglycerol amphiphiles, glycolipid amphiphiles, phosphatidylethanolamine amphiphiles, and urea-based amphiphiles, and derivatives or analogs thereof.

[0089] Ethylene oxide amphiphiles include C 12 (EO)2, C 12 (EO)4, C 12 (EO)5, and C 12(EO)6 and dialkylethylene oxide amphiphiles may be included. Monoacylglycerols may include monomyristolein, monoolein, monovaccenin, and monoerucin. Amphiphiles similar to monoacylglycerols may be suitable, including oleyl glycerate, phytanyl glycerate, glyceryl monooleyl ether, glyceryl phytanyl ether, phytantriol, and monononadecenoin. Glycolipids with sugar moieties that may be suitable 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 dodecylurea (DU), octadecylurea (ODU), oleylurea (OU), oleyl biuret (OBU), linoleylurea (LU), phytanylurea (PU), hexahydrofarnesylurea (HFU).

[0090] In embodiments, the one or more fusogenic amphiphilic lipids are 1-monoolein, 2-monoolein, cytreme, lactate oleoyl, oleamide, monoelaidin, linoleic acid, elaideic acid, monopalmitolein, monolinolein, phytantriol, diolein, triolein, dioleoylglycerol, didodecyldimethylammonium bromide, dioctadecyl(dimethyl)ammonium chloride (DOAC / DODMAC) or bromide (DODAB), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE ... The oleoyl-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 (tocopheryl) acetate, phytanoyl monoethanolamide, farnesoyl monoethanolamide, oleoyl monoethanolamide, linoleoyl monoethanolamide, and linolenoyl monoethanolamide.

[0091] Single-chain amphiphilic lipids include saturated C7-C16 fatty acids, oleic acid, elaide acid, linoleic acid, sodium / gadolinium oleate, oleamide, 1-glyceryl monooleyl ether (GME), GMO, 2-MO, lactated oleoyl, cytrem, diglycerol oleate (DGMO), lyso(1-oleoyl)-phosphatidylcholine, (Z)-octadec-9-enylferrocene, N-dodecyl-caprolactam (C12), vitamin K1, ubiquinone-10 (coenzyme Q10), vitamin E, vitamin E acetate, vitamin A palmitate, amic acid, alpha-tocopheryl PEO1000 succinate (vitamin E TPGS), PEG2000-MO, PEG-PT, and PEO. x -stearate (x=40-100), polysorbate-80.

[0092] Amphiphilic lipids with multiple alkyl chains include didodecyldimethylammonium bromide (DDAB), di(canola ethyl ester)dimethylammonium chloride (DEEDAC); dioctadecyl(dimethyl)ammonium chloride (DOAC / DODMAC) or bromide (DODAB); diolein; dioleoylglycerol (DOG), EDTA-bi-oleoyl; EDTA-bi-phytanyl; 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); 1,2-dioleoyl-phosphatidic acid (DOPA); 1,2-dioleyl-phosphatidylglycerol (DOPG), 1,2-distearoyl-phosphatidylglycerol (DOPG), and 1,2-distearoyl-phosphatidylglycerol (DOPA). 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Distearoyl-glycero-3-phosphoethanolamine (DSPE), 1,2-Dioleoyl-phosphatidylcholine (DOPC), 1-Palmitoyl-2-Dioleoyl-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-nC 10 ) may be selected from the group consisting of

[0093] In any embodiment herein, the fusogenic amphiphilic lipid can be monoolein and / or phytantriol.

[0094] In any embodiment herein, the lipid particles of the present disclosure may comprise MO or phytantriol in combination with one or more of cholesterol, DLPC, DSPC, DPPE, DPPS, DOPS, DPPC, DMPC, DMPS, and DLPS.

[0095] Monoacylglycerols are known to form antiphases across a large region of their phase diagram, with monoolein being the most prominent. Antiphase formation is favored due to the kink introduced by the cis-double bond. Longer acyl chains increase the hydrophobic chain volume, making monoolein more wedge-shaped and shifting it toward type 2 in the mesophase spectrum. As the double bond approaches the lipid terminus, its effect diminishes, resulting in less wedge-shaping. Acyl chain elongation is expected to drive mesophase formation toward type 2, and on this basis, it is not surprising that the H2 phase becomes the dominant phase in such a transition.

[0096] In embodiments, when the non-lamellar lyotropic liquid crystalline phase particles comprise at least two fusogenic amphiphilic lipids, at least one of the fusogenic amphiphilic lipids may be selected from monoolein and phytantriol.

[0097] In such embodiments, monoolein and / or phytantriol fusogenic amphiphilic lipids may be combined, individually or in combination, with one or more of triolein, vitamin E, and DOPE. If it is desired to present a charge on the non-lamellar lyotropic liquid crystalline phase particles, these combinations may be further combined with one or more cationic lipids selected from those which are themselves well known in the art and commercially available, including a wide variety of quaternary ammonium cationic compounds.

[0098] Certain lipids may be specifically selected for their effect on the internal curvature of the final lipid particle, e.g., DOPE. When such lipids are included in addition to a primary fusogenic lipid such as monoolein, they may be present at approximately 10-40 mol%.

[0099] Representative cationic lipids may be selected from the following non-limiting examples: 3-β[ 4 N( 1 N 8-diguanidinospermidine)-carbamoyl]cholesterol (BGSC); 3-β[N,N-diguanidinoethylaminoethane)-carbamoyl]cholesterol (BGTC); N,N 1 , N 2 , N 3Tetramethyltetrapalmitylspermine (Cellfectin); NtN'-butyl-N'-tetradecyl-3-tetradecyl-aminopropionamidine (CLONfectin); dimethyldioctadecylammonium bromide (DDAB); 1,2-myristoxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE); 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-p-lopanax(nitrotrifluoroacetate) ) (DOSPA); 1,3-dioleoyloxy-2-(6-carboxyspermyl)-propylamide (DOSPER); 4-(2,3-bis-palmitoyloxy-propyl)-1-methyl-1H-imidazole (DPIM); N,N,N',N'-tetramethyl-N,N'-bis(2-hydroxyethyl)-2,3-dioleoyloxy-1,4-butanediammonium 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 substituted with a cholesteryl group (in the case of ChOTB) DORI (DL-1,2-dioleoyl-3-dimethylaminopropyl (disclosed in WO 93 / 03709)-β-hydroxyethylammonium) or DORIE (DL-1,2-O-dioleoyl-3-dimethylaminopropyl)-β-hydroxyethylammonium) (DORIE) or its analogs. They are double-stranded (DOT) via the butanol spacer arm 1,2-dioleoyl-3-(4'-trimethylammonio)butanol-sn-glycerol (DOBT) or cholesteryl (4'-trimethylammonia) butanoate (ChOTB); 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC); tetraoctylammonium bromide (TOAB) as a cationic phase transfer agent; cholesteryl hemisuccinate ester (ChOSC);Mydonglycylspermine (DOGS) and dipalmitoylphosphatidylethanolamylspermine (DPPES) or cationic lipids disclosed in U.S. Pat. No. 5,283,185, such as cholesteryl-3β-carboxy-amido-ethylenetrimethylammonium chloride, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesterylcarboxylate iodide, cholesteryl-3-O-carboxyamidoethyleneamine, cholesteryl-3-β-oxysuccinamido-ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3-β-oxysuccinate iodide, 2-(2-trimethylammonio)-ethylmethylaminoethyl-cholesteryl 3-β-Oxysuccinate iodide, 3-β-N-(N',N'-dimethylaminoethane)carbamoylcholesterol (DC-chol), and 3-β-N-(polyethyleneimine)-carbamoylcholesterol; O,O-Dimyristyl-N-lysyl-aspartate (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-Dipaltoyl (dipal Toyl)-sn-glycero-3-ethylphosphocholine (DPEPC); 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSEPC); 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); dioleoyldimethyldimethylaminopropane (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).

[0100] In embodiments, particularly preferred cationic lipids are DOTAP and / or DODAB and / or tetraoctylammonium bromide (TOAB).

[0101] In embodiments, the non-lamellar lyotropic liquid crystalline phase particles may include a fatty acid in addition to one or more fusogenic lipids, such as, for example, oleic acid.

[0102] In embodiments, the non-lamellar lyotropic liquid crystalline phase particles may include at least one stabilizer, which may be selected from those known in the art.

[0103] Preferably, the stabilizer is a poloxamer or surfactant, or a PEGylated lipid stabilizer, or modified versions thereof.

[0104] In embodiments, the stabilizer is selected from PEG-PPO-PEG triblock copolymers and nonionic block copolymer surfactants and PEO copolymerized with a charged moiety. Poloxamer 407 and Pluron 127 may be suitable examples of stabilizers and may be incorporated into any of the embodiments of the first aspect described herein. PEO copolymerized with (3-acrylamidopropyl)trimethylammonium chloride or a similar charge-carrying moiety may also be suitable. PEGylated lipid stabilizers, including but not limited to PEG2000-MO, PEG-PT, DSPE-PEG(2000)amine, 18:0 PEG2000 PE, and DSPE-PEG(5000)amine, are also suitable. Many such stabilizers are known in the art.

[0105] The use of a stabilizer is preferred, and the nature of the stabilizer may be selected based on the nature of the lipid, although the selection and understanding of the compatibility of these ingredients may be based on information known in the art.

[0106] Many steric stabilizers reported to date can be divided into four groups: (i) amphiphilic block copolymers (i.e., Poloxamer™), (ii) PEGylated lipids, (iii) customized lipid-copolymers, and (iv) alternative steric stabilizers (e.g., bile salts, proteins). Ideally, the stabilizer selected prevents particle aggregation by providing an electrostatic barrier, or more generally, a steric barrier, between approaching particles. Stabilizers that can function optimally in the lipid particles of the present disclosure share similar properties, including (i) they are generally highly hydrophilic, with a high HLB (hydrophilic-lipophilic balance) value, due to the asymmetric amphiphilic polymer structure with larger hydrophilic domains. It is important that the hydrophilic portion of the molecule is not surrounded by hydrophobic regions. A high HLB can be achieved by using longer or multiple PEG chains, (ii) the presence of hydrogen bond acceptors and the absence of hydrogen bond donors, and (iii) electroneutrality. Those skilled in the art can select an appropriate stabilizer based on this information. Additionally, the following journal articles address important aspects of stabilizers that may be suitable for use with the lipid particles of the present disclosure and are incorporated herein by reference in their entirety: (i)JYTChong,X.Mulet,BJBoyd and CJDrummond;“Steric Stabilizers for Cubic Phase Lyotropic Liquid Crystal Nanodispersions(Cubosomes)”in “Advances in Planar Lipid Bilayers and Liposomes”,Vol 21,Chp 5, (2015) p.131-187, ISSN1554-4516, Elsevier; (ii) J. Zhai, B. Fan, SHThang, CJ Drummond “Novel amphiphilic block copolymers for the formation of stimuli-responsive non-lamellar lipid nanoparticles” Molecules,2021,26,3648-3664.DOI:10.3390 / molecules26123648;(iii)J.Zhai,R.Suryadinata,B.Luan,N.Tran,T.MHinton,J.Ratcliffe,X.Hao and C.J.Drummond “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 B.W.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)J.Y.T.Chong,X.Mulet,D.Keddie,L.J.Waddington,S.T.Mudie,B.J.Boyd and C.J.Drummond “Novel Steric Stabilisers for Lyotropic Liquid Crystalline Nanoparticles:Pegylated Phytanyl Copolymers” Langmuir,2015,31,2615-2629.DOI:10.1021 / la501471z;(vi)J.Y.T.Chong,X.Mulet,A.Postma,D.J.Keddie,L.J.Waddington,B.J.Boyd and C.J.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,C.J.Drummond and B.J.Boyd “Disposition and Association of the Steric Stabiliser Pluronic F127 in Lyotropic Liquid Crystalline Nanostructured Particle Dispersions” J.Colloid and Interface Science,2013,392,288-296.DOI:10.1016 / j.jcis.2012.09.051(viii)J.Y.T.Chong,X.Mulet,L.J.Waddington,B.J.Boyd and C.J.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)J.Y.T.Chong,X.Mulet,L.J.Waddington,B.J.Boyd and C.J.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.

[0107] A stabilizer may be present during the formation of the particles of the first embodiment.

[0108] The stabilizer may be present at 5 to 20 wt%, 6 to 18 wt%, 7 to 16 wt%, or 8 to 14 wt%.

[0109] In an embodiment, the lipids forming the particles of the first aspect comprise substantially: (a) monolein and / or phytantriol; (b) monolein and / or phytantriol, and DOPE; (c) monolein and / or phytantriol, and DOTAP; (d) monolein and / or phytantriol, and TOAB; (e) monolein and / or phytantriol, and oleic acid; (e) Monolein and / or phytantriol, and DOPE and DOTAP.

[0110] In embodiments in which the lipid particles of the first aspect include DOPE, it may be present at 10 to 40 mol %.

[0111] In embodiments in which the lipid particles of the first aspect include DOTAP, it may be present at 0.5 to 5 mol %, or 0.5 to 4 mol %.

[0112] In certain embodiments, the lipids forming the particles of the first aspect may comprise substantially: a) monoolein, or b) monoolein (80 to 99.9 mol%), triolein (0.1 to 20 mol%), or c) monoolein (80 to 99.9 mol%), vitamin E (0.1 to 20 mol%), or d) Monoolein (80-99.9 mol%), DOPE (0.1-20 mol%).

[0113] In certain embodiments, the lipids forming the particles of the first aspect may comprise substantially: a) monoolein (95 to 99.9 mol%), DOTAP (0.1 to 5 mol%), or b) monoolein (95 to 99.9 mol%), DODAB (0.1 to 5 mol%), or c) Any lipid from the list above with DOTAP (0.1-5 mol%).

[0114] The non-lamellar lyotropic liquid crystalline phase particles may consist of or consist essentially of monoolein and / or phytantriol and one or two amphiphilic lipids selected from the list of amphiphilic lipids above.

[0115] In embodiments, the one or more amphipathic lipids may be selected from those presenting a hydrophobic tail group selected from the group consisting of oleoyl, linoleoyl, linolenoyl, phytanoyl, farnesoyl, or an extended aliphatic hydrophobic chain.

[0116] In such embodiments, the head groups of the fusogenic amphiphilic lipids may be "non-conventional" head groups modified to display chemical moiety charges or other desired surface functionality.

[0117] In embodiments in which at least one fusogenic amphiphilic lipid exhibits a non-conventional head group, it may be a peptide or cationic head group that does not normally associate with the attached hydrophobic tail. In one non-limiting example, the head group may be an aminoglycoside-based head group.

[0118] In embodiments, the hydrophobic tail of the fusogenic amphiphilic lipid may be a "non-traditional" tail group modified to exhibit specific surface functionality or physical characteristics. In particular, the tail group may be modified to allow control over the final CPP value of the particle. Thus, in embodiments, the hydrophobic tail of the fusogenic amphiphilic lipid may be selected to provide a CPP value of about 1.0 to about 3.0, preferably about 1.0 to about 2.5, more preferably about 1.0 to about 2.0, even more preferably about 1.0 to about 1.75, and even more preferably about 1.0 to about 1.5.

[0119] In embodiments in which the non-lamellar lyotropic liquid crystalline phase particles comprise one or more charged amphiphiles or surfactants, elicitor or otherwise, they may be selected from the group consisting of DOTAP, DOTMA, DODAP, dioctadecyl(dimethyl)ammonium chloride (DOAC / DODMAC) or bromide (DODAB), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and CTAB.

[0120] In embodiments, an active agent may be selected from any substance that can affect or help identify any physical or biochemical property of an interaction relevant to a biological system, pathway, molecule, or organism, including, but not limited to, animals and humans. In particular, as used herein, a drug includes, but is not limited to, any substance intended for the diagnosis, cure, detection, mitigation, treatment, or prevention of disease in humans or other animals, or otherwise enhancing the physical or mental well-being of humans or animals. Examples of biologically active molecules include, but are not limited to, peptides, proteins, dyes, enzymes, and small molecule drugs. Classes of active agents suitable for use with the methods and compositions described herein include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, bactericides, metal-containing nanoparticles, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, anxiolytic agents, hormones, growth factors, steroids, gene expression modifiers, knockdown agents, siRNAs, RNAi agents, Dicer substrates, miRNAs, shRNAs, antisense oligonucleotides, aptamers, microbial toxins, antibodies and fragments thereof, including anti-tuberculosis antibody fragments, and the like.

[0121] Particularly preferred active agents may include any agent known to be effective against gram-negative bacteria and / or mycobacteria and / or fungi.

[0122] The active agent may be present at 0.1-30.0 mol%, or 0.1-25.0 mol%, or 0.1-20.0 mol%, or 0.1-10.0 mol%, or 0.1-5.0 mol%, or 0.1-4.0 mol%, or 0.1-3.5 mol%, or 0.1-3.0 mol%, or 0.5-30.0 mol%, or 0.5-25.0 mol%, or 0.5-20.0 mol%, or 0.5-10.0 mol%, or 0.5-5.0 mol%, or 0.5-4.0 mol%, or 0.5-3.5 mol%, or 0.5-3.5 mol%.

[0123] One advantage of the present invention is that it can use active agents that cannot otherwise be used as "free" active substances.For example, it is known that certain antibiotics bind to serum proteins to such an extent that their effectiveness is significantly reduced.Alternatively, some active agents may be active against their target when inside bacterial cells, but cannot pass through the bacterial membrane.This approach provides lipid carrier particles that protect active agents from degradation or avoid unnecessary binding to serum proteins, or provides improved delivery to bacterial or fungal cells.

[0124] It will be understood that the term "agent" or "active agent" may include the active compound in pharmaceutically effective or acceptable salt form.

[0125] In any embodiment herein, the active agent is not a metal nanocrystal.

[0126] Thus, in one embodiment, there are non-lamellar lyotropic liquid crystalline phase particles comprising one or more fusogenic amphiphilic lipids, and particles encapsulating an active agent, the particles having (i) an average CPP of about 1.0 to about 3.0, (ii) a zeta potential of greater than 0 mV, and (iii) a viscosity of about 10 Pas from 0° C. to 40° C. -1 ~approx. 1e6Pas -1 and a dynamic viscosity of

[0127] In such embodiments, the average CPP value can be from about 1.0 to about 2.5, more preferably from about 1.0 to about 2.0, even more preferably from about 1.0 to about 1.75, and even more preferably from about 1.0 to about 1.5.

[0128] In embodiments, non-lamellar lyotropic liquid crystalline phase particles comprising one or more amphiphilic lipids and particles encapsulating an active agent, the particles having a diameter of (i) about -0.10 nm -1 ~about -0.55nm -1 Internal curvature-guided spray

number

[0129] In embodiments, the non-lamellar lyotropic liquid crystal phase particles have a particle diameter of 50 nm to 450 nm, 80 nm to 400 nm, 100 nm to 300 nm, or 120 nm to 300 nm.

[0130] Preferably, the non-lamellar lyotropic liquid crystalline phase particles are cubosome particles.

[0131] Preferably, the active agent is one or more of a gram-negative bacterial active agent or a mycobacterial antibacterial active agent or an antifungal agent.

[0132] Suitably, the non-lamellar lyotropic liquid crystalline phase particles, which in one embodiment are cubosome nanocarrier particles, comprise one or more positively charged lipids or stabilizers.

[0133] In a second aspect, there is provided a pharmaceutical composition comprising the non-lamellar lyotropic liquid crystalline phase particles of the first aspect, a lipid vehicle which may be a pharmaceutically acceptable carrier, diluent and / or excipient.

[0134] Suitably, the pharmaceutically acceptable carrier, diluent, and / or excipient may be or comprise one or more of a diluent, solvent, pH buffer, binder, filler, emulsifier, disintegrant, polymer, lubricant, oil, fat, wax, coating agent, viscosity modifier, flow agent, and the like.

[0135] Diluents may include one or more of microcrystalline cellulose, lactose, mannitol, calcium phosphate, calcium sulfate, kaolin, dry starch, powdered sugar, etc. Binders may include one or more of povidone, starch, stearic acid, gum, hydroxypropyl methylcellulose, etc. Disintegrants may include one or more of starch, croscarmellose sodium, crospovidone, sodium starch glycolate, etc. Solvents may include one or more of ethanol, methanol, isopropanol, chloroform, acetone, methyl ethyl ketone, methylene chloride, water, etc. Lubricants may include one or more of magnesium stearate, zinc stearate, calcium stearate, stearic acid, sodium stearyl fumarate, hydrogenated vegetable oil, glyceryl behenate, etc. Flow agents may be one or more of colloidal silicon dioxide, talc, corn starch, etc. The buffering agent may include, but is not limited to, phosphate buffer, borate buffer, and carbonate buffer. The filler may include, but is not limited to, one or more gels, including gelatin, starch, and synthetic polymer gels. The coating agent may include one or more of a film-forming agent, a solvent, a plasticizer, and the like. Suitable film-forming agents may be one or more of hydroxypropylmethylcellulose, methylhydroxyethylcellulose, ethylcellulose, hydroxypropylcellulose, povidone, sodium carboxymethylcellulose, polyethylene glycol, acrylates, and the like. Suitable solvents may be one or more of water, ethanol, methanol, isopropanol, chloroform, acetone, methyl ethyl ketone, methylene chloride, and the like. The plasticizer may be one or more of propylene glycol, castor oil, glycerin, polyethylene glycol, polysorbate, and the like.

[0136] Reference is made to Handbook of Excipients 6th Edition, Eds. Rowe, Sheskey & Quinn (Pharmaceutical Press), which provides non-limiting examples of excipients that may be useful according to the present invention.

[0137] It will be understood that the choice of pharmaceutically acceptable carrier, diluent, and / or excipient 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.

[0138] Preferably, the pharmaceutical composition is a liquid dispersion of the particles of the first embodiment. The liquid dispersion can be an aqueous dispersion. The liquid dispersion can be encapsulated in a standard capsule known for delivering liquid formulations.

[0139] Suitably, the pharmaceutical composition is for the treatment or prevention of a disease, disorder or condition in a mammal, as further described herein.

[0140] In a third aspect, there is provided a method for controlled release of an active agent, comprising forming the non-lamellar lyotropic liquid crystalline phase particles of the first aspect and administering the non-lamellar lyotropic liquid crystalline phase particles to a target area.

[0141] In embodiments, the target area may be a gram-negative bacterial infection, or a mycobacterial infection, or a fungal infection. The infection may be in a mammal.

[0142] In a fourth aspect, there is provided a method of forming non-lamellar lyotropic liquid crystalline phase particles of the first aspect, comprising the steps of: (i) providing one or more fusogenic amphiphilic lipids; and (ii) exposing the one or more fusogenic amphiphilic lipids to a solution in the presence of an active agent.

[0143] The solution may be an aqueous solution.

[0144] In embodiments, the solution may be tailored to be flexible through the use of hydrophobic active agents, for example, a solvent mixture may be used.

[0145] As discussed, the particles of the first embodiment may provide a desirable release profile of the active agent due to the entrapment of the active agent within the complex internal architecture of the non-lamellar LLC particles.

[0146] The particle of the first aspect can be formed in various ways, depending on what exactly the desired composition is.In brief, the selected fusogenic lipid can be combined with suitable active agent, and then be exposed to, for example, aqueous solution to induce self-assembly.Usually, stabilizer is also included in aqueous solution.The stabilizer such as poloxamer and pluronics, including those mentioned above, can be suitable.

[0147] If it is desired to include an additional charged component within the particle, DOTAP, or a similarly charged species, may be included with the fusogenic lipid and active agent.

[0148] The properties of these components can be selected to affect the mesophase of the final particle product, which in turn affects the release profile of the active agent.

[0149] The target area can be any area to which it is desired to deliver an active agent. Typically, the target area will be within a biological sample, tissue, or fluid of a subject, such as a human subject. For example, the target area can be a tissue infected with a bacterial infection to which an antibacterial agent is delivered via the particles of the first embodiment.

[0150] It will be understood that the final release profile can be best achieved by using several different particles of the first embodiment with different release profiles of the same activity.For example, the above parameters can be selected to provide two different particle populations.One population can be a cubosome population, and the other can be a hexosome population, each carrying antibacterial properties.The different populations can be administered separately or together, and due to their different internal architectures and different fusogenic lipids, they can deliver antibacterial agents at different rates to achieve therapeutic effects over a longer time frame.

[0151] Alternatively, the different populations may comprise different antimicrobial agents which are tailored to the active agent with which the architecture of the particles of the first embodiment is associated.

[0152] In a fifth aspect, there is provided a method of treating or preventing a disease, disorder or condition, comprising administering to a subject in need thereof a therapeutically effective amount of non-lamellar lyotropic liquid crystalline phase particles of the first aspect, or a pharmaceutical composition of the second aspect, thereby treating or preventing the disease, disorder or condition.

[0153] In a sixth aspect, there is provided the non-lamellar lyotropic liquid crystalline phase particles of the first aspect, or the pharmaceutical composition of the second aspect, for use in the treatment or prevention of a disease, disorder or condition.

[0154] In a seventh aspect there is provided the use of non-lamellar lyotropic liquid crystalline phase particles of the first aspect in the manufacture of a medicament for the treatment of a disease, disorder or condition.

[0155] As generally used herein, terms such as "administer" or "administration" refer to the introduction of the relevant particle or composition into a mammal by a particular route, vehicle, etc. Routes of administration include topical, parenteral, and enteral, including, but not limited to, oral, buccal, sublingual, nasal, anal, gastrointestinal, subcutaneous, intramuscular, and intradermal routes of administration.

[0156] "Treating," "treatment," or "treating" refers to administering the relevant particle or composition to a subject to at least ameliorate, reduce, or inhibit existing signs or symptoms of a disease, disorder, or condition experienced by the subject, to the extent that the medical condition is improved according to clinically accepted criteria. For example, "treating a bacterial infection" means reducing the infection or eradicating the infection or alleviating the symptoms of the infection in a patient, where improvement and alleviation are assessed by clinically accepted standardized tests and / or empirical tests, including swab sample tests, etc.

[0157] "Prevent," "preventing," or "prophylactic" means the prophylactic administration of a particle or composition relevant to a subject that does not exhibit signs or symptoms of a disease disorder or condition, but is expected or predicted to be likely to exhibit such signs or symptoms in the absence of prevention. Prophylactic treatment may at least alleviate or partially ameliorate the expected symptoms or symptoms.

[0158] As used herein, "effective amount" or "therapeutically effective amount" refers to the administration of a sufficient amount of the relevant particle or composition to prevent the onset 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 as understood by those skilled in the art, given the patient's age, sex, weight, etc. Appropriate dosages or dosage regimes can be ascertained through routine testing or based on current treatment regimes for the active agent delivered via the particles of the first embodiment.

[0159] As used herein, the terms "subject" or "individual" or "patient" may refer to any subject for whom treatment is desired, particularly a vertebrate subject, and more particularly a mammalian subject. Suitable vertebrates include, but are not limited to, primates, birds, livestock animals (e.g., sheep, cattle, horses, donkeys, pigs), laboratory animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs), and captive wild animals (e.g., foxes, deer, dingoes). A preferred subject is a human in need of treatment for a disease, disorder, or condition described herein. However, it will be understood that the above terms do not necessarily imply that symptoms are present. In one embodiment, the subject is a human being treated for a bacterial infection, particularly a gram-negative bacterial infection.

[0160] As used herein, the terms "co-therapy" and "combination therapy" refer to the treatment of a subject in need thereof by administering one or more particles or compositions described herein and one or more agents for treating a disease, disorder, or condition simultaneously, sequentially, separately, or in a single pharmaceutical formulation or combination. When administered in separate dosage forms, the number of doses administered per day for each compound may be the same or different. The associated 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.

[0161] As discussed above, particles of the first embodiment exhibiting different architectures may be formed and used to deliver the same or different active agents. This may be particularly relevant, for example, when treating multi-strain bacterial infections, where two or more particles may be selected based on morphology that is optimal for delivery of the relevant active agents and / or ability to fuse with or adhere to particular strains.

[0162] In embodiments, the disease, disorder, or condition may be a disease, disorder, or condition associated with or caused by a microorganism selected from the group consisting of gram-negative bacteria, including mycobacteria, and fungi.

[0163] For purposes of this disclosure, mycobacteria are considered to be Gram-negative bacteria. Mycobacteria are considered to have cell walls that exemplify aspects of both Gram-positive and Gram-negative bacteria, although important mycobacteria, such as Mycobacterium tuberculosis, may be considered genomically closer to Gram-negative bacteria than to Gram-positive bacteria: https: / / doi.org / 10.1054 / tube.2002.0328; therefore, this nomenclature applies herein. Without wishing to be bound by theory, the major common component of the cell wall is peptidoglycan, which is found in nearly all bacteria and plays a role in protecting the integrity of the cell. The complex cell wall, a unique feature of mycobacteria, is a well-recognized drug target with unusually strong hydrophobic properties. The essential core cell wall structure is composed of three main components: arabinogalactan polysaccharides, peptidoglycan, and a long outer layer, mycolic acid. Approximately 60% of the mycobacterial cell membrane is composed of lipids. The mycobacterial cell wall is shown herein to be suitable for delivery of active agents using particles of the first aspect.

[0164] In a specific embodiment, the disease, disorder or condition is a disease, disorder or condition that responds to treatment with an antimicrobial agent, including an antibiotic and / or an antifungal agent, i.e., the disease, disorder or condition is a gram-negative bacterial infection and / or a fungal infection.

[0165] In one embodiment, the disease, disorder or condition is caused by or associated with a pathogen. The pathogen may be a gram-negative bacterium or fungus capable of infecting a mammal.

[0166] In embodiments, when the infection being treated is a gram-negative infection, the causative pathogen may be selected from the group consisting of Enterobacteriaceae, Pseudomonas, Vibrio, Campylobacter, Legionella, Neisseria, Mycobacterium, Hemophilus, and Bartonella.

[0167] In embodiments, the gram-negative bacteria may be selected from the group consisting of E. coli, Pseudomonas aeruginosa, Klebsiella, Acinetobacter baumannii, Neisseria gonorrhoeae, Enterobacteriaceae, Mycobacterium tuberculosis, and Mycobacterium smegmatis.

[0168] Non-limiting examples of fungi include, but are not limited to, Candida, Cryptococcus, and Aspergillus species. In embodiments, the fungus associated with or causing a disease, disorder, or condition is selected from the group consisting of C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. dublinensis, C. krusei, C. lusitaniae, C. auris, C. neoformans, and A. fumigatus.

[0169] The inventors have found that the particles of the present disclosure offer the advantageous property of undergoing fusion events with Gram-negative bacteria and fungi, but not following similar fusion events with Gram-positive bacteria, and thus some active agents can still be delivered to Gram-positive bacteria, which exhibit optimal efficacy against Gram-negative and fungal species.

[0170] In any aspect or embodiment herein, the active agent can be a hydrophobic active agent.

[0171] In embodiments, the active agent is a hydrophobic antibacterial or antifungal active. Preferably, the hydrophobic antibacterial active is a gram-negative hydrophobic antibacterial active.

[0172] Such active agents are well known in the art and are commonly used against gram-negative bacterial and fungal infections. The present disclosure provides improved delivery vehicles for such already known antibacterial and antifungal agents, and therefore, based on the present disclosure, one skilled in the art will have no difficulty in selecting an appropriate active agent for loading the particles of the present disclosure for subsequent delivery to the site of infection.

[0173] When the active agent is an antifungal, the antifungal may be selected from azoles, echinocandins, and amphotericin B. The former two classes are inhibitors of fungal cell wall components (ergosterol and glucans, respectively), while the latter binds to ergosterol.

[0174] In embodiments, the active agent is selected from the group consisting of rifampicin, fusidic acid, ampicillin, piperacillin, gentamicin, vancomycin, filipin, amphotericin B, benzylpenicillin, meropenem, clarithromycin, novobiocin, ethambutol, isoniazid, streptomycin, fluconazole, itraconazole, and pyrazinamide. It will be understood that such active agents are only representative.

[0175] It will be appreciated that the antibacterial or antifungal agent is released into the Gram-negative bacteria or fungus following a fusion event between the non-lamellar lyotropic liquid crystalline phase particles and the outer layer or membrane of the Gram-negative bacteria or fungus. That is, the treatment of the Gram-negative or fungal infection is not a diffuse release of the active agent near or adjacent to the pathogen. Rather, it is the fusion of the non-lamellar lyotropic liquid crystalline phase particles of the first embodiment with the pathogen outer wall, layer, or membrane that allows the active agent to be released directly into the pathogen.

[0176] In an eighth aspect, there is provided a method of delivering an active agent to a biological target, the method comprising administering non-lamellar lyotropic liquid crystalline phase particles of the first aspect.

[0177] In one embodiment, the active agent being delivered may be a detection agent, and thus the delivery method may be a detection method.

[0178] The detection agent can be a tag, a probe, or a dye.

[0179] In the first aspect, the particles of active agent and biological target may be as described above for any of the above aspects. The biological target is preferably a biological sample or an in vivo tissue, organ, or fluid from a patient. In embodiments, the patient may have a bacterial infection.

[0180] Encapsulation of an active agent within a particle of the first embodiment may provide one or more advantages, including improving the solubility of the active agent or masking solubility problems, protecting the active agent from destruction or modification in vivo, affecting circulation time within a subject, reducing the cytotoxicity of the encapsulated active agent, and reducing the required dosage due to more efficient delivery into target cells.

[0181] In embodiments in which the particles of the first aspect are used to treat Gram-negative bacterial infections, the particles do not allow bacteria to enter to any significant extent via endocytosis, in other words, when the particles of the first aspect are used to treat Gram-negative bacterial infections, the active agent is delivered following and due to fusion of the particles with the bacterial membrane.

[0182] In eukaryotic cells, uptake is regulated by several endocytic pathways that engulf and internalize many known nanocarriers or therapeutic agents. Despite internalization, cellular processes, including transport to lysosomes, often lead to degradation of both the carrier and any encapsulated therapeutic agent, resulting in low therapeutic efficacy. However, due to their unique lipid bilayer motif, particles of the first embodiment can subvert these processes by utilizing a different internalization mechanism: membrane fusion of the particle's bilayer motif with either the outer cell wall or internal endosomes. The direct affinity of particles of the first embodiment with cell membranes can be rationalized by their shared self-assembly properties. While polymeric or inorganic nanocarriers are constructed by bonded tethering molecules, nonlamellar LLC particles are objects formed by intermolecular interactions. Therefore, the former require significantly greater membrane perturbation for internalization. The increased surface curvature of the constituent lipids within nonlamellar LLC particles may specifically promote bilayer fusion. Structurally, yeast cells share a similar inner plasma lipid membrane with Gram-negative bacteria, which is coated by an outer layer of chitin, glucan, and protein.

[0183] In a ninth aspect of the present invention there is provided a method of diagnosing a disease, disorder or condition in a mammal, the method comprising the step of administering non-lamellar lyotropic liquid crystalline phase particles of the first aspect or a composition of the second aspect, wherein the active agent in the non-lamellar lyotropic liquid crystalline phase particles of the first aspect is a labeled active agent to the mammal or to a biological sample obtained from the mammal to facilitate diagnosis of the disease, disorder or condition in the mammal.

[0184] It will be appreciated from the results presented herein that dyes or labeling agents can be delivered to a range of mammalian cells, and therefore that the particles of the first aspect may be useful as detection agents. The particles may also be designed to incorporate molecular tags or targeting moieties so that they are targeted to certain cell types or receptors.

[0185] The following experimental section describes in more detail the characterization and efficacy of certain compounds of the invention, the intent being to illustrate certain specific embodiments of the compounds of the invention and their efficacy without limiting the invention in any way.

[0186] experiment [Table 1] Naming convention For figures and notations used in the following text, the following will be used: Constituent_lipid-modifier_lipid-drug, e.g., MO-DOTAP-1NOVO—denotes monolein as the main lipid, further modified by DOTAP and encapsulating 1 mol % of novobiocin as the active agent.

[0187] Plots / Data / Experimental Rules Concentration is in ug / ml and is the concentration of the drug in use. Viability is usually assessed by the percentage inhibition of cell growth at various concentrations, with higher numbers corresponding to more dead bacteria. In some cases, viability is assessed by colony forming unit (CFU) count, with lower numbers corresponding to more dead bacteria.

[0188] material Monoolein (MO) (97%, Sigma), Pluronic F127 (Sigma), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (99%, Avanti), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (99%, Avanti), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) (99%, Avanti), 1,2-dioleoyl-3-trimethylammonium-propane ( DOTAP (99%, Avanti), TOAB (99%, Sigma), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N (cyanine 5) (Cy-5), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N (lissamine rhodamine B sulfonyl) (99%, Avanti), octadecylrhodamine chloride (R18) (Thermofisher), ethanol (AR), and isopropanol (AR) were used as received without further purification. Peptidoglycan extract (from S. aureus) and lipoteichoic acid (from S. aureus) were used as received by preparing 1 mg / ml dispersions. Rifampicin (98%, Sigma), fusidic acid (98%+, Sigma), ampicillin (Sigma), piperacillin (Sigma), gentamicin (Sigma), vancomycin (Sigma), filipin (Sigma), amphotericin B (Sigma), benzylpenicillin (Sigma), meropenem (Sigma), clarithromycin (Sigma), novobiocin (Sigma), ethambutol (Sigma), isoniazid (Sigma), streptomycin (Sigma), and pyrazinamide (Sigma).

[0189] McFarland 0.5 Barium Sulfate Turbidity Standard: To standardize the inoculation density, a BaSO4 turbidity standard is used (0.5 McFarland standard). The procedure consists of the following steps: (1) Prepare this turbidity standard by adding 0.5 mL of 0.048 mol / L BaCl2 (1.175% w / v BaCl2 x 2H2O) to 99.5 mL of 0.18 mol / L H2SO4 (1% v / v). (2) Using a spectrophotometer equipped with a 1 cm light path and matching cuvettes, determine the absorbance, confirming the correct density of the turbidity standards. The absorbance at 625 nm should be 0.08-0.10 for the 0.5 McFarland standard. (3) Divide 4–6 mL into screw-cap tubes of the same size as those used to grow or dilute the broth culture inoculum. (4) Seal the tubes tightly and store them in the dark at room temperature. (5) The turbidity standard is vigorously mixed on a mechanical vortex mixer immediately before use. (6) Replace standards after three months of preparation or recheck their density.

[0190] Establishing the effect of activity on particles and optimizing particle design Particle preparation: Lipid components (e.g., monoolein, 50 mg) are mixed with an active agent (e.g., Cy5, 0.1 mg) in ethanol (0.5 ml). The solution is dried in a vacuum oven for at least 12 hours. The homogeneous lipid mixture is hydrated with a solution of a stabilizer (e.g., 0.5 wt% F-127 block copolymer). The mixture is dispersed by probe sonication (Branson Ultrasonicator 250, 50% duty cycle, 5 min), resulting in a 5 wt% dispersion of cubosomes. Variations in lipid composition and encapsulated cargo can be introduced during the lipid mixing stage. Stabilizers and altered lipid components can be used to minimize any undesirable phase transitions, release profiles, or general reductions in colloidal stability, as outlined below.

[0191] The colloidal properties of the particles are characterized prior to application. Particle size and zeta potential are determined by dynamic light scattering (DLS). The internal nanostructure of the particles is determined via SAXS. This characterization is performed on both the neat particles and each encapsulated formulation. The particles require a low free drug release rate before associating with the pathogen target. The release of the encapsulated drug is determined from dialyzed samples using UV absorbance measurements under infinite sink conditions. Response to biological media is determined by repeating colloid and release experiments in the presence of buffer (e.g., PBS), culture medium (DMEM), and LB broth to provide a context for routine laboratory experiments and a solution containing serum proteins to mimic in vivo conditions. The effect of specific proteins is then determined. Following exposure to biological media, protein characterization is performed. Proteins bound to the particles are screened by SDS-PAGE gel electrophoresis using at least three replicates for each particle / environment combination. Isothermal titration calorimetry (ITC) is used to quantify the affinity of individual biomolecules for the particles. A typical screen uses 2 μL per 360 seconds, but injection volume and wait time are optimized for each thermal signature. The effect of concentration is screened over a range (10-100 μM). ITC is repeated at temperatures outlined in Aim 1 and repeated at least three times per target. Dilution experiments are run as controls.

[0192] General Procedure: Cubosomes / hexosomes were prepared by mixing lipids, e.g., monoolein (approximately 50 mg total), in ethanol (1 ml). For charge loading, DOTAP or TOAB was included in the lipid mixture to obtain 1 mol%, e.g., (MO 49.5 mg, DOTAP 0.93 mg). For drug loading, antibiotics such as Rif (3.46 mg) and Fus (2.17 mg) were included in the lipid mixture for samples MO-DOTAP-3Rif and MO-DOTAP-3Fus, respectively. The solution was then dried in a vacuum oven for at least 12 hours. The homogeneous lipid mixture was hydrated with a solution of F-127 (1 mL, 5 mg / mL). The mixture was dispersed by probe sonication (Branson Ultrasonicator 250, 50% duty cycle, 3 seconds on with 5 seconds off, total duration 5 minutes), resulting in a cubosome dispersion of approximately 5% by weight.

[0193] Examples of DOPE loading: MO-10PE-1DOTAP-monoolein (40 mg), DOPE (9.91 mg), DOTAP (0.93 mg), MO-20PE-1DOTAP-monoolein (32.5 mg), DOPE (16.7 mg), DOTAP (0.93 mg), MO-30DOPE-1TAP-monoolein (26 mg), DOPE (24 mg), DOTAP (0.93 mg), MO-40DOPE-1TAP-monoolein (21.5 mg), DOPE E (28.2 mg), DOTAP (0.93 mg), MO-1DOTAP-1Rif-monoolein (49.5 mg), DOTAP (0.93 mg), rifampicin (1.15 mg), MO-1DOTAP-2Rif-monoolein (49.5 mg), DOTAP (0.93 mg), rifampicin (2.30 mg), MO-1DOTAP-3Rif-monoolein (49.5 mg), DOTAP (0.93 mg), rifampicin (3.46 mg), MO- 1DOTAP-4Rif-monoolein (49.5 mg), DOTAP (0.93 mg), rifampicin (4.62 mg), MO-1DOTAP-1NOVO-monoolein (49.5 mg), DOTAP (0.93 mg), novobiocin (0.86 mg), MO-1DOTAP-3NOVO-monoolein (49.5 mg), DOTAP (0.93 mg), novobiocin (2.58 mg), MO-1DOTAP-5NOVO-monoolein (49.5 mg) ), DOTAP (0.93 mg), novobiocin (4.29 mg), MO-1DOTAP-1PIP-monoolein (49.5 mg), DOTAP (0.93 mg), piperacillin (0.72 mg), MO-1DOTAP-3PIP-monoolein (49.5 mg), DOTAP (0.93 mg), piperacillin (2.17 mg), MO-1DOTAP-5PIP-monoolein (49.5 mg), DOTAP (0.93 mg), piperacillin (3.63 mg).

[0194] MTS-based cell viability assay for E. coli O157:H7 A pure culture of E. coli O157:H7 was obtained from the Microbial Culture Collection at RMIT University (Australia). Bacterial strains were maintained in nutrient broth (NB) and nutrient agar (NA) (Sigma-Aldrich (Australia)). Syringe filters (0.45 μm, PES) were purchased from Millipore (Australia). Sterile Technoplas Petri Dishes were purchased from Interpath Services (Australia). The MTS assay kit (Promega CellTiter 96 Aqueous One Solution) was purchased from Promega.

[0195] Cell viability assay Cell viability was determined based on the mean activity of mitochondrial succinate dehydrogenase using an MTS assay kit.

[0196] E. coli O157:H7 and Pseudomonas aeruginosa (ATCC2835) bacterial cells were cultured in nutrient broth (NB) in 10 mL sterile plastic screw-cap centrifuge tubes with an optical density (OD) of 0.5–0.6 and grown at 37°C with shaking at 150 rpm.

[0197] Cell viability assays against E. coli O157:H7 and Pseudomonas aeruginosa (ATCC2835) bacterial strains were performed in duplicate for all sets of samples. Each set contained either neat antibiotic or antibiotic-loaded nanoparticles. Neat antibiotic stock solutions were prepared in dimethyl sulfoxide (DMSO). A 1% bacterial inoculum was added to a positive control containing DMSO (1%) and a negative control containing 50 μg / mL of MO-based cationic (DOTAP 1 mol%) lipid nanoparticles. The nanoparticles loaded with different antibiotics were filtered using a sterile Millex-GP syringe filter (0.45 μm, PES, Millipore) and used in the experiments. Free antibiotic (5 mg / mL stock in DMSO) and lipid nanoparticles loaded with the intended concentration of antibiotic were added at the time of inoculation to 5 mL of culture medium (containing 1% inoculum) and incubated for 20–24 h, at which time bacterial growth reached stationary phase at 37°C with shaking at 150 rpm.

[0198] 100 μL of cell culture medium was taken from each sample and distributed into a 96-well plate. Bacterial cell viability was measured using an MTS assay kit. 10 μL of MTS solution was added to each 100 μL sample and incubated at 37°C for 2 hours. The absorbance at 490 nm was measured using a microplate reader (SpectraMax, Molecular Devices). The measured absorbance from the control sample was set relative to 100% cell viability. Therefore, all other sample data were adjusted relative to this value.

[0199] CFU-based cell viability assay for E. coli O157:H7 Minimum inhibitory concentration (MIC) measurement assay. Pure cultures of E. coli O157:H7 were obtained from the Microbial Culture Collection at RMIT University (Australia). Bacterial strains were maintained in nutrient broth (NB) and nutrient agar (NA) (Sigma-Aldrich, Australia). MIC determination assays against E. coli O157:H7 were performed in duplicate for all sets of samples, each containing either free rifampicin (or fusidic acid) or rifampicin (or fusidic acid)-loaded nanoparticles of the present disclosure. For each replicate, 50 μL of overnight culture of E. coli was diluted into 5 mL of nutrient broth (NB) and grown at 37°C with shaking at 150 rpm to an optical density (OD) of 0.5-0.6. Free rifampicin and fusidic acid stock solutions were prepared in DMSO. The positive control contained 1% bacterial inoculum with DMSO (1%), and the negative control contained 50 μg / mL MO-based cationic (DOTAP 1 mol%) lipid nanoparticles. The rifampicin- and fusidic acid-loaded nanoparticles were filtered using a sterile Millex-GP syringe filter (0.45 μm) before use.

[0200] The drug concentrations before and after filtration were evaluated by UV-Vis spectroscopy. A 4 mg / mL stock solution of rifampicin was prepared in ethanol. Ten standard serial dilutions with concentrations of 0, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015, 0.0078, and 0.00390 mg / mL were prepared in ethanol, and the absorbance at 340 nm was plotted against the relative concentration to obtain a standard calibration curve. 50 μL of rifampicin (3 mol% or 3.5 mg / mL)-loaded MO-based cationic (DOTAP 1 mol%) lipid nanoparticles were dissolved in 950 μL of ethanol solution before and after filtration. The rifampicin concentrations of both nanoparticle samples (before and after filtration) were determined from the rifampicin standard curve. After filtration, the rifampicin concentration was found to be approximately 5% lower than in the unfiltered sample. Free rifampicin (5 mg / mL stock in DMSO) and rifampicin (3 mol% or 3.5 mg / mL) drug-loaded MO-based cationic (DOTAP 1 mol%) lipid nanoparticles were added at concentrations ranging from 0.05 to 5 μg / mL to 5 mL of medium at the time of inoculation. Bacterial growth was measured by reading the absorbance at 600 nm and determining colony-forming units (CFU / mL) after 24 h of incubation, at which point bacterial growth reached stationary phase. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of free drug or nanoparticles that inhibited any growth of bacteria on nutrient agar plates (NA) by more than 90%.

[0201] Fungal cell viability assay Candida albicans (ATCC 10251) organisms were subcultured from sterile vials onto Sabouraud dextrose agar or potato dextrose agar to ensure purity and viability. The temperature throughout the incubation was 35°C.

[0202] Inoculum was prepared by selecting five colonies approximately 1 mm in diameter from a 24-h-old culture of Candida albicans. The colonies were suspended in 5 mL of sterile 0.145-mol / L saline (8.5 g / L NaCl, 0.85% saline). The resulting suspension was vortexed for 15 seconds, and the cell density was adjusted spectrophotometrically by adding enough sterile saline to increase the transmittance at 530 nm relative to that produced by a 0.5 McFarland standard (shown below).

[0203] This procedure yields a fungal cell stock suspension of 1 x 10 to 5 x 10 cells per mL. The working suspension is made by a 1:100 dilution of the stock suspension with RPMI 1640 broth medium, followed by a 1:20 dilution, which yields 5.0 x 10 to 2.5 x 10 cells per mL.

[0204] Cell viability assays for Candida albicans strains were performed in duplicate for all sets of samples. Each set contained either neat antibiotic or antibiotic-loaded nanoparticles. Neat antibiotic stock solutions were prepared in dimethyl sulfoxide (DMSO). 1% of the cell stock suspension was added to a positive control containing DMSO (1%) and to a negative control containing 50 μg / mL of MO-based cationic (DOTAP 1 mol%) lipid nanoparticles. Nanoparticles loaded with different antibiotics were filtered using a sterile Millex-GP syringe filter (0.45 μm, PES, Millipore) and used in the experiments. Free antibiotic (5 mg / mL stock in DMSO) and antibiotic-loaded lipid nanoparticles at the intended concentration were added to 5 mL of medium (containing 1% of the cell stock suspension) at the time of inoculation and incubated for 24–30 h, during which time fungal cell growth reached stationary phase at 35–37 °C.

[0205] 100 μL of cell culture medium was taken from each sample and distributed into a 96-well plate. Fungal cell viability was measured by using an MTS assay kit. 10 μL of MTS solution was added to each 100 μL sample and incubated at 37°C for 2 hours. The absorbance at 490 nm was measured using a microplate reader (SpectraMax, Molecular Devices). The measured absorbance from the control sample was set relative to 100% cell viability. Therefore, all other sample data were adjusted relative to this value.

[0206] result Interaction with Gram-positive bacteria Cubosome uptake was assessed in two Gram-positive bacterial species, Bacillus cereus (B. cereus) and Staphylococcus aureus (S. aureus). The outermost layer of peptidoglycan is estimated to be approximately 50 nm and 23 nm thick for these species, respectively. Total internal reflection fluorescence microscopy (TIRF) was used to reveal cubosome interactions in situ. Bacterial cells were visualized by λ , shown in blue. ex Cubosomes can be observed by their intrinsic fluorescence from excitation at λ = 405 nm, while cubosomes are visualized by the phospholipid dye 18:1-Cy5 (λ = 405 nm), which is shown in red. ex = 647 nm). The bacteria themselves are fluorescently tagged with light at this wavelength (λ ex A representative fluorescence image of bacteria in the absence of the fluorescent tag is shown in Figure 1.

[0207] A time-lapse sequence showing the uptake of fluorescently tagged cubosomes into B. cereus is shown in Figure 2a. From the time-lapse in Figure 2a, transient cubosomes in solution are highlighted with white arrows. Cubosomes, visible as bright circular spots, were observed to move throughout the solution between frames at t = 8.7 s and 14.5 s, and then attach to the B. cereus surface at approximately t = 14.9 s. The mean square displacement (Δr 2) was determined by particle tracking shown in Figure 2b. The sudden deviation in the slope at approximately 8.7 seconds (indicated by the dashed line) coincides with the attachment of cubosomes to the bacterial surface. Note that in the displacement data, t = 0 corresponds to the appearance of the cubosomes (closest to frame 8.7 seconds in the time-lapse in Figure 2a). Once attached, the cubosomes remained on the bacterial surface even while the cubosomes moved into solution. This is also indicated by the displacement tracking shown in Figure 2b, where the reduced slope once attached indicates the movement of significantly larger colloids, i.e., bacteria-cubosome complexes. The time-lapse in Figure 2c shows that over a longer time period (approximately 3 hours), the cubosomes appeared as bright red spots (λ ex = 647 nm) as the blue bacterial surface (λ ex = 405 nm). The cubosomes maintained their size and shape over a period of several minutes, demonstrating long-term stability.

[0208] The extensive time-lapse in Figure 2c-d shows that after several hours, the initially localized fluorescence source from the cubosome gradually spreads to cover the entire B. cereus cell. This is qualitatively observed in this time-lapse by a steady increase in the red signal. This behavior is quantified in Figure 2e-f. The fluorescence intensity (I) of individual cubosomes (Figure 2e) decayed exponentially over time. Plotting the data on a logarithmic scale reveals two distinct regimes: the first approximately 15 s is characterized by a slope of -1 / 9 and a dominant slope of -1 / 2 between approximately 15 s and 400 s, with I approximately t for each regime. -1 / 9 Or about t -1 / 2 This longevity is in stark contrast to the uptake behavior observed in eukaryotic cells, where cubosomes appear to fuse within 10 seconds. It is expected that during the early stages of attachment, cubosomes may wet the surface, i.e., the outer peptidoglycan layer of B. cereus. The dominant scaling observed for the latter part (after 15 seconds) correlates with that expected for diffusion of a point source emission in one dimension, C maxabout t -0.5d wherein C max is the maximum concentration at time t and d is the dimensionality. This can be conceptually rationalized in terms of cubosome component lipids diffusing inward through a thick peptidoglycan layer.

[0209] The cumulative effect of many cubosomes interacting with bacteria is reflected in the fluorescence intensity throughout the cell, which is shown to steadily increase (Figure 2f). An increase in fluorescence intensity was observed across all cubosome compositions. The magnitude and rate of the fluorescence intensity increase were reasonably similar between MO and PT, but there was a significant acceleration and larger magnitude for positively charged cubosomes. For example, after 3 h of incubation, the intensity observed for MO-DOTAP cubosomes (ζ = +31 mV) was twice that of MO cubosomes (ζ = -12 mV). This increase can be attributed to an increased number of successful collisions with bacteria, facilitated by the attractive potential between the negatively charged B. cereus membrane and the positive cubosome surface.

[0210] The uptake of cubosomes into S. aureus bacteria is shown in Figure 3. While the small, spherical bacteria were somewhat difficult to observe, they remained consistently bound to the surface, allowing for clearer particle tracking. Two representative time lapses are shown in Figure 3a and b. In the first series, individual cubosomes can be observed hitting the surface before gradually dissipating over the course of approximately 4 minutes. In the second series, an increase in the number of cubosomes was observed, including the simultaneous attachment of three individual cubosomes to the bottom bacterium. In this series, cubosomes were clearly observed landing and dissipating over a period of approximately 2–7 minutes. This time lapse shows that cubosomes generally retain their shape but shrink in size after attachment. However, the bottommost cubosome in Figure 3b shows a clear contraction in the cubosome lateral footprint.

[0211] Within the same time course (Figure 3b), there was some variation in the size and duration of attachment, likely due to changes in particle size. Larger cubosomes likely had larger footprints with increased intensity and required longer periods to fully decay. Particles appeared to dissipate after 2–10 min. The peak fluorescence intensity over time for individual cubosomes was again tracked in the representative plot shown in Figure 3c. In the inset of Figure 3c, the logarithmic scale data show a correlation with a slope of approximately -1 / 3, with some points approaching -1 / 2. Interestingly, this scaling is reasonably consistent with that observed for B. cereus. The similarity between the scaling of both Gram-positive bacteria suggests that the interaction is governed by the presence of an outer peptidoglycan layer. The variation in the slope between -1 / 3 and -1 / 2 may result from geometric differences. The reduction in the surface area available for S. aureus relative to B. cereus could even lead to interactions between fusing cubosomes. Nevertheless, the dominant scaling observed again correlates with a 1-d point-source release of material through the outer layer of peptidoglycan.

[0212] We further evaluated the interaction between cubosomes and the outer peptidoglycan layer of bacteria using a peptidoglycan extract from S. aureus. Based on the similarity of the extract to S. aureus itself, the extract provided a peptidoglycan shell that preserved the original bacterial shape. In this case, imaging resolution was somewhat clearer than with live S. aureus (Figure 3e). Spheroid particles typically existed in planar sheets or stacks at the interface. From the time-lapse (Figure 3e), many cubosomes were observed to accumulate around the periphery of the cluster. Over time, red fluorescence was observed to gradually spread throughout the cluster domain. In contrast to in vitro experiments, cubosomes did not appear to reduce the footprint or intensity.

[0213] Mimetics were investigated in similar experiments by including additional cell wall components. In this case, peptidoglycan extracts were incubated with lipoteichoic acid (LTA) extracts (50% w / w LTA:peptidoglycan). In stark contrast to the previous results, no cubosome attachment to the combined extracts was observed. A time-lapse of cubosome attachment to the combined extracts is shown in Figure 3h. The relative uptake between clusters of S. aureus, peptidoglycan, and peptidoglycan + LTA extracts is quantified in Figure 3d. In general, S. aureus itself showed steady uptake, with several peaks resulting from the addition of new cubosomes. This gradual increase once again reflects uptake of cubosome material into S. aureus, correlating with the earlier results shown by B. cereus. In contrast, peptidoglycan and peptidoglycan + LTA extracts showed enhanced and inhibited uptake, respectively. The presence of LTA, which is found widely in the peptidoglycan layer, appears to interfere with cubosome attachment, although the exact mechanism of this remains to be elucidated.

[0214] To investigate adhesion at higher resolution, SEM was performed on peptidoglycan extracts and S. aureus (Figure 3f, g). Again, the extracts were geometrically similar to immobilized S. aureus. SEM images show that after treatment with cubosomes, the roughness between the extract bodies was clearly eliminated, indicating that the cubosome material had spread and engulfed the extract (Figure 3f). In comparison, no significant geometric contrast was observed between treated and untreated S. aureus bacteria. This may reflect particle degradation by live bacteria or the timing of cubosome interactions, as SEM captures only a single snapshot in time. Next, for cubosomes treated with immobilized S. aureus, SEM revealed a bimodal distribution of bacteria decorated by neat bacteria and smaller circular features toward the ends of the bacteria. Two representative images are shown in Figure 3g, which highlight the external spherical features relative to the cells. The size of this feature (lateral diameters of 290 nm and 220 nm in the left and right panels) is consistent with the size of cubosomes. Additionally, the location is consistent with the uptake location observed during TIRF measurements. The apparent contact angles between the spherical feature and S. aureus bacteria in Figure 3g are approximately 132° and 120°, respectively. The high contact angle is consistent with the minimal spreading of cubosomes in TIRF measurements, but the angle may have been altered by physical treatment during fixation. The physical stability of cubosomes in vacuum is due to the crosslinking of unsaturated bonds and hydroxyls in MO with osmium and glutaraldehyde. Details of the internal structure are not resolved by SEM, especially considering the 5 nm sputter coating. Note that polymerization and negative staining have previously enabled TEM of mesostructures. These results demonstrate the difficulty in approaching Gram-positive bacteria.

[0215] Interaction with Gram-negative bacteria Interactions with Gram-negative bacteria were investigated using Escherichia coli (E. coli). Similar to S. aureus, E. coli remained well attached to the interface. From the time-lapse shown in Figure 4a, cubosomes were observed to continuously attach over approximately 30 minutes. Once attached, the cubosomes gradually decayed and shrank, appearing to behave primarily as previously described for Gram-positive species. While these single-cubosome interactions were most common, interactions involving multiple cubosomes were also observed, as seen in the time-lapse in Figure 4b, spanning a longer period of approximately 3 hours. Between 0 and approximately 47 minutes, a single cubosome was observed to attach to the bacterial surface. Following this, secondary adsorption was observed on top of the previously attached cubosome. At approximately 90 minutes, new cubosomes were observed to land, encouraging fluorescence diffusion throughout a significant portion of the cell. By the 94-minute frame, the entire cell was brightly fluorescent at the cubosome wavelength. Later frames in this sequence show this process being repeated (approximately 175 minutes). These sequences suggest that cubosome-contaminated regions of bacteria are preferred landing sites for new cubosomes, likely enabled by the inclusion of fusogenic lipids in the cell membrane. Furthermore, cubosome material can spread rapidly throughout E. coli.

[0216] Single channel (λ exBy examining uptake at λ = 647 nm, the rapid uptake event can be resolved more clearly. The short sequence in Figure 4c highlights this for MO cubosomes. In the top panel, a small circular feature is resolved to be approximately 100-200 nm in diameter at 20 seconds. Suddenly, after 3 minutes, this feature appeared to induce a burst of fluorescence throughout the cell body. A similar event is shown for MO-DOTAP cubosomes in Figure 4d, where cubosome attachment can be inferred by the bright spot in the center right of the cell body. As mentioned above, in the case of Gram-negative bacteria, cubosomes first interact with the outer plasma membrane rather than the peptidoglycan layer. In this sense, the interaction between cubosomes and Gram-negative bacteria is more comparable to interactions with mammalian membranes. The rapid spreading of material may reflect the ability of cubosomal material to fuse with the outer membrane. The movement of material through the plasma membrane is perhaps most clear in Figure 4e. The initial frame highlights the location of the bacteria at λ. ex The image series below shows the signal channel (λ = 405 nm). ex At a wavelength of 647 nm (λ = 647 nm), two cubosomes could be observed landing at 10 and 20 seconds, respectively. By 30 seconds, the local fluorescence had dissipated throughout the bacterium. Interestingly, an increased signal was observed around the cell periphery, consistent with the location of the dye within the lipid membrane. In this series, spatiotemporal gradients of material in the membrane were resolved, allowing for rapid interactions and the combination of large amounts of cubosome material.

[0217] The SEM images shown in Figures 4f-m revealed images of cubosome attachment remarkably similar to those shown in Figure 4a. From the series of images, several distinct spherical asperities can be observed on the surface of E. coli. The size of the protrusions in the images varies from approximately 400 nm to approximately 50 nm in diameter. Upon contact with the E. coli surface, larger cubosomes appeared to exhibit higher contact angles with the bacterial surface. This is most evident in Figures 4g and 4l, where the contact angle appears to exceed 90°. In contrast, smaller cubosomes appear to be more spread out and engulfed in the bacterial surface. From the size variation, it can be inferred that each cubosome is trapped at various stages of internalization into bacteria that have been effectively frozen by the fixation process.

[0218] The uptake rate by individual E. coli cells was assessed by quantifying fluorescence intensity over time. The number of individual cubosome uptake events can be seen in Figures 5a and 5b, which are representative results for MO and MO-DOTAP cubosomes, respectively. Numerous peaks in intensity are observed, which can be attributed to cubosome attachment to cells. Between the two plots, it is immediately clear that MO-DOTAP results in an increased frequency of peaks due to cubosome attachment. This is generally consistent with earlier results for B. cereus and previous results for liposomes in which the incorporation of positive charges into the liposome minimizes disturbance. Focusing on a narrower time span in Figure 5c, the red curve shows a peak intensity curve with two prominent peaks from cubosome attachment. The gray dots represent the fluorescence intensity recorded over the entire cell area. After each cubosome attachment event, the peak intensity decays, but there is a stepwise increase in area intensity. These combined results indicate the transfer of donated fluorescence from the cubosome to the bacteria. Furthermore, the results clearly demonstrate the quantized delivery of the loading component from the cubosomes.

[0219] By tracking the intensity over time for individual cubosomes attached to E. coli, further differences were observed relative to that of Gram-positive species. While still showing an exponential decay, two regimes were observed on the logarithmic scale, as indicated by the two slopes -1 / 6 and -1 (Figure 5d). One representative result is plotted in Figure 5e, and the corresponding time series of images is plotted in Figure 5f. These two slopes suggest that the interaction of cubosomes with E. coli occurs in two stages. Given the homogeneous distribution of fluorophores within the cubosomes, the intensity I max is scaled by the cubosome size (R), and the mass (m) balance is

number

number

number

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[0220] It is anticipated that antimicrobial agents, including traditional antibiotics (e.g., beta-lactams, ansamycins) or antimicrobial peptides, can be delivered in a manner similar to the phospholipid dyes studied herein. Assuming no leakage of encapsulated material, delivery should be quantified by the number of attached cubosomes. Delivery to Gram-positive bacteria, scaling with exponential −1 / 2, is consistent with 1-D point release. For Gram-negative bacteria, the two-step process involves initial fusion with exponential −1 / 6 followed by release of the cargo as a point source in 2-D with exponential −1, suggesting that delivery is more effective than in Gram-positive species. This is further supported by the observation that delivered compounds can rapidly spread throughout Gram-negative bacteria, suggesting that the Gram-negative bacterial plasma membrane aids in the integration of cubosome material and the cell wall. This ability to evade the outer membrane may prove essential for the delivery of therapeutic agents to increasingly resilient Gram-negative species. Furthermore, the inclusion of fusogenic lipids, which lower the energetic barrier to fusion, should further facilitate this process.

[0221] The intermittent occurrence of rapid fluorescence expansion shown in Figure 4c-d caused deviations from the two-step model. A subset of intensity versus time curves for cubosomes that exhibited rapid spreading during attachment is shown in Figure 6. Generally, the intensity curves follow the scaling shown previously in Figure 6a-b, but each curve here also has a sudden drop in intensity, marked by an arrow in the plot. This drop corresponds to the point at which the localization of fluorescence spread throughout the bacterium. The time required for this drop to occur was sporadic and did not occur for all cubosomes. In each case, peak fluorescence decreased by 40-50%, and the duration of the drop was faster than the normal decay (seconds versus minutes). As a result, scaling analysis for this intermittent process was not feasible due to the reduced number of data points. That is, the suggested slope of the drop shown in the log plot varied between approximately -2.13 and 2.33. Given that these events were irregular and that the entire bacterium became fluorescent, it is possible that these particular cubosomes (or portions thereof) had traversed the entire cell wall. Deviations from the two-step transport model may reflect the influence of enzymes secreted by bacteria: Thorn and colleagues recently demonstrated that the presence of lipases promotes burst release of hydrophobic cargo. 8

[0222] Fusion behavior between cubic nanocarriers and supported lipid bilayers (SLBs) using total internal reflection fluorescence (TIRF) is shown in Figure 7a. This series shows the fusion of cubosome material, initially observed as bright spots with radially diffusing fluorescence over a period of approximately 6.5 seconds. Using the same approach in eukaryotic cells, it is possible to directly observe and quantify the dynamics of these interactions. Experiments show cubosomes colocalizing with bacteria over a 12-hour period. Results reveal distinct regimes and differences in cubosome transport to Gram-negative versus Gram-positive bacteria, highlighted in Figures 7b and 7c, respectively. Snapshots in Figures 7d and 7e show representative results from in situ surface-sensitive measurements, allowing for differentiation between cubosomes and bacteria and the observation of individual cubosome-bacteria interactions.

[0223] In the top image of Figure 7f, a ring of red fluorescence around the fungus can be observed. As the fungus undergoes mitosis, the fungal membrane deforms, allowing the attached cubosomes to fuse with the plasma membrane. As shown in the bottom image of Figure 7f, extensive internalization of red fluorescence from the cubosomes is observable. It is reasonable to expect that the same design criteria for bacteria apply to fungi.

[0224] Delivery of active agents Novobiocin Novobiocin inhibits enzymes involved in DNA synthesis. Figures 8 and 9 and Table 2 show the inhibition of P. aeruginosa and E. coli by cubosome formulations containing the antibiotic novobiocin, as presented in Table 1. The plot shows the inhibition (y-axis) of each formulation against the concentration of novobiocin (x-axis). The total concentration is comparable between each formulation.

[0225] Against P. aeruginosa, all encapsulated formulations of novobiocin had significantly more inhibition than the freely dissolved one (solid black line).

[0226] Inhibition increased as the concentration of drug per cubosome (or drug molecules per particle) increased. Coverage range (1-5 mol%). This result indicates that fewer particles with higher drug loadings are more effective than more particles with lower drug loadings.

[0227] For example, at 80 μg / ml, MO-DOTAP-1NOVO, MO-DOTAP-3NOVO, and MO-DOTAP-5NOVO showed 51%, 55%, and 85% inhibition, respectively, while the free antibiotic achieved 13% inhibition.

[0228] The inclusion of the secondary drug fusidic acid, an antibiotic / bacteriostatic agent that inhibits protein synthesis, also further increased the inhibition (plus vs. pentagon).

[0229] In fact, P. aeruginosa is normally highly resistant to novobiocin due to limited permeability across the outer plasma membrane. The increased inhibition upon encapsulation is attributed to the fusion mechanism demonstrated here by cubic phase nanoparticles.

[0230] The inclusion of a "fusogenic" lipid promotes the fusion uptake mechanism. The inclusion of the highly curved lipid, DOPE at 10 mol%, increased inhibition by an average of 57% across the entire concentration range. For example, MO-DOTAP-3NOVO and MO-DOTAP-10PE-3NOVO at 60 μg / ml resulted in approximately 48% and 85%, respectively.

[0231] Novobiocin is not often used in practice due to its high inactivation in serum due to binding to serum proteins. In contrast, when novobiocin was encapsulated in cubosomes, inhibition appeared to improve in the presence of serum albumin.

[0232] Inhibition was assessed using MO-DOTAP-3NOVO in the presence of human serum albumin (HSA), bovine serum albumin (BSA), and fetal bovine serum (FBS). Figure 10 shows that encapsulation preserves antibiotic activity against freely dissolved antibiotics (black line). The dashed line reflects the performance of MO-DOTAP-3NOVO in the absence of serum proteins at an equivalent loading (20 μg / ml). [Table 2] [Table 3]

[0233] Piperacillin Piperacillin is a beta-lactam antibiotic that inhibits peptidoglycan cross-linking, which leads to cell rupture. Figures 11 and 12 and Table 4 show the inhibition of P. aeruginosa and E. coli by cubosome formulations containing the antibiotic piperacillin, as presented in Table 3.

[0234] Against P. aeruginosa (FIG. 11), all encapsulated formulations of piperacillin had significantly more inhibition than the free dissolved drug (solid line).

[0235] Inhibition increased as the concentration of drug per cubosome (or drug molecules per particle) increased.

[0236] For example, MO-DOTAP-1Pip, MO-DOTAP-3Pip, and MO-DOTAP-5Pip achieved 52%, 59%, and 75%, respectively.

[0237] Incorporation of a positive charge by inclusion of the cationic lipid DOTAP significantly increased inhibition, as can be observed by clustering of particles with approximately 80%+ inhibition (diamond, cross, plus) and without cationic lipid with approximately 50% (inverted triangle, pentagon, square).

[0238] Similar inhibition was achieved by inclusion of TOAB as the cationic moiety (cross vs. plus).

[0239] The same trend was observed for E. coli (Figure 12): encapsulated piperacillin exceeded the performance of the free antibiotic when the encapsulation concentration was above 1 mol%.

[0240] Table 4 shows the inhibition for piperacillin against E. coli when encapsulated in cubosomes of different key component lipids, monoolein or phytantriol. Performance between formulations was similar. [Table 4] [Table 5]

[0241] Meropenem Meropenem is a beta-lactam antibiotic that inhibits cell wall synthesis, leading to cell rupture and death. Figures 13 and 14 show the inhibition of P. aeruginosa and E. coli by cubosome formulations containing the antibiotic piperacillin, as presented in Table 5.

[0242] Against P. aeruginosa and E. coli, all encapsulated formulations of meropenem had significantly more inhibition than the free dissolved drug (solid black line).

[0243] Inhibition increased with increasing concentration of drug per cubosome (or drug molecules per particle). In the case of meropenem, this effect appeared to saturate between 3 and 5 mol%.

[0244] Incorporation of a positive charge by inclusion of the cationic lipid DOTAP significantly increased inhibition, as can be observed in the group of particles without cationic lipids, which had approximately 80% inhibition (diamond-cross-plus) and approximately 50% (square, triangle, pentagon). [Table 6]

[0245] Clarithromycin Clarithromycin inhibits protein synthesis. Figure 15 shows the inhibition of P. aeruginosa and E. coli by cubosome formulations containing this antibiotic, as presented in Table 6.

[0246] Incorporation of a positive charge by inclusion of the cationic lipid DOTAP increased inhibition. When encapsulated in cationic cubosomes at 1 and 3 mol%, encapsulated clarithromycin resulted in increased inhibition of E. coli compared to the free, dissolved antibiotic.

[0247] In the absence of cationic lipid, performance was essentially equivalent between formulations.

[0248] The enhancement was less pronounced with clarithromycin, likely due to precipitation of the drug, as shown in Figure 16. This may be addressed by additional variations to the formulation that minimize this effect. [Table 7]

[0249] Gentamicin Gentamicin is an aminoglycoside that disrupts protein synthesis and may have some effects on bacterial cell membranes. Figures 17 and 18 show the inhibition of P. aeruginosa and E. coli by cubosome formulations containing this antibiotic, as presented in Table 7.

[0250] Inhibition was greatly enhanced when encapsulated, achieving peak inhibition at significantly reduced concentrations compared to the free dissolved antibiotic.

[0251] A 10-20% increase in inhibition was observed when the charged lipids DOTAP or OA were incorporated into the cubosome particles, which increased the zeta potential and negative curvature, respectively.

[0252] The striking contrast may be due to the enhanced delivery coupled with the mode of action of gentamicin, a concentration-dependent antibiotic, which requires high concentrations as opposed to the continuous presence of low concentrations. [Table 8]

[0253] Dicloxacillin Dicloxacillin is a narrow spectrum beta-lactam antibiotic that inhibits cell wall synthesis, leading to cell rupture and death. Dicloxacillin has limited activity against gram-negative bacteria.

[0254] Figure 19 shows the inhibition of E. coli by cubosome formulations containing the antibiotic dicloxacillin, as shown in Table 8. Dual encapsulation with tazobactam (a beta-lactamase inhibitor) effectively doubled the inhibition (squares vs. triangles), thereby demonstrating that even antibiotics that are typically not accepted against gram-negative bacteria can be useful in the disclosed approach when used in combination with a second active agent. [Table 9]

[0255] benzylpenicillin Benzylpenicillin is a narrow-spectrum beta-lactam antibiotic that inhibits cell wall synthesis, leading to cell rupture and death. Benzylpenicillin has activity against Gram-negative bacteria due to their inherent resistance to beta-lactamase antibiotics. Figure 20 shows the inhibition of E. coli by cubosome formulations containing the antibiotic benzylpenicillin, as shown in Table 9.

[0256] Benzylpenicillin resulted in increased inhibition when encapsulated at 3 mol% compared to the free antibiotic, thereby demonstrating that simple changes in formulation such as drug concentration / loading can be used to improve outcomes.

[0257] CFU testing revealed a further reduction in colony counts of 10-84% when encapsulated, generally increasing with concentration (Table 10). [Table 10] [Table 11]

[0258] Rifampin Rifampicin inhibits RNA synthesis. Figures 21 and 22 show the inhibition of E. coli by cubosome formulations containing the antibiotic rifampicin, as presented in Table 11.

[0259] As the concentration of drug per cubosome (or drug molecules per particle) increased, the inhibition increased. This result indicates that fewer particles with a higher drug load can be more effective than more particles with a lower individual drug load. This result also indicates that encapsulation significantly increases the effectiveness of the same amount of drug.

[0260] It was noted that at 4 mol% loading, inhibition was reduced from that at 3 mol%. This loss of functionality is attributed to the loss of ordered structure in the nanoparticles, or more specifically, the loss of mesostructuring of the fusogenic cubic phase with higher drug concentrations. [Table 12]

[0261] Increasing curvature While not wishing to be limited by theory, it is proposed that delivery of the antibiotic active agents described herein occurs via fusion of the self-assembled nanoparticles and bacterial membranes. Thus, the inclusion of a fusogenic lipid should facilitate the fusion delivery pathway by further reducing the energetic barrier for fusion to occur. In the case of rifampicin, this is demonstrated with increasing concentrations of DOPE, highly fusogenic lipids (Table 12), and the presence of cationic lipids (Table 13) across the range of formulations.

[0262] Between 0 and 20% dope, the particles are in a cubic phase. At 30-40% dope, the particles exhibit hexagonal symmetry. With increasing dope from 0 to 20 (i.e., from 0 to 20%), the lattice parameter of the particles decreased from 146 to 99.6 Å, reflecting an increase in particle curvature. The average spontaneous curvature was -0.161 nm. -1 to -0.216 nm -1At the same time, an increase in inhibition by 1 mol% rifampicin was observed (Figures 22 and 23). For example, at 2 μg / ml, the inhibition was 14%, 36%, and 60%, respectively.

[0263] These results indicate that the inclusion of fusogenic lipids increases the effectiveness of cubosomes in delivering antibiotics. This trend was also observed with cationic nanoparticles (Figure 24). Increased inhibition was also observed from formulations with 40% DOPE compared to 30% DOPE (Figures 26 and 27). This increase was somewhat less pronounced with cationic nanoparticles (Figures 28 and 29). [Table 13] [Table 14] [Table 15] [Table 16]

[0264] Ampicillin Ampicillin is a beta-lactam antibiotic that inhibits peptidoglycan cross-linking, which leads to cell rupture. Table 17 shows the inhibition of P. aeruginosa by cubosome formulations containing the antibiotic ampicillin, as presented in Table 16.

[0265] Between 0.5 and 10 μg / ml, the encapsulated formulation showed 50–70% more inhibition than the free antibiotic (e.g., 62% compared to 39% at 0.5 μg / ml). The enhancement plateaued at higher concentrations with a time-dependence of drug vs. concentration dependence. [Table 17] [Table 18]

[0266] Vancomycin Vancomycin is a glycopeptide antibiotic that inhibits cell wall synthesis and is generally inactive against Gram-negative strains (eg, P. aeruginosa) due to diffusion barriers across membranes and altered targeting.

[0267] Table 19 shows the inhibition of P. aeruginosa by cubosome formulations containing the antibiotic vancomycin, as presented in Table 18. At concentrations of 0.5 to 20 μg / ml, encapsulated vancomycin resulted in at least 2.7-fold greater inhibition. At the lowest concentration (0.5 μg / ml), encapsulation resulted in over 16-fold greater inhibition. This demonstrates the advantage of this approach, allowing for the effective delivery and use of drugs that may not normally be effective against Gram-negative bacteria due to the difficulty of diffusion across the bacterial membrane. [Table 19] [Table 20]

[0268] Double drug load The periodic three-dimensional topology of the lipid particles of the present disclosure facilitates the encapsulation of multiple drug cargoes within the same particle, as demonstrated below for the combination of Novo-Fus, Rif-Fus, and Pip-Fus. [Table 21] [Table 22] [Table 23]

[0269] Fusidic acid Fusidic acid is a bacteriostatic agent that interferes with protein synthesis. It is highly hydrophobic and therefore has minimal utility against Gram-negative bacteria. When used in the lipid particles of the present disclosure, a significant decrease in E. coli colony-forming units (CFU) was observed, particularly as the concentration increased, as seen in Figure 30. A significant decrease in CFU count was observed at Fus concentrations above 0.1 mg / ml, with a reduction of more than half at 0.2 mg / ml. The respective minimum inhibitory concentrations (MICs), indicated by * on the plot, were 0.5 mg / ml and 1.5 mg / ml for the encapsulated and free drugs, respectively. The MIC refers to the minimum concentration that achieves 90% inhibition of cell growth. Again, it is noted that Fus has limited activity against Gram-negative bacteria, typically due to poor transport across the outer membrane due to its molecular size and hydrophobicity.

[0270] Control The toxicity of nanoparticles without loaded active agents was examined against E. coli and P. aeruginosa. The results, shown in Figure 31, indicate that lipid cubosome particles have a measurable effect on bacterial viability when no antibiotic is encapsulated.

[0271] Anti-TB drug formulations were loaded onto MO-based cubosomes. Commercially available TB drugs have a short shelf life and rapid clearance, which limits their effectiveness. To increase the efficacy of anti-TB drugs and improve the success rate of TB treatment, an effective and robust delivery system, such as the lipid particle delivery system of the present disclosure, is needed.

[0272] This study evaluated the potential of monoolein (MO)-based cubosomes for encapsulation and delivery of single antituberculosis drugs (rifampicin, ethambutol, isoniazid, pyrazinamide, and streptomycin) to Mycobacterium smegmatis and Mycobacterium tuberculosis H37Ra in vitro culture models.

[0273] To test this, five anti-TB drugs, rifampicin, isoniazid, pyrazinamide, ethambutol, and streptomycin, were encapsulated in MO cubosomes. Encapsulation of the hydrophobic drugs rifampicin and isoniazid (octanol-water partition coefficients of 4.24 and -0.70, respectively) resulted in an increase in the lattice parameter of the Im3m phase. Drug loadings of up to 4 mol% were achieved for rifampicin and 15 mol% isoniazid. The results are shown in Figures 32-35. Figure 32 shows the MIC determination for Mycobacterium smegmatis, and Figure 33 shows the cell death at the MIC for Mycobacterium smegmatis. The reported MIC values ​​of Rif for intracellular bacteria are 4-5 μg / mL, and the MIC value for intracellular bacteria in the presence of MO-Rif (1 mol%) is 3 μg / mL, whereas the MIC value for intracellular bacteria in the presence of MO-DOTAP (0.1 mol%) was found to be 1 μg / mL. The reported values ​​for cell death are approximately 20-24 hours. MO-Rif reduced the incubation time at the MIC value from 24 hours to 15 hours, while MO-DOTAP (0.1 mol%)-Rif (1 mol%) reduced the incubation time at the MIC value from 24 hours to 6 hours.

[0274] Figure 34 shows the MIC determination for Mycobacterium tuberculosis H37Ra, and Figure 35 shows the cell death at MIC for Mycobacterium tuberculosis H37Ra. The reported MIC values ​​of Rif for intracellular bacteria are 0.04-0.05 μg / mL, and the MIC value in the presence of MO-Rif (1 mol%) against intracellular bacteria was found to be 0.03 μg / mL, while the MIC value in the presence of MO-DOTAP (0.1 mol%) for intracellular bacteria was found to be 0.015 μg / mL. The reported cell death at MIC is approximately 8-9 days. MO-Rif reduced the incubation time at MIC to 7-8 days, while MO-DOTAP (0.1 mol%)-Rif (1 mol%) reduced the incubation time at MIC from 8 days to 5 days.

[0275] Therefore, rifampicin encapsulated in cubosomes has a significantly greater killing effect compared to the free drug, potentially shortening the life cycle duration of axenic bacterial cultures. Furthermore, incorporating a positive charge through the addition of the cationic lipid DOTAP (0.1 mol%) significantly increased the killing effect. Encapsulation of the anti-TB drug rifampicin in cationic cubosomes improved drug bioavailability in in vitro studies, reducing the time required to reduce the bacillus load from 3 days to 1 day. This could reduce dosing frequency and potentially resolve the challenges of poor patient compliance resulting from extended TB drug treatment regimes. [Table 24] [Table 25]

[0276] Interaction with fungi Philippine and amphotericin B The efficacy of antifungal drugs was examined against the fungal strain Candida albicans. Filipin and amphotericin B are antifungal drugs that act by removing ergosterol from the plasma membrane, promoting cell leakage and death. Both compounds are hydrophobic, and several lipid-complexed products are available. All of the disclosed encapsulated formulations of filipin resulted in increased inhibition relative to the free drug (Figure 36). Incorporation of a positive charge through the inclusion of the cationic lipid DOTAP significantly increased inhibition. This can be observed by the clustering of particles with cationic properties (plus and pentagon symbols) and those without (triangle and square symbols).

[0277] All encapsulated formulations of amphotericin provided at least equivalent inhibition to the free, dissolved drug (Figure 37). Reinclusion of cationic lipid significantly increased inhibition. Compared to the free, dissolved drug, the concentration required to achieve 98% inhibition was reduced 100-fold, from 10 μg / ml to 0.1 μg / ml. [Table 26] [Table 27]

[0278] Fluconazole Fluconazole controls fungal diseases by impairing the synthesis of ergosterol, an important structural component of the fungal cell membrane. However, systemic therapy with fluconazole poses two serious problems: drug toxicity and drug resistance.

[0279] Synthesis of ionizable aminolipid: Aminolipid morpholine oleate (MOE / lipid-5) was synthesized using the esterification reaction between 4-(2-hydroxyethyl)morpholine and oleic acid (OA). Briefly, aminoalcohol (1.1 equiv.) was added to a cooled solution of OA (1.0 equiv.) in DCM, EDCl (1.1 equiv.), and DMAP (0.2 equiv.). The reaction mixture was stirred at room temperature for 24 h. The solvent was removed using a rotary evaporator, and the resulting crude material was purified on a flash silica column. The synthesized lipid structure was confirmed by nuclear magnetic resonance (NMR) imaging (1H) analysis. Fluconazole-loaded and empty nanoparticles without fluconazole were prepared by the dry lipid hydration method. Dry films were prepared by dissolving MO (20 mg / 1 mL ethanol) and MOE (20 mg / 1 mL ethanol), mixing them in a 70:30 ratio, and removing the ethanol by placing them in a vacuum oven for 12 hours. Empty nanoparticles without drugs were prepared by hydrating the formed dry lipids with 1 mL of F127 (2 mg in 1 mL DI water) solution. The resulting mixture was sonicated for 5 minutes in pulsed mode using a probe sonicator (Q Sonica) to obtain an opaque dispersion. Encapsulation formulations were prepared by hydrating the formed dry lipids with 1 mL of DI water containing F127 and fluconazole (2 mg each). The resulting mixture was sonicated for 5 minutes in pulsed mode using a probe sonicator (Q Sonica) to obtain an opaque dispersion.

[0280] Staining and growth conditions: The fungus, fluconazole-resistant Cryptococcus neoformans, was obtained from SA Pathology and stored in potato dextrose (PD) broth at -80°C. The fungus was grown overnight in potato dextrose (PD) plates at 37°C before use. An inoculation loop was used to suspend the cells in PD broth, and the optical density at 600 nm (OD600) was adjusted to approximately 2 using a UV-vis spectrometer. Five μl of the fungal suspension was added to a 96-well plate in a final volume of 100 μl, yielding a final OD600 concentration of approximately 0.1.

[0281] Antimicrobial Assay: Using a 96-well plate, the minimum inhibitory concentrations of antimicrobial agents (fluconazole-free formulation, drug-free control nanoparticles, and fluconazole-encapsulated lipid nanoparticles) were determined to inhibit the growth of fluconazole-resistant C. neoformans by 50% (MIC-50) and 90% (MIC-90). The 96-well plate was incubated in a UV-vis spectrophotometer for 24 hours, and spectra were collected from 220 to 1000 nm. Briefly, the plate contained a range of concentrations of antimicrobial agents incubated with fungi, including a fungal control, a PD broth control, and an antibacterial control. The experiment was performed at pH 5.2 and pH 7.1.

[0282] result O2ME is neutral at pH 7.4 and positively charged at lower pH (4-6.0), causing headgroup extension and charge repulsion at lower pH levels. Incorporation of 30% by weight O2ME into MO at pH 7.4 resulted in hexosomes with an internal hexagonal phase. Due to headgroup extension and charge repulsion at lower pH values, membrane curvature decreased and the mesophase changed from hexagonal to cubic. Due to fluconazole resistance, the MIC-90 was not reached by treating infections with fluconazole alone; however, an encapsulated formulation with a cubic structure and positive charge had an MIC-90 value of 123.015 μg / ml at pH 5.17 compared to 183.843 μg / ml at pH 7.08. SEM and confocal images support the disruption of the fungal wall, as seen in Figure 38. [Table 28] [Table 29]

[0283] Reference list 1.Santos,R.S.,Figueiredo,C.,Azevedo,N.F.,Braeckmans,K.& De Smedt,S.C.Nanomaterials and molecular transporters to overcome the bacterial envelope barrier:Towards advanced delivery of antibiotics.Adv.Drug Deliv.Rev.136,28-48(2018). 2.Sahay,G.et al.Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling.Nat.Biotechnol.31,653(2013). 3.Oh,N.& Park,J.-H.Endocytosis and exocytosis of nanoparticles in mammalian cells.Int.J.Nanomedicine 9,51(2014). 4.Deshpande,S.& Singh,N.Influence of Cubosome Surface Architecture on Its Cellular Uptake Mechanism.Langmuir 33,3509-3516(2017). 5.Prange,J.A.et al.Overcoming Endocytosis Deficiency by Cubosome Nanocarriers.ACS Appl.Bio Mater.(2019). 6.Vandoolaeghe,P.,Barauskas,J.,Johnsson,M.,Tiberg,F.&Nylander,T.Interaction between lamellar(vesicles) and nonlamellar lipid liquid-crystalline nanoparticles as studied by time-resolved small-angle X-ray diffraction.Langmuir 25,3999-4008(2009). 7.Chang,D.P.et al.Non-lamellar lipid liquid crystalline structures at interfaces.Adv.Colloid Interface Sci.222,135-147(2015). 8.Thorn,C. R.,Clulow,A.J.,Boyd,B.J.,Prestidge,C.A.& Thomas,N.Bacterial lipase triggers the release of antibiotics from digestible liquid crystal nanoparticles.J.Control.Release(2019).

Claims

1. A pharmaceutical composition for the treatment or prevention of diseases, disorders, or conditions related to Gram-negative bacteria or fungi, comprising non-lamellariotropic liquid crystal phase particles containing an antibacterial agent or antifungal agent, wherein the non-lamellariotropic liquid crystal phase particles are One or more fusionable amphiphilic lipids selected from those using unsaturated hydrophobic substances and / or isoprenoid-type hydrophobic substances, A further fusionable amphiphilic lipid which is at least one cationic lipid and / or phosphatidylethanolamine A pharmaceutical composition containing the following:

2. The pharmaceutical composition according to claim 1, wherein the disease, disorder, or condition is an infection caused by Gram-negative bacteria or fungi.

3. The pharmaceutical composition according to claim 1 or 2, wherein the non-lamellariotropic liquid crystal phase particles encapsulate the antibacterial agent or antifungal agent within their channels or folds.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the non-lamellariotropic liquid crystal phase particles are formed by the self-assembly of one or more fusionable amphiphilic lipids.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the non-lamellariotropic liquid crystal phase particles comprise at least two fusionable amphiphilic lipids.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the non-lamellar otropic liquid crystal phase particles have a bulk phase selected from the group consisting of a cubic phase, a hexagonal phase, and a sponge phase.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the non-lamellariotropic liquid crystal phase particles are cubosomes.

8. The non-lamellar otropic liquid crystal phase particles are -0.05 nm －１ Internal curvature-induced sprays less than [Math 1] A pharmaceutical composition according to any one of claims 1 to 7, having the following characteristics.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the non-lamellar otropic liquid crystal phase particles have a particle diameter greater than 50 nm.

10. The pharmaceutical composition according to any one of claims 1 to 9, comprising at least one stabilizer present in the non-lamellariotropic liquid crystal phase particles in an amount of 6 to 18% by weight of the particles.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the non-lamellariotropic liquid crystal phase particles contain one or more positively charged lipids in an amount of 0.1 to less than 20 mol%.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the non-lamellariotropic liquid crystal phase particles further comprise a non-amphiphilic and / or non-fusionable charged compound.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the one or more fusionable amphiphilic lipids are selected from those exhibiting hydrophobic tail groups selected from the group consisting of oleoyl, linoleoyl, linolenoyl, phytanoyl, farnesoyl, and elongated aliphatic hydrophobic.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the one or more fusionable amphiphilic lipids are selected from those that exhibit a head group selected from the group consisting of alcohol, carboxyl, polyol, sugar, amide, amine, lactic acid, glyceryl, diglyceryl, coordination complex, caprolactam, ether, acetic acid, quinone, and combinations thereof.

15. The one or more fusionable amphiphilic lipids mentioned above are 1-monoolein, 2-monoolein, citreme, lactic oleoyl, oleamide, monoelaidin, linoleic acid, elaidic acid, monopalmitrein, monolinolein, phytantriol, diolein, triolein, dioleoylglycerol, didodecyldimethylammonium bromide, dioctadecyl(dimethyl)ammonium chloride (DOAC / DODMAC) or bromide (DODAB), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-phosphati A pharmaceutical composition according to any one of claims 1 to 14, selected from the group consisting of diglycerol (DOPG), oleic acid, lyso-hydroxy-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-dihexyl-phosphocholine, vitamin E tocopherol, vitamin E (tocopherol) acetate, phytanoyl monoethanolamide, farnesoyl monoethanolamide, oleoyl monoethanolamide, linoleoyl monoethanolamide, and linolenoyl monoethanolamide.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the non-lamellariotropic liquid crystal phase particles comprise at least two fusionable amphiphilic lipids, and at least one of the fusionable amphiphilic lipids is selected from monoolein and phytantriol.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the antibacterial agent is a Gram-negative antibacterial agent.

18. The pharmaceutical composition according to any one of claims 1 to 17, wherein the antibacterial agent or antifungal agent is hydrophobic.

19. The pharmaceutical composition according to any one of claims 1 to 18, wherein the antibacterial agent or antifungal agent is present in an amount of 0.1 to 30.0 mol% of the particles.

20. The pharmaceutical composition according to any one of claims 1 to 19, wherein the non-lamellar liotropic liquid crystal phase particles include monoolein.

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