Antimicrobial lipid nanoparticles

Abstract

Disclosed herein are lipid nanoparticles that demonstrate effective antimicrobial effects. An example lipid nanoparticle includes an unsaturated monoglyceride, a saturated monoglyceride, and a glycerol behenate. Also disclosed herein are microfluidic methods of making the lipid nanoparticles, pharmaceutical compositions that include the lipid nanoparticles, methods of modulating a microorganism's activity, and methods of promoting beneficial health effects in an animal.

Description

Attorney Docket No. 218760-0002-W001ANTIMICROBIAL LIPID NANOPARTICLESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 691 ,193 filed on September 5, 2024, which is incorporated fully herein by reference.TECHNICAL FIELD

[0002] The present disclosure relates to lipid nanoparticles and microfluidic methods of making the same.INTRODUCTION

[0003] Certain medium-chain, saturated monoglycerides such as glycerol monolaurate (GML) are recognized as having antimicrobial activities due to membrane-disruptive activities. Biophysical studies have established that such monoglycerides are active above their corresponding critical micelle concentration (CMC) values at which they begin to form micelles and can inhibit membrane-enveloped viruses and bacteria. The latter antibacterial functionality is significant given the urgent need to find sustainable alternatives to bacteria-inhibiting antibiotics. On the other hand, medium-chain monoglycerides are generally inactive below CMC where they exist as monomers, which is a key challenge for industrial applications that often involve sample dilution, e g., of nanoparticles. Accordingly, improved lipid nanoparticles and methods of making the same could be beneficial for monoglyceride use in antibacterial applications.SUMMARY

[0004] In one aspect, disclosed herein are lipid nanoparticles including: an unsaturated monoglyceride; a saturated monoglyceride; and a glycerol behenate.

[0005] In another aspect, disclosed herein are lipid nanoparticles including: glycerol monooleate; glycerol monolaurate; and a glycerol behenate selected from the group consisting of glycerol monobehenate, glycerol dibehenate, and a combination thereof.Attorney Docket No. 218760-0002-W001

[0006] In another aspect, disclosed herein are methods of making an organic solvent-free lipid nanoparticle, the method including: microfluidically mixing a lipid mixture, the lipid mixture including a lipid solvent, an unsaturated monoglyceride, a saturated monoglyceride, and a glycerol behenate, with an aqueous mixture, the aqueous mixture including an aqueous buffer and a stabilizer, wherein the lipid mixture and the aqueous mixture are both substantially free of an organic solvent

[0007] In another aspect, disclosed herein are methods of modulating an activity of a microorganism, the method including contacting the microorganism with an effective amount of a lipid nanoparticle as disclosed herein.

[0008] In another aspect, disclosed herein are methods of promoting a beneficial health effect in an animal, the method including administering to the animal an effective amount of a lipid nanoparticle as disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 A shows chemical structures of example lipids.

[0010] FIG. 1 B is a schematic of an example microfluidic diagram.

[0011] FIG. 1 C is a series of pie charts showing the ratio of components in example lipid nanoparticles based on mass ratio.

[0012] FIG. 2 is a set of graphs showing nanoparticle size and zeta potential characterization. Column graph summary of (A) size and (B) zeta potential of example lipid nanoparticles. Data are reported as the mean ± standard deviation from n=3 independent replicates.

[0013] FIG. 3 is a plot showing antibacterial testing of example lipid nanoparticles to inhibit S. aureus bacterial growth. Relative bacterial cell viability levels are reported as a function of nanoparticle mass concentration for the following nanoparticle compositions: (A) GMO, (B) GMO-GML, (C) GMO-GML-GMB, and (D) GMO-GML-GDB. Data are reported relative to bacteria-only controls, and each data point is reported as the mean ± standard deviation from n=3 independent replicates. MICso values were computed based on variable slope analysis (four-parameter logistic curve) of relative bacterial cell viability levels as a function of nanoparticle mass concentration.

[0014] FIG. 4A is a plot showing time-resolved QCM-D change in frequency (Af) characterization of example nanoparticle interactions with supported lipid bilayer platforms.Attorney Docket No. 218760-0002-WQ01Arrow 1 corresponds to nanoparticle injection starting from around t=10 min and arrow 2 corresponds to buffer washing starting from around f=40 min. Baseline signals at t=0 min correspond to initially fabricated supported lipid bilayer (SLB) platforms in equivalent buffer solution.

[0015] FIG. 4B is a plot showing time-resolved QCM-D change in dissipation (AD) signals of example nanoparticle interactions with SLB platforms. Arrow 1 corresponds to nanoparticle injection starting from around f=10 min and arrow 2 corresponds to buffer washing starting from around t=40 min. Baseline signals at t=0 min correspond to initially fabricated SLB platforms in equivalent buffer solution.

[0016] FIG. 4C is a time-independent plot of the QCM-D Af and AD signals for each nanoparticle interaction in FIG. 4A and FIG. 4B.

[0017] FIG. 4D is a bar graph summarizing final Af and AD shifts for example nanoparticle interactions with the SLB platforms. Net shifts are reported relative to the SLB platform values prior to nanoparticle addition and are represented as the mean ± standard deviation from n=3 independent replicates.

[0018] FIG. 5 is a set of plots showing electrochemical impedance spectroscopy (EIS) characterization of example nanoparticle interactions with tethered lipid bilayer platforms. Time- resolved EIS (A) Gmand (B) Cmsignals are presented for nanoparticle interactions with tBLM platforms. Baseline signals at t=0 min correspond to initially fabricated tBLM platforms in equivalent buffer solution.

[0019] FIG. 6A is a Bode plot of example nanoparticle interactions with tethered lipid bilayers for GMO nanoparticles. For FIGS. 6A-D, Baseline refers to tBLM prior to nanoparticle addition, while Treatment and Post-Wash refer to during nanoparticle interaction and after buffer washing, respectively.

[0020] FIG. 6B is a Bode plot of example nanoparticle interactions with tethered lipid bilayers for GMO-GML nanoparticles.

[0021] FIG. 6C is a Bode plot of example nanoparticle interactions with tethered lipid bilayers for GMO-GML-GMB nanoparticles.

[0022] FIG. 6D is a Bode plot of example nanoparticle interactions with tethered lipid bilayers for GMO-GML-GDB nanoparticles.

[0023] FIG. 7 is a set of plots showing QCM-D characterization of SLB platform formation. Time-resolved QCM-D (A) Af and (B) AD signals are presented SLB formation with arrow 1 corresponding to solvent (50 / 50 volume% IPA / water) injection starting from around t=5 min upon achieving baseline signal in PBS buffer, arrow 2 corresponding to lipid (70 / 30 mol%Attorney Docket No. 218760-0002-WQ01DOPC / DOPG) injection, arrow 3 corresponding to PBS buffer exchange and arrow 4 corresponding to washing with PBS buffer that contains 0.031 mM bile salt (equivalent to the concentration in the 0.2 mg / mL lipid nanoparticle test samples).

[0024] FIG. 8A is a plot showing time-resolved QCM-D Af characterization of example nanoparticle interactions with supported lipid bilayer platforms, with arrow 1 corresponding to nanoparticle injection starting from around f=10 min and arrow 2 corresponding to buffer washing starting from around f=40 min. Baseline signals at t=0 min correspond to initially fabricated SLB platforms in equivalent buffer solution.

[0025] FIG. 8B is a plot showing time-resolved QCM-D AD signals for example nanoparticle interactions with SLB platforms, with arrow 1 corresponding to nanoparticle injection starting from around t=10 min and arrow 2 corresponding to buffer washing starting from around t=40 min. Baseline signals at f=0 min correspond to initially fabricated SLB platforms in equivalent buffer solution.

[0026] FIG. 8C shows time-independent plots of the QCM-D Af and AD signals for each nanoparticle interaction of FIG. 8A and FIG. 8B.

[0027] FIG. 9 is a set of plots showing QCM-D characterization of buffer exchange step. The insets in both (A) and (B) present magnified views of the signals for the period after the SLB formed in PBS buffer (f=25 min to f=30 min) and after the subsequent addition of PBS buffer containing 0.031 mM bile salt (equivalent to the concentration in the 0.2 mg / mL lipid nanoparticle test samples) (f=30 min to f=35 min). There were no changes in the signals during and after that period.

[0028] FIG. 10 is a bar graph showing stability of different formulations.

[0029] FIG. 11 is a plot of MIC values for Salmonella and S. aureus with a GDB4-res1 formulation.

[0030] FIG. 12 is a schematic of an example microfluidic process that can be used to make disclosed lipid nanoparticles.

[0031] FIG. 13 is a schematic of a lipid nanoparticle cubosome and different parameters that can be used to control lipid nanoparticle structure.

[0032] FIG. 14A is a scanning electron microscopy (SEM) image of example lipid nanoparticles having a cubosome structure. Scale bar is 2 pm.

[0033] FIG. 14B is a SEM image of example lipid nanoparticles having a cubosome structure. Scale bar is 2 pm.

[0034] FIG. 14C is a SEM image of example lipid nanoparticles having a cubosome structure. Scale bar is 1 pm.Attorney Docket No. 218760-0002-W001

[0035] FIG. 14D is a SEM image of example lipid nanoparticles having a cubosome structure. Scale bar is 5 pm.DETAILED DESCRIPTION

[0036] Disclosed herein are monoglyceride-based nanoparticles with potent antibacterial activity against, e.g., Staphylococcus aureus bacteria as a model pathogen, which can be prepared by a solvent-free microfluidic fabrication strategy. Dynamic light scattering (DLS) and zeta potential measurements verified that the fabricated nanoparticles had ~250- to 350-nm diameters and high colloidal stability due to negative surface charge while the antibacterial activity of the nanoparticles can be influenced by the nanoparticle composition. Nanoparticles composed of glycerol monooleate (GMO) alone were inactive while the inclusion of antimicrobial glycerol monolaurate (GML) slightly improved antibacterial activity. Surprisingly, the additional inclusion of a small amount of glycerol monobehenate (GMB) or glycerol dibehenate (GDB) - long considered to be inactive structural components in the nanoparticle composition boosted antibacterial potency by up to 270-times. Biophysical experiments further showed that nanoparticle compositions with more potent antibacterial activity caused greater membrane disruption according to quartz crystal microbalance-dissipation (QCM-D) and electrochemical impedance spectroscopy (EIS) measurements. These capabilities provide a microfluidic route to prepare antimicrobial lipid nanoparticles without requiring organic solvents and demonstrate how precision tuning of molecular components can dramatically boost the antibacterial activity of monoglyceride-based nanoparticles.1. Definitions

[0037] 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. In case of conflict, the present document, including definitions, will control. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. Methods and materials similar or equivalent to those described herein can be used in practice or testing of the disclosed technology. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0038] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and”Attorney Docket No. 218760-0002-W001 and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0039] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9-1.1. Other meanings of “about” may be apparent from the context, such as rounding off, so, for example “about 1” may also mean from 0.5 to 1.4.

[0040] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0041] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated, and for the range 1.5-2, the numbers 1.5, 1.6, 1.7, 1.8, 1.9, and 2 are contemplated.

[0042] The term “effective dosage” or “therapeutic dosage” or “therapeutically effective amount” or “effective amount,” as used herein, refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results, to modulate a biological process, and / or treat a disease or one or more of its symptoms and / or to prevent or reduce the risk of the occurrence or reoccurrence of the disease or disorder or symptom(s) thereof. A therapeutically effectiveAttorney Docket No. 218760-0002-W001 amount is also one in which any toxic or detrimental effects of substance are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount. In reference to antimicrobial activity an effective or therapeutically effective amount can include an amount sufficient to, among other things, inhibit the growth of the microorganism.

[0043] The term “glyceride,” as used herein, refers to lipophilic compounds comprising a glycerol molecule bonded to fatty acid groups. Monoglycerides are glycerol molecules bonded to a single fatty acid group; diglycerides are glycerol molecules bonded to two fatty acid groups; and triglycerides are glycerol molecules bonded to three fatty acid groups.

[0044] The term “fatty acid,” as used herein, refers to carboxylic acids with lipophilic chains comprising carbon and hydrogen atoms. Specific fatty acids can be identified by counting the number of carbon atoms and determining other chemical properties, such as the presence and location of double bonds between the carbon atoms, any branching of carbon atoms off the main lipophilic chain, and the presence of other atomic species in the chain. Fatty acids may be described as “saturated” (no double or triple bonds between the carbon atoms), “monounsaturated” (one double or triple bond between the carbon atoms), or “polyunsaturated” (more than one double or triple bond between the carbon atoms). In some instances, the number of carbon atoms in a fatty acid is indicated by the prefix “Cx-y” or “Cx-Cy-”, wherein x is the minimum and y is the maximum number of carbon atoms in the fatty acid. Thus, for example, “C6-22 fatty acid” or “C6-C22 fatty acid” refers to a fatty acid containing from 6 to 22 carbon atoms.

[0045] The term “microorganism,” as used herein, refers to an organism of microscopic size, which may exist in its single-celled form or as a colony of cells. A microorganism may be a pathogenic microorganism. Example microorganisms include, but are not limited to, a bacterium, a fungus, a virus, and a parasite.

[0046] “Subject” and “patient,” as used herein, interchangeably refers to any vertebrate, including, but not limited to, a mammal that wants or is in need of the herein described lipid nanoparticles or methods. The subject may be a human or a non-human. The subject may be a vertebrate. The subject may be a mammal. The mammal may be a primate or a non-primate. The mammal can be a non-primate such as, for example, cow, pig, camel, llama, hedgehog, anteater, platypus, elephant, alpaca, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse. The mammal can be a primate such as a human. The mammal can be a nonAttorney Docket No. 218760-0002-W001 human primate such as, for example, monkey, cynomolgous monkey, rhesus monkey, chimpanzee, gorilla, orangutan, and gibbon. The subject may be of any age or stage of development, such as, for example, an adult, an adolescent, or an infant. The subject may be male. The subject may be female. In some embodiments, the subject has a specific genetic marker. The subject may be undergoing other forms of treatment.

[0047] The terms “treatment” or “treating” refer to the medical management of a patient with the intent to heal, cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.2. Lipid Nanoparticles

[0048] Provided herein are lipid nanoparticles that have advantageous antimicrobial properties. The lipid nanoparticle includes an unsaturated monoglyceride, a saturated monoglyceride, and a glycerol behenate. The lipid nanoparticle has an advantageous composition, which can improve interactions with a membrane of a microorganism. This ultimately can allow the disclosed lipid nanoparticles to effectively disrupt the membrane of a microorganism, thereby providing an antimicrobial effect.

[0049] The lipid nanoparticle can have a varying particle size (e.g., diameter). For example, the lipid nanoparticle can have a diameter of about 100 nm to about 500 nm, such as about 100 nm to about 400 nm, about 110 nm to about 390 nm, about 150 nm to about 350 nm, about 175 nm to about 325 nm, about 200 nm to about 300 nm, about 100 nm to about 300 nm, about 200 nm to about 400 nm, or about 200 nm to about 450 nm. In some embodiments, the lipid nanoparticle has a diameter of no less than 100 nm, no less than 125 nm, no less than 150 nm, no less than 175 nm, no less than 200 nm, no less than 225 nm, or no less than 250 nm. In some embodiments, the lipid nanoparticle has a diameter of no greater than 500 nm, no greater than 450 nm, no greater than 400 nm, no greater than 375 nm, no greater than 350 nm, noAttorney Docket No. 218760-0002-W001 greater than 325 nm, no greater than 310 nm, no greater than 300 nm, or no greater than 275 nm.

[0050] The zeta potential of a lipid nanoparticle may be used to indicate the electrokinetic potential of the composition. For example, the zeta potential may describe the surface charge of a lipid nanoparticle. The lipid nanoparticle can have a zeta potential of about -40 mV to about - 10 mV, such as about -38 mV to about -12 mV, about -35 mV to about -15 mV, about -35 mV to about -20 mV, about -35 mV to about -25 mV, about -40 mV to about -25 mV, or about -30 mV to about -10 mV. In some embodiments, the lipid nanoparticle has a zeta potential of no less than -40 mV, no less than -38 mV, no less than -36 mV, no less than -35 mV, no less than -34 mV, no less than -33 mV, no less than -32 mV, no less than -31 mV, no less than -30 mV, or no less than -25 mV. In some embodiments, the lipid nanoparticle has a zeta potential of no greater than -10 mV, no greater than -12 mV, no greater than -14 mV, no greater than -16 mV, no greater than -18 mV, no greater than -20 mV, no greater than -22 mV, or no greater than -25 mV.

[0051] Lipid nanoparticles may be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) may be used to examine the morphology and size distribution of a lipid nanoparticle. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) may be used to measure zeta potentials. Dynamic light scattering may also be used to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) may also be used to measure multiple characteristics of a lipid nanoparticle, such as particle size, polydispersity index, and zeta potential.

[0052] By using microfluidics to make the disclosed lipid nanoparticles, desired shapes can be achieved. For example, the lipid nanoparticle can have a cubosome structure. Cubosomes based on the cubic phase of monoolein or phytantriol can have a unique nanostructure, which can include a lipid bilayer and two water channels. This unique structure can offer high encapsulation of a drug or its active components due to their high surface area.A. Unsaturated Monoglycerides

[0053] The lipid nanoparticle includes an unsaturated monoglyceride. An unsaturated monoglyceride is glycerol molecule bonded to a single fatty acid group, where the fatty acid has at least one multiple bond (e.g., double or triple bond). The lipid nanoparticle can include one type of unsaturated monoglyceride (e.g., an individual unsaturated monoglyceride) or can include at least 2, at least 3, at least 4, or at least 5 different types of unsaturatedAttorney Docket No. 218760-0002-W001 monoglycerides. In some embodiments, the lipid nanoparticle includes 2 to 5 different types of unsaturated monoglycerides.

[0054] The unsaturated monoglyceride can include a fatty acid having a varying hydrocarbon chain length. For example, the unsaturated monoglyceride can include a C4- C3o fatty acid, such as a Ce - C28 fatty acid, a Cs - C26 fatty acid, a C10 - C26 fatty acid, a C12 - C24 fatty acid, a C14 - C22 fatty acid, a C16 - C20 fatty acid, a C4- C20 fatty acid, or a C16 - C30 fatty acid. In some embodiments, the unsaturated monoglyceride includes a Cis - C20 fatty acid.

[0055] In some embodiments, the unsaturated monoglyceride includes a hydrocarbon chain of no more than 22 carbon atoms, no more than 21 carbon atoms, no more than 20 carbon atoms, no more than 19 carbon atoms, or no more than 18 carbon atoms. In some embodiments, the saturated monoglyceride includes a hydrocarbon chain of no less than 10 carbon atoms, no less than 12 carbon atoms, no less than 14 carbon atoms, no less than 16 carbon atoms, or no less than 18 carbon atoms.

[0056] Example unsaturated monoglycerides include, but are not limited to, glyceryl (e.g., glycerol) monooleate, glyceryl monolinoleate, glyceryl monolinolenate, glyceryl monoeicosenoate, glyceryl monoerucate, glyceryl monopalmitoleate, and glyceryl monovaccenate. In some embodiments, the unsaturated monoglyceride includes glyceryl monooleate, glyceryl monolinoleate, glyceryl monolinolenate or a combination thereof. In some embodiments, the unsaturated monoglyceride includes glyceryl monooleate. In some embodiments, the unsaturated monoglyceride is glyceryl monooleate.

[0057] The lipid nanoparticle can include the unsaturated monoglyceride in a varying amount. For example, the lipid nanoparticle can include the unsaturated monoglyceride at about 20 wt% to about 40 wt%, such as about 22 wt% to about 38 wt%, about 24 wt% to about 36 wt%, about 26 wt% to about 34 wt%, about 26 wt% to about 30 wt%, about 22 wt% to about 32 wt%, about 20 wt% to about 30 wt%, or about 26 wt% to about 40 wt%. In some embodiments, the lipid nanoparticle includes the unsaturated monoglyceride at no less than 20 wt%, no less than 21 wt%, no less than 22 wt%, no less than 23 wt%, no less than 24 wt%, no less than 25 wt%, no less than 26 wt%, no less than 27 wt%, no less than 28 wt%, no less than 29 wt%, or no less than 30 wt%. In some embodiments, the lipid nanoparticle includes the unsaturated monoglyceride at no more than 40 wt%, no more than 39 wt%, no more than 38 wt%, no more than 37 wt%, no more than 36 wt%, no more than 35 wt%, no more than 34 wt%, no more than 33 wt%, no more than 32 wt%, no more than 31 wt%, no more than 30 wt%, no more than 29 wt%, or no more than 28 wt%. The foregoing percentages in reference to the unsaturated monoglyceride denote percentage by weight of the lipid nanoparticle.Attorney Docket No. 218760-0002-W001B. Saturated Monoglycerides

[0058] The lipid nanoparticle includes a saturated monoglyceride. A saturated monoglyceride is a glycerol molecule bonded to a single fatty acid group, where the fatty acid includes no multiple bonds (e.g., double or triple bond). The lipid nanoparticle can include one type of saturated monoglyceride (e.g., an individual saturated monoglyceride) or can include at least 2, at least 3, at least 4, or at least 5 different types of saturated monoglycerides. In some embodiments, the lipid nanoparticle includes 2 to 5 different types of saturated monoglycerides.

[0059] The saturated monoglyceride can include a fatty acid having a varying hydrocarbon chain length. For example, the saturated monoglyceride can include a C4 - C30 fatty acid, such as a Ce - C28 fatty acid, a Cs - C26 fatty acid, a C10 - C26 fatty acid, a C12 - C24 fatty acid, a C4 - Cis fatty acid, a C4 - C16 fatty acid, a Ce - C20 fatty acid, a Ce - C16 fatty acid, a Cs - C20 fatty acid, a Cs - C16 fatty acid, a C10 - C14 fatty acid, a C14 - C22 fatty acid, a C16 - C20 fatty acid, or a C16 - C30 fatty acid. In some embodiments, the saturated monoglyceride includes a Ce - Cis fatty acid. In some embodiments, the saturated monoglyceride includes a Cs - C16 fatty acid. In some embodiments, the saturated monoglyceride includes a C4 - C12 fatty acid.

[0060] In some embodiments, the saturated monoglyceride includes a hydrocarbon chain of no more than 12 carbon atoms, no more than 11 carbon atoms, no more than 10 carbon atoms, no more than 9 carbon atoms, or no more than 8 carbon atoms. In some embodiments, the saturated monoglyceride includes a hydrocarbon chain of no less than 4 carbon atoms, no less than 5 carbon atoms, no less than 6 carbon atoms, no less than 7 carbon atoms, or no less than 8 carbon atoms.

[0061] Example saturated monoglycerides include, but are not limited to, glyceryl monostearate, glyceryl monopalmitate, glyceryl monomyristate, glyceryl monolaurate, glyceryl monocaprate, glyceryl monocaprylate, glyceryl monoarachidate, and glyceryl monolignocerate. In some embodiments, the saturated monoglyceride includes glyceryl monostearate, glyceryl monopalmitate, glyceryl monomyristate, glyceryl monolaurate, or a combination thereof. In some embodiments, the saturated monoglyceride includes glyceryl monolaurate. In some embodiments, the saturated monoglyceride is glyceryl monolaurate.

[0062] The lipid nanoparticle can include the saturated monoglyceride in a varying amount. For example, the lipid nanoparticle can include the saturated monoglyceride at about 50 wt% to about 70 wt%, such as about 52 wt% to about 68 wt%, about 54 wt% to about 66 wt%, about 56 wt% to about 64 wt%, about 58 wt% to about 62 wt%, about 55 wt% to about 65 wt%, about 56 wt% to about 66 wt%, about 59 wt% to about 63 wt%, about 50 wt% to about 62 wt%, or about 60 wt% to about 70 wt%. In some embodiments, the lipid nanoparticle includes the saturatedAttorney Docket No. 218760-0002-W001 monoglyceride at no less than 50 wt%, no less than 51 wt%, no less than 52 wt%, no less than 53 wt%, no less than 54 wt%, no less than 55 wt%, no less than 56 wt%, no less than 57 wt%, no less than 58 wt%, no less than 59 wt%, no less than 60 wt%, no less than 61 wt%, or no less than 62 wt%. In some embodiments, the lipid nanoparticle includes the saturated monoglyceride at no more than 70 wt%, no more than 69 wt%, no more than 68 wt%, no more than 67 wt%, no more than 66 wt%, no more than 65 wt%, no more than 64 wt%, no more than 63 wt%, no more than 62 wt%, no more than 61 wt%, or no more than 60 wt%. The foregoing percentages in reference to the saturated monoglyceride denote percentage by weight of the lipid nanoparticle.C. Glycerol Behenates

[0063] The lipid nanoparticle includes a glycerol behenate. A glycerol behenate is a glycerol molecule bonded to a fatty acid group that is behenic acid (e.g., C22H44O2). Example glycerol behenates include glycerol monobehenate, glycerol dibehenate, and glycerol tribehenate. In some embodiments, the lipid nanoparticle includes glycerol monobehenate, glycerol dibehenate, or a combination thereof. In some embodiments, the lipid nanoparticle only includes a glycerol monobehenate. In other embodiments, the lipid nanoparticle only includes a glycerol dibehenate.

[0064] The lipid nanoparticle can include the glycerol behenate in a varying amount. For example, the lipid nanoparticle can include the glycerol behenate at about 0.1 wt% to about 20 wt%, such as about 0.5 wt% to about 18 wt%, about 1 wt% to about 15 wt%, about 0.5 wt% to about 15 wt%, about 0.5 wt% to about 12 wt%, about 0.1 wt% to about 15 wt%, about 0.1 wt% to about 12 wt%, about 0.8 wt% to about 12 wt%, or about 2 wt% to about 15 wt%. In some embodiments, the lipid nanoparticle includes the glycerol behenate at no less than 0.1 wt%, no less than 0.2 wt%, no less than 0.3 wt%, no less than 0.4 wt%, no less than 0.5 wt%, no less than 0.6 wt%, no less than 0.7 wt%, no less than 0.8 wt%, no less than 0.9 wt%, no less than 1 wt%, no less than 2 wt%, no less than 3 wt%, no less than 4 wt%, or no less than 5 wt%. In some embodiments, the lipid nanoparticle includes the glycerol behenate at no more than 20 wt%, no more than 18 wt%, no more than 16 wt%, no more than 15 wt%, no more than 14 wt%, no more than 13 wt%, no more than 12 wt%, no more than 11 wt%, no more than 10 wt%, no more than 9 wt%, no more than 8 wt%, no more than 7 wt%, no more than 6 wt%, or no more than 5 wt%. The foregoing percentages in reference to the glycerol behenate denote percentage by weight of the lipid nanoparticle.D. Additional Components

[0065] The lipid nanoparticle can also include additional components that can aid, e.g., in particle formation, stability, and / or other useful properties. For example, the lipid nanoparticleAttorney Docket No. 218760-0002-W001 can include a stabilizer. Example stabilizers include, but are not limited to, a bile salt (e.g., deoxycholic acid), a cholesterol, and a polyethylene glycol (PEG) conjugated lipid. In some embodiments, the stabilizer includes a bile salt. In some embodiments, the stabilizer is a bile salt. In addition to stabilizing the lipid nanoparticle, bile salts (e.g., cholic acid) can be used to reduce delivery of the lipid nanoparticle to liver cells in vitro and can improve delivery in a variety of other cell types, including T cells, B cells, and epithelial cells. There is also a potential benefit to enhance mRNA therapeutic applications such as for gastrointestinal or immune cell delivery.

[0066] The lipid nanoparticle can also include a tall oil rosin. A tall oil rosin is a natural resin that can be obtained as a byproduct of the kraft pulping process, e.g., used in the paper industry, particularly when processing pine wood. Tall oil rosin can include a mixture of fatty acids, rosin acids, and other compounds. For example, the tall oil rosin can include a monosaturated fatty acid, a diunsaturated fatty acid, a triunsaturated fatty acid, a resin acid, or a combination thereof. In some embodiments, the lipid nanoparticle includes a resin acid. Example resin acids include, but are not limited to, abietic acid, dehydroabietic acid, and pimaric acid. In some embodiments, the resin acid includes abietic acid, dehydroabietic acid, pimaric acid, or a combination thereof.

[0067] Because the lipids of the lipid nanoparticle themselves can provide antimicrobial properties, the lipid nanoparticle does not require further antimicrobial agents to be included. For example, in some embodiments, the lipid nanoparticle does not include a drug or therapeutic, such as a small molecule drug or peptide.E. Example Embodiments

[0068] In some embodiments, the lipid nanoparticle includes by weight of the lipid nanoparticle: about 20 wt% to about 40 wt% of an unsaturated monoglyceride; about 50 wt% to about 70 wt% of a saturated monoglyceride; and about 0.1 wt% to about 20 wt% of a glycerol behenate.

[0069] In some embodiments, the lipid nanoparticle includes glycerol monooleate; glycerol monolaurate; and a glycerol behenate selected from the group consisting of glycerol monobehenate, glycerol dibehenate, and a combination thereof.

[0070] In some embodiments, the lipid nanoparticle includes, by weight of the lipid nanoparticle: about 20 wt% to about 40 wt% of glycerol monooleate; about 50 wt% to about 70 wt% of glycerol monolaurate; and about 0.1 wt% to about 20 wt% of a glycerol behenate selected from the group consisting of glycerol monobehenate, glycerol dibehenate, and a combination thereof.Attorney Docket No. 218760-0002-W001

[0071] In some embodiments, the lipid nanoparticle does not include any other diglycerides or triglycerides other than the glycerol behenate.3. Methods of Making

[0072] The present disclosure also provides methods of making the lipid nanoparticles. The method can take advantage of microfluidics to make stable, uniform lipid nanoparticles. For example, the method can include microfluidically mixing a lipid mixture with an aqueous mixture. Two individual inlets can be used to introduce the lipid mixture and the aqueous mixture into a chamber. The lipid mixture and the aqueous mixture can both be individually heated (e.g., about 30 °C to about 50 °C, such as about 40 °C) prior to flowing through the inlets. In the chamber, the lipid mixture and the aqueous mixture can be microfluidcally mixed and the lipid nanoparticles are provided from this microfluidic mixing. The method can use a chaotic microfluidic platform.

[0073] Microfluidically mixing can include introducing the lipid mixture and the aqueous mixture both at varying individual flow rates. For example, the lipid mixture and the aqueous mixture can be individually introduced at a flow rate of about 0.1 mL / min to about 20 mL / min, such as about 0.2 mL / min to about 18 mL / min, about 0.5 mL / min to about 15 mL / min, about 0.75 mL / min to about 12 mL / min, or about 0.8 mL / min to about 11 mL / min. In some embodiments, the lipid mixture is introduced at a flow rate of about 0.1 mL / min to about 5 mL / min, such as about 0.5 mL / min to about 2 mL / min, about 0.75 mL / min to about 1 .5 mL / min, or about 0.8 mL / min to about 1.2 mL / min. In some embodiments, the aqueous mixture is introduced at a flow rate of about 1 mL / min to about 20 mL / min, such as about 4 mL / min to about 16 mL / min, about 6 mL / min to about 12 mL / min, or about 8 mL / min to about 10 mL / min. The flow rate of the lipid mixture and the aqueous mixture combined can be referred to as the total flow rate. The total flow rate of the method can be the combined flow rates of the lipid mixture and the aqueous mixture as disclosed herein

[0074] Microfluidically mixing can include introducing the lipid mixture and the aqueous mixture at a varying flow rate ratio. For example, the lipid mixture and the aqueous mixture can be introduced at a flow rate ratio of about 10:1 to about 2:1 (aqueous mixture: lipid mixture), such as about 10:1 to about 4:1 , about 10:1 to about 6:1 , or about 9.5:1 to about 8.5:1. In some embodiments, the lipid mixture and the aqueous mixture are introduced at a flow rate ratio of about 9:1 (aqueous mixture: lipid mixture).

[0075] Microfluidically mixing can be performed on a microfluidic device (e.g., NeonanoTM). The microfluidic device can have pumps that can control flow rates and microfluidic mixing. TheAttorney Docket No. 218760-0002-W001 microfluidic device can include a chamber. The chamber can include wells (e.g., fluidic traps), which can aid in microfluidic mixing. In some embodiments, the chamber includes 1 to 12 wells, such as 1 to 10 wells, 1 to 7 wells, 1 to 5 wells, or 1 to 3 wells. In some embodiments, the wells are in series. In some embodiments, the wells are in a serpentine pattern.

[0076] The lipid mixture can include a lipid solvent, the unsaturated monoglyceride, the saturated monoglyceride, and the glycerol behenate. In some embodiments, the lipid mixture includes a lipid solvent, glycerol monooleate, glycerol monolaurate, and a glycerol behenate selected from the group consisting of glycerol monobehenate, glycerol dibehenate, and a combination thereof. The lipid solvent can include a combination of lipids, such as medium chain mono- and diglycerides, propylene glycol monocaprylate, propylene glycol monolaurate, and combinations thereof. An example lipid solvent includes, but is not limited to, Capmul MCM, Capmul PG-8, and Capmul PG-12. In some embodiments, the lipid solvent includes a medium chain mono- and / or diglyceride, a propylene glycol monocaprylate, and a propylene glycol monolaurate at a molar ratio of 6:2:2 (medium chain mono- and / or diglyceride : propylene glycol monocaprylate : propylene glycol monolaurate). The lipid solvent can further include a tall oil rosin or a resin acid as disclosed herein.

[0077] The lipid mixture can have a varying total mass concentration. For example, the lipid mixture can include a total mass concentration of about 50 mg / mL to about 150 mg / mL, such as about 75 mg / mL to about 125 mg / mL, about 80 mg / mL to about 120 mg / mL, or about 90 mg / mL to about 110 mg / mL. In some embodiments, the lipid mixture includes a total mass concentration of no less than 50 mg / mL, no less than 60 mg / mL, no less than 70 mg / mL, no less than 80 mg / mL, no less than 90 mg / mL, or no less than 100 mg / mL. In some embodiments, the lipid mixture includes a total mass concentration of no more than 150 mg / mL, no more than 140 mg / mL, no more than 130 mg / mL, no more than 120 mg / mL, no more than 110 mg / mL, or no more than 100 mg / mL.

[0078] The aqueous mixture can include an aqueous buffer and a stabilizer. An example aqueous buffer includes, but is not limited to, phosphate buffered saline. The aqueous buffer can have a pH of about 7.1 to about 7.7, such as about 7.4 The stabilizer is discussed elsewhere herein. As an example, the stabilizer can be a bile salt.

[0079] The lipid mixture and the aqueous mixture can both individually be substantially free of an organic solvent. In some embodiments, the lipid mixture and the aqueous mixture are both individually free of an organic solvent. In some embodiments, the lipid mixture and the aqueous mixture both individually include an organic solvent at no more than 1% by mass, no more than 0.9% by mass, no more than 0.8% by mass, no more than 0.7% by mass, no more than 0.6%Attorney Docket No. 218760-0002-W001 by mass, no more than 0.5% by mass, no more than 0.4% by mass, no more than 0.3% by mass, no more than 0.2% by mass, no more than 0.1% by mass, or no more than 0.01% by mass. Mass percentage is based on the to total mass of the lipid mixture or the aqueous mixture. Because the disclosed method does not require the use of an organic solvent to solubilize the monoglycerides, in some embodiments, the method does not include dialysis or a dialysis step. In some embodiments, the method does not include a drying step (e.g., for removing an organic solvent).

[0080] The description of the lipid nanoparticles, unsaturated monoglyceride, saturated monoglyceride, glycerol behenate, and additional components can also be applied to the methods of making the lipid nanoparticles disclosed herein.4. Pharmaceutical Compositions

[0081] Further disclosed herein are pharmaceutical compositions that include one or more lipid nanoparticles. The pharmaceutical composition can further include a pharmaceutically acceptable excipient. The term “pharmaceutically acceptable excipient,” as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable excipients are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, citrate buffers, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. The route by which the composition is administered and the form of the composition can dictate the type of excipient to be used.

[0082] The route by which the disclosed lipid nanoparticles and pharmaceutical compositions thereof are administered can dictate the type of excipient to be used. The lipid nanoparticle andAttorney Docket No. 218760-0002-W001 pharmaceutical composition thereof may be in a variety of forms, suitable, for example, for systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral) or topical administration (e.g., dermal). The term “parenterally,” as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. In some embodiments, the lipid nanoparticle or pharmaceutical composition thereof is administered orally, intravenously, subcutaneously, transdermally, intramuscularly, transnasally, topically, sebaceously, transfollicularly, or intraperitoneally. In some embodiments, the lipid nanoparticle or pharmaceutical composition thereof is not administered ocularly.

[0083] The disclosed lipid nanoparticles and pharmaceutical compositions thereof may be topically administered. Topical compositions that may be applied locally to the skin may be in any form including solids, solutions, oils, creams, ointments, gels, lotions, shampoos, leave-on and rinse-out hair conditioners, milks, cleansers, moisturizers, sprays, skin patches, and the like. Topical compositions can include a disclosed lipid nanoparticle and an excipient. The excipient of the topical composition may aid penetration of the lipid nanoparticles into the skin. The excipient may further include one or more optional components. In some embodiments, the lipid nanoparticle or pharmaceutical composition thereof is administered as part of a wound dressing, a topical cream, or a combination thereof.

[0084] The pharmaceutically acceptable excipient may make up greater than 50% of the total mass or volume of a pharmaceutical composition including a lipid nanoparticle(s). For example, the pharmaceutically acceptable excipient may make up about 50%, about 60%, about 70%, about 80%, about 90%, or more of a pharmaceutical composition. In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the pharmaceutically acceptable excipient is approved for use in humans and for veterinary use. In some embodiments, the pharmaceutically acceptable excipient is approved by United States Food and Drug Administration. In some embodiments, the pharmaceutically acceptable excipient is pharmaceutical grade. In some embodiments, the pharmaceutically acceptable excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0085] General guidelines for the formulation and manufacture of pharmaceutical compositions and agents are available, for example, in Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006, which is incorporated by reference herein in its entirety. Conventional excipients and accessoryAttorney Docket No. 218760-0002-W001 ingredients may be used in any pharmaceutical composition, except insofar as any conventional excipient or accessory ingredient may be incompatible with one or more components of a lipid nanoparticle. An excipient or accessory ingredient may be incompatible with a component of a lipid nanoparticle if its combination with the component may result in any undesirable biological effect or otherwise deleterious effect.

[0086] In some embodiments, the pharmaceutically acceptable excipient includes buffering agents, solubilizers, solvents, antimicrobial preservatives, antioxidants, suspension agents, a tablet or capsule diluent, a tablet disintegrant, or a combination thereof. In some embodiments, the pharmaceutically acceptable excipient includes buffering agents, solubilizers, solvents, antimicrobial preservatives, antioxidants, suspension agents, a tablet or capsule diluent, or a tablet disintegrant.

[0087] The pharmaceutical compositions may be suitable for administration to a subject (such as a patient, which may be a human or non-human) well known to those skilled in the pharmaceutical art. The pharmaceutical composition may be prepared for administration to a subject. Such pharmaceutical compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular subject, and the route of administration.

[0088] The composition may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.

[0089] As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight, the severity of the affliction, and subjects treated, the particular compounds employed, and the specific use for which these compounds are employed. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine methods, for example, human clinical trials, in vivo studies and in vitro studies.

[0090] Dosage amount and interval may be adjusted individually to provide plasma levels of the biologically active agent which are sufficient to maintain the modulating effects, or minimal effective concentration (MEC). The MEC will vary for each agent but can be estimated from in vivo and / or in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, assays well known to those in the art can be used to determine plasma concentrations. Dosage intervals can also be determined usingAttorney Docket No. 218760-0002-W001MEC value. Compositions can be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, such as between 30-90% or between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the lipid nanoparticle may not be related to plasma concentration.

[0091] The pharmaceutical composition can be administered at varying dosages depending on, e.g., different characteristics of the subject and the route of administration. In some embodiments, pharmaceutical compositions of the disclosure may be administered at dosage levels sufficient to deliver about 0.0001 mg / kg to about 10 mg / kg, about 0.001 mg / kg to about 10 mg / kg, about 0.005 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 2 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg, about 0.0001 mg / kg to about 5 mg / kg, about 0.001 mg / kg to about 5 mg / kg, about 0.005 mg / kg to about 5 mg / kg, about 0.01 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 5 mg / kg, about 2 mg / kg to about 5 mg / kg, about 0.0001 mg / kg to about 1 mg / kg, about 0.001 mg / kg to about 1 mg / kg, about 0.005 mg / kg to about 1 mg / kg, about 0.01 mg / kg to about 1 mg / kg, or about 0.1 mg / kg to about 1 mg / kg in a given dose, where a dose of 1 mg / kg provides 1 mg lipid nanoparticle per 1 kg of subject body weight. In some embodiments, a dose of about 0.005 mg / kg to about 5 mg / kg of lipid nanoparticle of the disclosure may be administrated.

[0092] It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the symptoms to be treated and the route of administration. Further, the dose, and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.5. Methods of Use

[0093] Also disclosed herein are methods of using the lipid nanoparticles and pharmaceutical compositions thereof. The description of the lipid nanoparticles, unsaturated monoglyceride, monosaturated glyceride, glycerol behenate, additional components, and pharmaceutical compositions can also be applied to the methods of modulating and promoting disclosed herein.A. Methods of Modulating an Activity of a MicroorganismAttorney Docket No. 218760-0002-W001

[0094] Further provided are methods of modulating an activity of a microorganism. The method can include contacting a microorganism with an amount, such as an effective amount, of a lipid nanoparticle or pharmaceutical composition thereof as disclosed herein. Modulating the activity of the microorganism can include preventing, inhibiting, and / or reducing the activity of the microorganism. In some embodiments, modulating the activity of the microorganism includes killing the microorganism.

[0095] The microorganism can be any type of microorganism that can interact with the lipid nanoparticle (e.g., through membrane interactions with the lipid nanoparticle), such that the interaction can modulate the microorganism’s activity. Example microorganisms include, but are not limited to, a bacterium, a fungus, a virus, and a parasite. In some embodiments, the microorganism is a bacterium. Example bacterium include, but are not limited to, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter, and Escherichia coli. In some embodiments, the bacterium is selected from the group consisting of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter, Escherichia coli., and a combination thereof.

[0096] The bacterium can be in an in vitro or an in vivo environment. For example, the bacterium can be contacted outside of the subject. Alternatively, the bacterium can be in the subject. In some embodiments, the subject is a human or an animal.B. Methods of Promoting Beneficial Health Effects

[0097] Also provided are methods of promoting beneficial health effects, e.g., in an animal. The method can include administering to the animal an amount, such as an effective amount, of a lipid nanoparticle or pharmaceutical composition thereof. The lipid nanoparticle or pharmaceutical composition thereof can be administered orally, e.g., as part of an animal’s diet. The method can advantageously be used to promote beneficial health effects in animals used in food or meat production. Example animals include, but are not limited to, cattle, pigs, chicken (e.g., broiler chicken), sheep, and goats.

[0098] The lipid nanoparticle or pharmaceutical composition thereof can improve microorganism population within the digestive track of the animal. This in turn, can provide a beneficial health effect for the animal. For example, the beneficial health effect can include increased weight gain, decreased colonization of a microorganism, decreased mortality rate, or a combination thereof.6. ExamplesAttorney Docket No. 218760-0002-W001Example 1Materials and Methods for Example 2

[0099] Reagents. Glycerol monooleate (GMO), glycerol monolaurate (GML), glycerol monobehenate (GMB), glycerol dibehenate (GDB), Capmul MCM (MCM), Capmul PG-8 (PG8), and Capmul PG-12 (PG12). 1 ,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2- dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) were obtained from Avanti Polar Lipids (Alabaster, AL). All other reagents were of analytical grade, and deionized water was prepared using a Milli-Q purification system (MilliporeSigma, Burlington, MA).

[0100] Microfluidic preparation of lipid nanoparticles. Nanoparticles were fabricated using the Neonano™ system (Nanofluidics, CA, USA) with ISMATEC pump control via microfluidic mixing. The lipid phase was prepared by combining GMO, GML, GMB, and / or GDB in a lipid solvent (MCM:PG8:PG12 in a 6:2:2 molar ratio) to achieve a final concentration of 100 mg / mL of the lipid combination and adjusting volumes to achieve the specified ratio. The solvent was initially formulated by mixing the components and heating to 40 °C, followed by addition of the monoglycerides at the same temperature using a water bath (Grant Instruments, Ltd., Cambridge, UK). The lipid phase, maintained at 40 °C, was then blended with an aqueous phase including 1 mM deoxycholic acid (bile salts) in phosphate-buffered saline (PBS) at pH 7.4 through the microfluidic cartridge. Mixing parameters were set at a 9:1 ratio between the aqueous and lipid phases, with a total flow rate of 10 mL / min. Both phases were preheated to 40 °C in a water bath before mixing.

[0101] Nanoparticle characterization. The particle size was determined by dynamic light scattering (DLS) with a Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK). Before measurement, the samples were diluted to one-tenth of the original concentration (6.6 mg / mL) using PBS buffer at pH 7.4. Each sample was analyzed in triplicate using disposable plastic cuvettes, maintaining a consistent temperature of 25 °C throughout the measurements.

[0102] Bacterial cell culture. Staphylococcus aureus bacteria (ATCC 25923) (American Type Culture Collection, Manassas, VA) were cultured in Mueller Hinton broth (MHB) overnight at 37 °C in a JB Series incubator (Grant Instruments, Cambridge, UK). The suspension of the overnight culture was inoculated in fresh MHB broth and cultured under aerobic conditions until reaching an optical density at 600-nm wavelength (OD600) value of ~0.3 (exponential growth phase), as determined by spectrophotometric measurements (Jenway 6310, Barloworld Scientific Ltd, Dunmow, UK). The bacterial cells were harvested by centrifugation at 1500xg for 10 min, washed three times with PBS, and resuspended in MHB. The bacterial cell suspensionsAttorney Docket No. 218760-0002-W001 were diluted to an OD600 value of 0.1 , which corresponds to 1 x 107CFU / mL for S. aureus and used within 15 min for experiments.

[0103] Antimicrobial assay. The minimum inhibitory concentration (MIC) values of the antimicrobial lipid nanoparticles were determined by the broth microdilution method according to the standards set by the National Committee for Clinical Laboratory Standards (NCCLS) and British Society for Antimicrobial Chemotherapy (BSAC). Briefly, two-fold serial dilutions of the nanoparticle solutions in PBS were initially prepared in a 96-well microtiter plate and covered a concentration range from 40 pg / mL to 10 mg / mL. Each sample had a volume of 100 pL. Subsequently, a 5 pL inoculum of S. aureus (1 x 105CFU / mL) suspended in MHB was added to each well, which resulted in a final cell density of 5 x 104CFU per well. The samples were then incubated for 24 hr at 37 °C and the relative bacterial cell viability as a function of nanoparticle concentration was determined by OD600 measurements using a microplate reader (SpectraMax M5, Molecular Devices, UK). The MICso value for each nanoparticle sample was determined by a four-parameter logistic (4PL) curve fit of the concentration-dependent OD600 data and corresponds to the nanoparticle concentration that is required to inhibit bacterial cell viability by 50%.

[0104] Quartz crystal microbalance-dissipation. The interactions between antimicrobial lipid nanoparticles and supported lipid bilayer (SLB) platforms were investigated by utilizing the QSense E4 instrument (Biolin Scientific AB, Sweden). All experiments were performed using silica-coated quartz crystal sensor chips (QSX 303, Biolin Scientific AB), and changes in the resonance frequency (Af) and energy dissipation (AD) signals were tracked as functions of time. All samples were introduced into the measurement chamber at a volumetric flow rate of 100 pL / min. A stable baseline was first established with buffer solution, then 0.2 mg / mL of the nanoparticle samples was injected for 30 min, followed by a buffer washing step. Data were recorded at multiple odd overtones, and presented data are from the fifth overtone.

[0105] Electrochemical impedance spectroscopy. The effects of the antimicrobial lipid nanoparticles on the electrochemical properties of tethered bilayer lipid membrane (tBLM) platforms were investigated using the tethaPod instrument (product code: SDx-R1 , SDx Tethered Membranes, Sydney, Australia). The measurements were performed in alternating current (AC) mode and swept-frequency impedance spectroscopy with a 25 mV AC excitation and frequency range of 0.1 Hz to 2000 Hz was used. There was no offset voltage. Experiments were conducted using gold electrode slides (product code: SDx-BG, SDx Tethered Membranes) that were supplied with a benzyl-disulfide ethylene glycol T10 monolayer consisting of 90% spacer (hydroxyl terminated benzyldisulphide tetra-ethylene glycol) and 10% tetherAttorney Docket No. 218760-0002-W001(benzyldisulphide polyethylene glycol phytanyl). A slide was inserted into the tethaPlate cartridge (product code: SDx-T10, SDx Tethered Membranes) and then a solvent-exchange protocol was implemented to form the tBLM. The membrane composition including 70% zwitterionic C20 diphytanyl-diether-phosphatidylcholine lipid and 30% C20 glycerol diphytanyl ether (DPEPC) lipid (product code: SDx-S1 , SDx Tethered Membranes). Changes in the membrane conductance (Gm) and capacitance (Cm) signals were tracked as functions of time.Example 2Example Lipid Nanoparticles - I

[0106] Microfluidic Fabrication Strategy: A chaotic mixing-based microfluidic platform was employed to fabricate the lipid nanoparticles (FIG. 1 B). There were two independently flowing inlet streams and each inlet sample was preheated at 40 °C. In one inlet, the liquid-phase mixture of monoglycerides at the defined ratio was injected at a total mass concentration of 100 mg / mL. In the other inlet, the aqueous phase containing PBS (pH 7.4) with 1 mM deoxycholic acid (bile salt) was injected and the bile salt was included as a nanoparticle stabilizer. The total flow rate was 10 mL / min and the flow rate ratio was 9:1 (aqueous to lipid phases). The resulting lipid nanoparticles were dispersed in PBS at a total mass concentration of 10 mg / mL. A novel element of the present strategy is that no organic solvent is needed to solubilize the monoglycerides and hence no dialysis or drying step is needed post-fabrication.

[0107] Within the microfluidic chamber, the two inlet streams passed through a series of 12 wells (fluidic traps), which were organized in series of three wells followed by a serpentine turn. The microfluidic design also included flow bifurcation whereby some flowing liquid bypassed each well and remixed subsequently. Past experiments with dyed fluids as well as finite element analysis simulations support that high mixing efficiency (>90%) is achieved by the seventh well in the design and that the overall mixing is controlled and reproducible.

[0108] FIG. 1A presents chemical structures of the different lipids used in the nanoparticle design. The base component was glycerol monooleate (GMO), which is the monoglyceride derivative of oleic acid and has an 18-carbon long chain with one degree of unsaturation. When dispersed in aqueous solution, GMO is known to self-assemble into lyotropic liquid crystal nanoparticles depending on the water fraction and stabilizer concentration. Glycerol monolaurate (GML), which is a monoglyceride derivative of lauric acid and has a 12-carbon long saturated chain, was also used because it is known to be one of the most potent antimicrobial lipids and has widely documented antibacterial activities. In addition to these main lipids, a small fraction of glycerol behenate were included, which includes mono- and diglycerides that have aAttorney Docket No. 218760-0002-W00122-carbon long saturated chain and is often used as a structural component in solid lipid nanoparticles. Two versions of glycerol behenate were used that were mainly composed of glycerol monobehenate (GMB, monoglyceride) or glycerol dibehenate (GDB, diglyceride). Four main nanoparticle compositions including (1) GMO, (2) GMO and GML (GMO-GML), (3) GMO, GML, and GMB (GMO-GML-GMB), and (4) GMO, GML, and GDB (GMO-GML-GDB) were fabricated using the microfluidic platform and the compositional breakdowns are provided in FIG. 1C.

[0109] Nanoparticle Characterization: The size and zeta potential of the fabricated nanoparticles were then characterized. GMO nanoparticles had a mean diameter around 342 ± 31 nm while GMO-GML nanoparticles had a smaller diameter around 282 ± 23 nm (FIG. 2 at A). Similar sizes were also observed for GMO-GML-GMB and GMO-GML-GDB nanoparticles, which had mean diameters around 252 ± 40 nm and 253 ± 15 nm, respectively. The saturated chains of GML, GMB, and GDB can likely have strong intermolecular interactions that contribute to tight packing within the nanoparticles, as compared to GMO nanoparticles that have only unsaturated lipids and more disordered packing. The nanoparticles were negatively charged and had zeta potential values in the range of -23 to -33 mV, which indicate high colloidal stability (FIG. 2 at B).

[0110] Antibacterial Testing-. Since one of the main nanoparticle components, GML, exhibits significant inhibitory activity against Gram-positive bacteria, Staphylococcus aureus (S. aureus) was selected as a model species for antibacterial activity testing (FIG. 3). A fixed inoculum of S. aureus was incubated with different nanoparticle concentrations for 24 h at 37 °C before bacterial cell growth density was measured by optical density measurements. The relative level of growth inhibition was determined compared to bacterial-only controls.

[0111] Up to 5.2 mg / mL mass concentration, GMO nanoparticles had largely negligible inhibitory activity while they were able to inhibit S. aureus bacteria by over 99% at a high concentration of 10.4 mg / mL. Similarly, GMO-GML nanoparticles were largely inactive up to a 2.6 mg / mL concentration but reduced S. aureus bacteria by 51 % at 5.2 mg / mL concentration. In addition, at 10.4 mg / mL mass concentration, the GMO-GML nanoparticles almost completely inhibited S. aureus bacteria by over 99%.

[0112] In marked contrast, the inclusion of GMB in the nanoparticle significantly potentiated antibacterial potency. Down to 0.16 mg / mL mass concentrations, GMO-GML-GMB nanoparticles caused >99% bacterial inhibition while 0.08 and 0.04 mg / mL concentrations caused around 13% and 10% inhibition, respectively. The nanoparticles were inactive at lower concentrations. The potentiating effect of GDB inclusion was even greater. Down to 0.04 mg / mLAttorney Docket No. 218760-0002-WQ01 mass concentrations, GMO-GML-GDB nanoparticles caused complete bacterial inhibition while 0.02 and 0.01 mg / mL concentrations caused around 10% and 6% inhibition, respectively. Lower nanoparticle concentrations were inactive. Together, these data support that GMO-GML-GMB and GMO-GML-GDB nanoparticles were appreciably more potent than GMO and GMO-GML nanoparticles. The data further support that GMO-GML-GDB nanoparticles were inhibitory at concentrations where GMO-GML nanoparticles were almost inactive.

[0113] Based on these results, the extrapolated minimum inhibitory concentration (MICso) values at which each nanoparticle composition caused 50% inhibition of S. aureus bacteria were computed. The GMO and GMO-GML nanoparticles had MICso values of 10.8 and 6.7 mg / mL, respectively. These values indicate that these nanoparticle compositions have nearly negligible antibacterial activities. By contrast, the GMO-GML-GMB and GMO-GML-GDB nanoparticles had MICso values of 0.15 and 0.04 mg / mL, respectively, which indicate high antibacterial activity. The antibacterial activity of the different nanoparticles can be ranked as follows: GMO-GML-GDB > GMO-GML-GMB > GMO-GML > GMO. Of note, the MICso values further indicate that the GMO-GML-GDB nanoparticles had over 3-fold greater antibacterial potency than GMO-GML-GMB nanoparticles.

[0114] QCM-D Testing of Supported Lipid Bilayer Interactions: Motivated by the observed antimicrobial activities and ability of GML-based compositions to disrupt bacterial cell membranes, the membrane-disruptive properties of the different nanoparticle compositions were tested using negatively charged 70 / 30 mol% DOPC / DOPG SLB platforms. The SLB platforms were fabricated on silica-coated sensor chip surfaces by utilizing the solvent-assisted lipid bilayer (SALB) technique and QCM-D measurements confirmed high-quality SLB formation based on Af and AD shifts of around -24 ± 1 Hz and < 1 x 10-6, respectively, relative to buffer baseline signals (FIG. 7). After SLB formation, the Af and AD signals were reset to zero and the appropriate nanoparticle composition was injected into the SLB-coated measurement chamber under continuous flow conditions at a fixed mass concentration of 0.2 mg / mL for direct comparison.

[0115] FIG. 4A and FIG. 4B present the corresponding QCM-D measurement data for the time-resolved Af and AD signals. The measurement responses indicate that all nanoparticle compositions attached to the SLB interface as seen by an initial decrease in the Af signal that signals a mass increase. In tandem, the AD signal also increased by around 4 to 6 x 10’6, which is evidence that the nanoparticle adsorption process caused the SLB interface to transform from a rigid adlayer into a softer, more viscoelastic adlayer.Attorney Docket No. 218760-0002-W001

[0116] Notably, depending on the nanoparticle composition, the Af signal response over the entire interaction time course revealed marked differences in the membrane-interaction behavior (of. FIG. 4A). GMO nanoparticles initially attached until reaching an Af shift value of - 14 Hz and then the Af signal gradually increased to around -10 Hz. After buffer washing, the Af signal stayed around -8 Hz indicating modest nanoparticle attachment. Similarly, GMO-GML nanoparticles initially attached to the SLB interface until reaching a Af shift value of -21 Hz but there was a sharp uptick in the Af signal to around -12 Hz, suggesting more pronounced membrane-disruption behavior that aligns with the antibacterial data. However, the Af signal eventually stabilized at around -8 Hz and buffer washing only caused a slight increase to around -6 Hz.

[0117] On the other hand, the addition of GMO-GML-GMB nanoparticles caused a rapid decrease in the Af signal to around -16 Hz before rising to -6 Hz. The signal continued to rise to around -3 Hz and remained around -2 Hz even after buffer washing. These responses indicated that mass changes on the sensor surface were relatively negligible and the complex interaction kinetics are suggestive of a more dynamic interaction process involving membrane disruption. Supporting this view, the addition of GMO-GML-GDB nanoparticles caused intense membrane disruption that was markedly distinct from the other compositions. Initially, the Af signal rapidly decreased to around -21 Hz before steadily rising to around +12 Hz. A subsequent buffer washing step removed weakly attached adsorbate material, resulting in a net Af shift of +16 Hz that indicates extensive mass loss from the SLB interface that is likely related to nanoparticle- induced membrane disruption.

[0118] To further analyze the QCM-D data, time-independent curves of the Af vs. AD signals were plotted for each nanoparticle composition, which is a useful way to analyze structural transformations of the SLB platform due to nanoparticle-induced membrane disruption. In general, larger Af vs. AD responses indicate greater disruption while slope changes in the corresponding curves point to structural transformations / rearrangements, similar to spontaneous and peptide-mediated vesicle-to-bi layer transformations. FIG. 4C depicts the Af vs. AD responses and showed that the GMO-GML-GDB nanoparticles caused the greatest membrane disruption, followed by the GMO-GML-GMB and GMO-GML nanoparticle compositions. In all three cases, structural rearrangements are evident from slope changes in the respective curves, which support that all three compositions caused nanoparticle-induced membrane disruption to varying extents. By contrast, GMO nanoparticles caused nearly negligible structural rearrangements, reinforcing that nanoparticle adsorption was the main interaction outcome in that case.Attorney Docket No. 218760-0002-W001

[0119] A summary of the final Af and AD shifts relative to the initial SLB values based on multiple runs is also provided in FIG. 4D and supports that the GMO-GML-GDB nanoparticles caused the greatest membrane disruption in general. GMO and GMO-GML nanoparticles caused net Af shifts of around -8 Hz and -3 Hz, respectively. GMO-GML-GMB nanoparticle addition also caused a negligible net Af shift. In marked contrast, GMO-GML-GDB nanoparticles caused a dramatic net Af shift of around +15 Hz. In all cases, the corresponding net AD shifts were around 2 to 3 10’6, indicating that all tested nanoparticles interacted with the SLB interfaces while the extent of membrane disruption depended on the nanoparticle composition.

[0120] In addition to the lipid components of the nanoparticles, the other formulation components in the nanoparticle composition were found to be useful in the preparation of GMO- GML-GDB nanoparticles. For example, GMO-GML-GDB nanoparticles that lacked bile salt stabilizers in the formulation media did not disrupt the SLB platform (FIG. 8A, FIG. 8B, and FIG. 8C). Furthermore, free bile salt at an equivalent concentration to the bile salt amount in the formulation media did not affect the SLB platform as well (FIG. 9). Together, these findings support that the nanoparticle compositions disrupted SLBs in the following order: GMO-GML- GDB > GMO-GML-GMB > GMO-GML > GMO.

[0121] EIS Testing of Tethered Lipid Bilayer Permeabilization: These results to further investigation the membrane-disruptive properties of the different nanoparticle compositions by conducting EIS measurements with tethered lipid bilayer membrane (tBLM) platforms. tBLMs composed of branched chain phytanyl lipids with high electrical sealing properties were initially fabricated on functionalized gold electrode surfaces according to a solvent-exchange type process and the resulting membranes had low conductance (Gm) and capacitance (Cm) values of <1 pS and ~1 pF / cm2, respectively. After tBLM formation was verified, nanoparticles were injected into the tBLM measurement chambers at a fixed mass concentration of 0.2 mg / mL for direct comparison and time-resolved changes in the Gmand Cmparameters were tracked.

[0122] Marked differences in the membrane-permeabilizing behavior of different nanoparticle compositions were observed based on the Gmsignal, as reflected in FIG. 5 at A. The greatest permeabilizing activity occurred with the GMO-GML-GDB nanoparticles, which caused a maximum Gmsignal of around 115 pS that indicates extensive permeabilization. After a buffer washing step, the Gmsignal remained around 17 pS, which indicates irreversible membrane disruption. GMO-GML and GMO-GML-GMB nanoparticles also caused extensive membrane permeabilization as indicated by maximum Gmsignals of around 45-47 pS and the signals remained at around 11 pS even after buffer washing. By contrast, GMO nanoparticles only caused a maximum Gmsignal of around 7 pS and that dropped back down to around 2 pS afterAttorney Docket No. 218760-0002-W001 buffer washing. A similarly low level of inactivity was also observed for the negative control GMO-GML-GDB nanoparticle lacking bile salt in the formulation media. In addition to the Gmsignal changes, there were nearly negligible changes in the Cmsignal for all tested nanoparticle compositions (FIG. 5 at B). This finding indicates that the nanoparticle interaction mainly caused enhanced membrane permeabilization of the tethered lipid bilayers, which is indicated by the increased conductance.

[0123] To further assess the structural changes caused by nanoparticle-induced membrane permeabilization, Bode plots of the EIS measurement data were also analyzed, which represent the measured phase vs. applied frequency from the EIS runs (FIG. 6A, FIG. 6B, FIG. 6C, and FIG. 6D). The measured phase exhibits frequency-dependent behavior whereby a phase minimum occurs at a specific frequency and variations in the location of this phase minimum can indicate changes to the membrane properties. GMO nanoparticle addition caused the smallest changes in the Bode plot signatures while GMO-GML-GDB nanoparticle addition caused the largest and most irreversible changes. In accordance with the Gmand Cmparameters, the GMO-GML-GMB and GMO-GML nanoparticle compositions showed intermediate behavior, indicating a similarly moderate level of irreversible membrane permeabilization. Together, the EIS data support that the nanoparticle compositions permeabilized tBLMs in the following order: GMO-GML-GDB > GMO-GML-GMB ~ GMO-GML > GMO.

[0124] This example supports including additional monoglyceride components like GML and GMB along with GDB to increase the interfacial activity of GMO-based nanoparticles. While GML can be an antimicrobial lipid, it only increased antibacterial potency by around 60%. Interestingly, the further incorporation of GMB or GDB into the GMO-GML base composition dramatically boosted antibacterial potency by 72- or 270-times, respectively, compared to GMO nanoparticles. Since GMB and GDB have high melting points (> 50 °C). Furthermore, the results demonstrate that the antimicrobial lipid nanoparticles developed in this study are active in physiological pH range. As GDB is widely used in solid lipid nanoparticle design, it is anticipated that the GMO-GML-GDB nanoparticle composition has particularly high relevance for translational application possibilities by combining high antibacterial activity and use of industrially relevant lipid molecules, especially when considered alongside the capability to produce them using solvent-free microfluidics without requiring dialysis or other downstream purification processes.Example 3Attorney Docket No. 218760-0002-W001Example Lipid Nanoparticles Including Tall Oil Fatty Acid

[0125] Unmodified Tall Oil Rosin (TOR) product (CAS number: 8050-09-7) was used to make cubosome lipid nanoparticles with a slightly elevated content of abietic-type rosin acids. Details of the TOR are described below in Table 1 and Table 2.Table 1. Analysis of unmodified tall oil rosin (TOR).Analysis Value MethodAcid value, mg (KOH) / 1g 176 SCAN-T 11Color, rosin (Gardner) 1.7 ASTM D 6166, mod.Free rosin acids, % 91 SCAN-T 14Softening point (Metter cup & ball), °C 68 ASTM D 6090Unsaponificables, % 3 SCAN-T 13Table 2. Fatty acid analysis of unmodified TOR.TOFA: Degree of fatty acid saturation Value from the manufacturerSaturated 2.8Monounsaturated 31.1Diunsaturated - conjugated linoleic acids 55.2- 8.6Triunsaturated - pinolenic (columbinic) acid 10.2- 7.1Others 0.7

[0126] Preparing TOR. Melting a solid rosin required temperatures above 150 °C to avoid crystallization. For storage, thermally insulated tanks and equipment were used to maintain temperatures between 150 °C and 200 °C. Hot molten rosin had limited stability and did not undergo prolonged storage.

[0127] Solubility in solvent (step before lipid phase preparation). Resin was added to the lipid solvent before microfluidics step. Resin was melted in a water bath. After 24 hr, the resin was observed to remain melted in the solvent. A maximum solubility of approximately 35 mg / mL was observed.

[0128] Microfluidics. See FIG. 1B for a microfluidics diagram showing an inlet 1 , an inlet 2, and an outlet. In addition, FIG. 12 and FIG. 13 show example equipment and parameters for performing microfluidic-based lipid nanoparticle preparation. Resin was added to a lipid phase before being provided to microfluidic inlet 1 . A water phase with 1 mM Bile salts in PBS (pH 7.4 at 25 °C) was provided to microfluidic inlet 2. Cubosomes (FIG. 13, FIG. 14A, FIG. 14B, FIG. 14C, and FIG. 14D) with resin were observed at a microfluidic outlet.Table 3. Formulations of GDB4 with FOR87 (resin).Attorney Docket No. 218760-0002-W001%w - weight percent based on total weight of the lipid nanoparticle%m - mass percent based on total weight of the lipid nanoparticleTable 4. Physical-chemistry characterization of formulations, including particle size, polydispersity index (PDI), and zeta potential (ZP).

[0129] All formulations were stable, with no significant differences observed across the various tested compositions (FIG. 10). The addition of resin significantly enhanced the bactericidal efficacy of the formulation against of the formulation against Salmonella, while maintaining its antimicrobial activity against S. aureus (FIG. 11). The GDB4-res1 formulation demonstrated superior minimum inhibitory concentration (MIC) results, and effectively inhibited both tested bacterial strains.Example 4 Mechanistic Study of Lipid Interactions Methods

[0130] The initial lipid bilayer structure of each system was generated using the CHARMM- GUI membrane builder. In systems which were to contain GMCY - glycerol monocaprylate (C8Attorney Docket No. 218760-0002-W001MG), GMC - glycerol monocaprate (C10 MG), or GML - glycerol monolaurate (C12MG) a Tcl script was developed to modify the structure of glycerol monostearate to the necessary chain length, which was executed in VMD. Table 1 reports the asymmetric lipid distributions of each system. POPC refers to 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine. GMS refers to glycerol monostearate (C18 MG).Table 1 : Initial lipid species and lipid count present in the upper leaflet (UL) and lower leaflet (LL) in the initial conformation of each system. "_A" represents an asymmetrical number of MGs between the upper and lower leaflet in the starting structure. "_S" represents an equal number of MGs between the upper and lower leaflet in the starting structure.System Name Membrane lipid G Count (UI MG Count (IL)

[0131] All models were solvated by adding 40 A of TIP3P water either side of the membrane, and NaCI at a concentration of 0.15 mol / L. The CHARMM36m all-atom forcefield was used in the simulations. All systems underwent an initial energy minimization for 10 ns using coIvar restraints which restrained the membrane and were reduced in strength as the simulation continued. Following this, the NPT ensemble was used for 90 ns of equilibration and 500 ns of production runs. The production trajectories were executed at physiological conditions with constant temperature (298.15 K) and pressure (1.01325 bar) with a 2 fs timestep; 12A VdW cutoff and PME were employed together.

[0132] All models were simulated for 600 ns each. The last 500 ns of each system was used for analysis, while the first 100 ns was for equilibration and to allow relaxation of the membrane system. This proved to be sufficient for MCMG loaded systems, however the GMS-loaded system saw fluctuation across the whole production run, suggesting this system does not full equilibrate within the time frame achievable using all-atom simulations. This 100 ns period wasAttorney Docket No. 218760-0002-W001 excluded from data analysis presented below. All simulations were performed using NAMD3 CUDA version.Analysis

[0133] Area per lipid (APL), membrane thickness, and tail order parameters were calculated using VMD membrane analysis tool MEM BPLUGIN version 1.1.32. For APL calculation, the atoms selected to represent the lipid species in the system were 02, 01 and 03 for POPO and 03 for each MG. The 2D thickness across the XY plane was calculated every 20 steps of the trajectory using the membrane thickness map. Alignment of phospholipid tails with the membrane normal was calculated as lipid tail order parameters (SOD) for the sn1 chain of POPO. Mass density profiles for each system were calculated using the Density Tool plugin in VMD. and was calculated for the water, ions, POPO and MG for the upper and lower leaflets individually, and the whole membrane (combined POPO and MG) for each system. All data was processed and visualized using Python.Results and Discussion

[0134] To assess the effect of MG chain length, the systems with 100 GMCY (C8 MG), GMC (C10 MG), GML (C12 MG), or GMS (C18 MG) molecules were inserted inside the POPO bilayer asymmetrically into the upper leaflet. These medium chain monoglycerides (MCMGs) were chosen due to their reported bactericidal activity, which has not been reported for the longer chain GMS.

[0135] All MG loaded membrane systems saw an overall decrease in area per lipid, with an increase in APL in the upper leaflet and a decrease in the lower leaflet (Table 2). Within the individual leaflets, there was a correlation between the length of the acyl tail within the MCMGs, with GML producing the largest changes, though overall APL was similar between the three MCMGs. GMS caused the most significant APL reduction of the four MGs, though while this resulted in a 57.3% decreased in APL in the upper leaflet it only resulted in a 1 .7% increase in APL in the lower leaflet, in comparison to a 16.1% increase in the GML system. The significant reduction in APL in the upper leaflet of the 100GMS_A system is likely due to the much larger size of GMS in comparison to the MCMGs, similar in length to the sn1 tail of POPC, taking up more space within the leaflet than the MCMGs.Attorney Docket No. 218760-0002-W001Table 2: Averaged membrane thickness across the whole membrane (WM) and APL values for whole membrane (WM), upper leaflet (UL) and lower leaflet (LL). Brackets show standard deviation. All values given in Angstrom (A).System APL (UL) APL (LL) APL (WM) ThicknessControl 64.1 (1.1) 64-1 (1.1) 64.1 (1.1) 39.3 (0.5)100G CY A 43.4 (0.7) 71.0 (1.0) 53.3 (0.8) 36.9 (0.6)100G C A 42.2 (2.1) 73.2 (8.51 53.7 (0.9) 37.4 (0.6)100G L_A 41.4 (1-1) 744 (3.5) 53-1 (0.9) 38.2 (0.5)100G S A 36.7 (1.5) 65.2 (.2.9) 47.0 (1.2) 43.3 (0.7)

[0136] Each MCMG-loaded membrane displayed a decrease in average bilayer thickness as shown in Table 2. The reduction in bilayer thickness is inversely correlated with the MCMG acyl chain length. GML causes the smallest deviation from the control with a 2.8% decrease in thickness. In contrast, GMS loading experienced a significant 13.2% increase in bilayer thickness. The observed bilayer thinning is also likely to be due to the structure of the MGs. The shorter tail length of MCMGs disrupt the packing of monoglycerides, allowing the tails of POPC to splay out, particularly towards the hydrophobic core of the membrane. As GMS takes up a similar size to the sn1 chain of POPC this is likely to instead fit tighter with the existing packing of POPC, causing tighter packing and therefore forcing greater alignment of POPC with the membrane normal.

[0137] Evidence of this can be found in both calculated density profiles for these systems and the calculated tail order parameters for POPC. For example, it was found that when MCMGs are present in the upper leaflet of the membrane there is a notable thinning of not only the upper leaflet of the membrane, where they are primarily located, but also the lower leaflet, indicating the insertion of MCMGs alters the structure of the entire bilayer. Additionally increased density at the center of the membrane is observed in these systems. Increased density towards the hydrophobic core region of the membrane is associated with changes to the packing of membrane lipids, primarily due to the MCMGs sitting closer to the membrane center. Changes to bilayer density by MCMGs was similar between the three types of molecule, though there was a consistent small converse correlation between tail length and the degree of disruption. Order parameters for these systems also indicate an overall decrease in alignment of POPC with the membrane normal, particularly in the upper leaflet of the membrane. Water density profiles for each system were also asymmetrical unlike the control system, highlighting the permeation enhancing effects of MCMGs.Attorney Docket No. 218760-0002-W001

[0138] In contrast, GMS did not significantly influence the thickness of the lower leaflet of the membrane in comparison to the control but caused a large thickening of the upper leaflet at around 4 A, which aligns with the overall thickness increase for this system. GMS occupies a much larger area of the upper leaflet than the MCMGs, having a near flat distribution from +20 A to +10 A.

[0139] Lipid tail order parameter (SCD) profiles of the various membranes were calculated. Insertion of MGs into the upper leaflet led to notable increase in tail order within the leaflet that was strongly correlated with chain length. Notably, GMS induced significantly greater order than MCMG loading. Larger MGs also had a more substantial impact on the order of carbons towards the tail end of POPC chain Sn1. The effects of GMCY became negligible from C13 onwards while GML and GMS produced an increase in order along the entire acyl chain. The opposite effect was seen in the lower leaflet, with all systems except GMS displaying reduced order compared to the control, again proportional to chain length. Whole membrane SCD profiles indicate that GMS loading causes an overall increase in chain order within the system, while GMCY and GMC cause a decrease in order while GML does not significantly alter order in comparison to the control. The changes to membrane packing correlate with GMS’s classification as a stabiliser, as well as the antimicrobial potential for MCMGs as membrane fluidity is linked to permeability, potentially explaining the cellular component leakage observed in cells treated with MCMGs.

[0140] Plotted thickness maps indicate that the structure of the bilayer also becomes less stable when MGs are present within it. In the control map, there was a 1 .5 A difference between maximum and minimum thickness (39.8 to 38.3), which indicates lower variation in thickness across the membrane than GMCY (37.9 to 34.5), GMC (37.7 to 35), GML (39.1 to 36.8), or GMS (45.4 to 38.3). Between the MG loaded systems, GMS had the largest variation at 7.1 A, while GML had the lowest at 2.3 A. Thickness maps for these systems additionally indicate that the control system has the most uniform distribution of thickness while other systems, particularly GMS (6 B) and GMS (6 E) where regions with significant differences in membrane thickness were observed, explaining the larger variation in thickness in these systems and appear to be a large driving force in changes to average membrane thickness within these systems in comparison to the control. Changes to bilayer thickness can signify the induction of curvature due to phase changes, which significantly alter bilayer fluidity.

[0141] The frequency of lipid translocation events is inversely proportional to the MG chain length as show in Table 3. The 100GMCY_A system experienced significantly more translocation events than other MCMG systems while GMS reported no changes to MG leafletAttorney Docket No. 218760-0002-W001 concentration across the entire trajectory (Table 3). The differences in translocation and expulsion frequency between MGs may explain the differences in the reported antimicrobial efficacies of these MG. GMS may have higher energy barriers required for entry into the membrane making it harder for a potential localized concentration threshold for destabilization to be reached. Conversely, the rapid movement of GMCY may indicate that GMCY molecules are not stable enough in a leaflet to produce persistent disruptive effects over a longer timescale.Table 3: Counts of MG flip-flop and exit events for asymmetric MG loaded systems. UL indicates the final count of monoglyceride in upper leaflet. LL indicates the final count of monoglyceride in lower leaflet. Brackets indicate percentage of total monoglyceride.System Flip-flop Exit MG count (OL) MG count (LL)

[0142] It should be noted that the POPC model bilayer cannot fully capture the full complexity and diversity of bacterial membranes, and thus the inverse correlation between the extent of membrane changes and MG chain length may not fully align with experimental reports on monoglyceride antimicrobial efficacy. Additionally, it is difficult to compare monoglycerides at equal concentration within a model membrane experimentally due to differences in stability and critical micellar concentration required to form micelles between MGs.

[0143] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure.

[0144] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the technology, may be made without departing from the spirit and scope thereof.

[0145] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

[0146] Clause 1. A lipid nanoparticle comprising: an unsaturated monoglyceride; a saturated monoglyceride; and a glycerol behenate.Attorney Docket No. 218760-0002-W001

[0147] Clause 2. The lipid nanoparticle of clause 1 , wherein the unsaturated monoglyceride comprises a C4 - C30 fatty acid.

[0148] Clause 3. The lipid nanoparticle of clause 1 or 2, wherein the unsaturated monoglyceride comprises glycerol monooleate.

[0149] Clause 4. The lipid nanoparticle of any one of clauses 1-3, comprising the unsaturated monoglyceride at about 20 wt% to about 40 wt% by weight of the lipid nanoparticle.

[0150] Clause 5. The lipid nanoparticle of any one of clauses 1-4, wherein the saturated monoglyceride comprises a C4 - C30 fatty acid.

[0151] Clause 6. The lipid nanoparticle of any one of clauses 1-5, wherein the saturated monoglyceride comprises glycerol monolaurate.

[0152] Clause 7. The lipid nanoparticle of any one of clauses 1-6, comprising the saturated monoglyceride at about 50 wt% to about 70 wt% by weight of the lipid nanoparticle.

[0153] Clause 8. The lipid nanoparticle of any one of clauses 1-7, wherein the glycerol behenate is selected from the group consisting of glycerol monobehenate, glycerol dibehenate, and a combination thereof.

[0154] Clause 9. The lipid nanoparticle of any one of clauses 1-8, comprising the glycerol behenate at about 0.1 wt% to about 20 wt% by weight of the lipid nanoparticle.

[0155] Clause 10. A lipid nanoparticle comprising: glycerol monooleate; glycerol monolaurate; and a glycerol behenate selected from the group consisting of glycerol monobehenate, glycerol dibehenate, and a combination thereof.

[0156] Clause 11. The lipid nanoparticle of any one of clauses 1-10, further comprising a tall oil rosin.

[0157] Clause 12. The lipid nanoparticle of clause 11 , wherein the tall oil rosin comprises a monounsaturated fatty acid, a diunsaturated fatty acid, a tri unsaturated fatty acid, and a resin acid.

[0158] Clause 13. The lipid nanoparticle of any one of clauses 1-10, further comprising a resin acid.

[0159] Clause 14. The lipid nanoparticle of clause 12 or 13, wherein the resin acid comprises abietic acid, dehydroabietic acid, pimaric acid, or a combination thereof.

[0160] Clause 15. The lipid nanoparticle of any one of clauses 1-14, having a diameter of about 100 nm to about 400 nm.

[0161] Clause 16. The lipid nanoparticle of any one of clauses 1-15, having a cubosome structure.Attorney Docket No. 218760-0002-W001

[0162] Clause 17. The lipid nanoparticle of any one of clauses 1-16, wherein the lipid nanoparticle does not comprise a drug.

[0163] Clause 18. The lipid nanoparticle of any one of clauses 1-17, further comprising a stabilizer.

[0164] Clause 19. The lipid nanoparticle of clause 18, wherein the stabilizer is a bile salt.

[0165] Clause 20. A pharmaceutical composition comprising: one or more lipid nanoparticles according to any one of clauses 1-19; and a pharmaceutically acceptable excipient.

[0166] Clause 21. A method of making an organic solvent-free lipid nanoparticle, the method comprising: microfluidically mixing a lipid mixture, the lipid mixture comprising a lipid solvent, an unsaturated monoglyceride, a saturated monoglyceride, and a glycerol behenate, with an aqueous mixture, the aqueous mixture comprising an aqueous buffer and a stabilizer, wherein the lipid mixture and the aqueous mixture are both substantially free of an organic solvent.

[0167] Clause 22. The method of clause 21 , wherein the lipid mixture comprises a lipid solvent, glycerol monooleate, glycerol monolaurate, and a glycerol behenate selected from the group consisting of glycerol monobehenate, glycerol dibehenate, and a combination thereof.

[0168] Clause 23. The method of clause 21 or 22, wherein the lipid mixture further comprises a tall oil rosin or a resin acid.

[0169] Clause 24. The method of any one of clauses 21-23, wherein the stabilizer is a bile salt.

[0170] Clause 25. A method of modulating an activity of a microorganism, the method comprising contacting the microorganism with an effective amount of a lipid nanoparticle according to any one of clauses 1-19 or the pharmaceutical composition of claim 20.

[0171] Clause 26. The method of clause 25, wherein modulating the activity of the microorganism comprises preventing, inhibiting, and / or reducing the activity of the microorganism.

[0172] Clause 27. The method of clause 25 or 26, wherein the microorganism is a bacterium.

[0173] Clause 28. The method of clause 27, wherein the bacterium is selected from the group consisting of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter, Escherichia coli. , and a combination thereof.

[0174] Clause 29. The method of any one of clauses 25-28, wherein the lipid nanoparticle is administered orally, intravenously, subcutaneously, transdermally, intramuscularly, transnasally, topically, sebaceously, transfol licularly, or intraperitoneally.Attorney Docket No. 218760-0002-W001

[0175] Clause 30. The method of any one of clauses 25-29, wherein the lipid nanoparticle is not administered ocularly.

[0176] Clause 31. The method of any one of clauses 25-30, wherein the lipid nanoparticle is administered as part of a wound dressing, a topical cream, or a combination thereof.

[0177] Clause 32. The method of any one of clauses 28-31 , wherein the bacterium is in a subject.

[0178] Clause 33. The method of clause 32, wherein the subject is a human or an animal.

[0179] Clause 34. A method of promoting a beneficial health effect in an animal, the method comprising administering to the animal an effective amount of a lipid nanoparticle according to any one of clauses 1-19 or the pharmaceutical composition of claim 20.

[0180] Clause 35. The method of clause 34, wherein the beneficial health effect comprises increased weight gain, decreased colonization of a microorganism, decreased mortality rate, or a combination thereof.

Claims

Attorney Docket No. 218760-0002-W001CLAIMSWhat is claimed is:

1. A lipid nanoparticle comprising: an unsaturated monoglyceride; a saturated monoglyceride; and a glycerol behenate.

2. The lipid nanoparticle of claim 1 , wherein the unsaturated monoglyceride comprises a C4- C3o fatty acid.

3. The lipid nanoparticle of claim 1 , wherein the unsaturated monoglyceride comprises glycerol monooleate.

4. The lipid nanoparticle of claim 1 , comprising the unsaturated monoglyceride at about 20 wt% to about 40 wt% by weight of the lipid nanoparticle.

5. The lipid nanoparticle of claim 1 , wherein the saturated monoglyceride comprises a C4- C30 fatty acid.

6. The lipid nanoparticle of claim 1 , wherein the saturated monoglyceride comprises glycerol monolaurate.

7. The lipid nanoparticle of claim 1 , comprising the saturated monoglyceride at about 50 wt% to about 70 wt% by weight of the lipid nanoparticle.

8. The lipid nanoparticle of claim 1 , wherein the glycerol behenate is selected from the group consisting of glycerol monobehenate, glycerol dibehenate, and a combination thereof.

9. The lipid nanoparticle of claim 1 , comprising the glycerol behenate at about 0.1 wt% to about 20 wt% by weight of the lipid nanoparticle.

10. The lipid nanoparticle of claim 1 , further comprising a tall oil rosin.Attorney Docket No. 218760-0002-W00111 . The lipid nanoparticle of claim 10, wherein the tall oil rosin comprises a monounsaturated fatty acid, a diunsaturated fatty acid, a triunsaturated fatty acid, and a resin acid.

12. The lipid nanoparticle of claim 1 , further comprising a resin acid.

13. The lipid nanoparticle of claim 12, wherein the resin acid comprises abietic acid, dehydroabietic acid, pimaric acid, or a combination thereof.

14. The lipid nanoparticle of claim 1 , having a diameter of about 100 nm to about 500 nm.

15. The lipid nanoparticle of claim 1 , having a cubosome structure.

16. The lipid nanoparticle of claim 1 , wherein the lipid nanoparticle does not comprise a drug.

17. The lipid nanoparticle of claim 1 , further comprising a stabilizer.

18. The lipid nanoparticle of claim 17, wherein the stabilizer is a bile salt.

19. A lipid nanoparticle comprising: glycerol monooleate; glycerol monolaurate; and a glycerol behenate selected from the group consisting of glycerol monobehenate, glycerol dibehenate, and a combination thereof.

20. A pharmaceutical composition comprising: one or more lipid nanoparticles according to claim 1 ; and a pharmaceutically acceptable excipient.Attorney Docket No. 218760-0002-W00121 . A method of making an organic solvent-free lipid nanoparticle, the method comprising: microfluidically mixing a lipid mixture, the lipid mixture comprising a lipid solvent, an unsaturated monoglyceride, a saturated monoglyceride, and a glycerol behenate, with an aqueous mixture, the aqueous mixture comprising an aqueous buffer and a stabilizer, wherein the lipid mixture and the aqueous mixture are both substantially free of an organic solvent.

22. The method of claim 21 , wherein the lipid mixture comprises a lipid solvent, glycerol monooleate, glycerol monolaurate, and a glycerol behenate selected from the group consisting of glycerol monobehenate, glycerol dibehenate, and a combination thereof.

23. The method of claim 21, wherein the lipid mixture further comprises a tall oil rosin or a resin acid.

24. The method of claim 21 , wherein the stabilizer is a bile salt.

25. A method of modulating an activity of a microorganism, the method comprising contacting the microorganism with an effective amount of a lipid nanoparticle according to claim 1.

26. The method of claim 25, wherein modulating the activity of the microorganism comprises preventing, inhibiting, and / or reducing the activity of the microorganism.

27. The method of claim 25, wherein the microorganism is a bacterium.

28. The method of claim 27, wherein the bacterium is selected from the group consisting of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter, Escherichia coli. , and a combination thereof.

29. The method of claim 25, wherein the lipid nanoparticle is administered orally, intravenously, subcutaneously, transdermally, intramuscularly, transnasally, topically, sebaceously, transfol licularly, or intraperitoneally.Attorney Docket No. 218760-0002-W00130. The method of claim 25, wherein the lipid nanoparticle is not administered ocularly.31 . The method of claim 25, wherein the lipid nanoparticle is administered as part of a wound dressing, a topical cream, or a combination thereof.

32. The method of claim 27, wherein the bacterium is in a subject.

33. The method of claim 32, wherein the subject is a human or an animal.

34. A method of promoting a beneficial health effect in an animal, the method comprising administering to the animal an effective amount of a lipid nanoparticle according to claim 1.

35. The method of claim 34, wherein the beneficial health effect comprises increased weight gain, decreased colonization of a microorganism, decreased mortality rate, or a combination thereof.

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