WO2024112807A1 - Nanoparticle dispersions comprising therapeutic agents - Google Patents

Abstract

Disclosed herein are dense nanolipid fluid (DNLF) dispersions comprising desirable characteristics for incorporating bioactive agents such as peptides into lipid phase of the dispersion for biodelivery of the agents for their typical purpose. Continuous methods for preparing the DNLF dispersions are also disclosed herein to include formation of a crude mill base and passing the base through a twin screw extruder. Dispersions disclosed herein can express a particle size of less than 150 nm under stable storage conditions, while forming lamellar structures after exposure to heat and/or evaporation of the aqueous components of the dispersion.

Description

NANOPARTICLE DISPERSIONS COMPRISING THERAPEUTIC AGENTS

RELATED APPLICATIONS

This application is being filed on November 21, 2023, as a PCT International Patent Application and claims the benefit of and priority to U.S. Provisional Application Ser. No. 63/384,585, filed November 21, 2022; US Patent Application 63/384,584, filed November 21, 2022; U.S. Provisional Application Ser. No. 63/486,132, filed February 21, 2023, and U.S. Provisional Application Ser. No. 63/511,992, filed July 5, 2023, each of which are incorporated in their entirety herein by reference.

BACKGROUND OF THE INVENTION

Peptide compounds, water soluble drugs and hydrophilic bioactive compounds can be difficult to administer to living organisms in an effective manner because of poor bioavailability. Poor bioavailability can result from oral administration because the peptide is degraded in the gastrointestinal tract or interference from a variety of conditions such as congenital defects in the digestive or absorptive system, intestinal disease/resection, drug interaction or chronic alcohol use. Peptides and hydrophilic compounds in general are also poorly absorbed through the skin stratum corneum. Poor transdermal bioavailability can result from inability to permeate the skin stratum corneum.

Peptides are an important class of drugs which activate and moderate biological functions including signaling, that is transcription and translation of cellular DNA, as agonists and antagonists for cellular function, hormones, neurotransmitters, growth factors, antibacterial agents, and many others.

Important biologically active peptides include monopeptides comprising a single amino acid residue, oligopeptides comprising from two to about 25 amino acid residues linked in a polypeptide chain, and proteins comprising greater than about 25 amino acid residues. Peptides can comprise only amino acid residues or they can be chemically modified, for example by lipidation including acylation of peptides and proteins with long- chain, saturated lipids. Peptides also are subject to degradation under conditions that are typically employed in synthesis of lipid nanoparticles. For example, insulin shows unfolding of the globular structure and aggregation under Couette flow at relatively low shear rates of 200 sec'¹ [Bekard IB, Dunstan DE. Shear-induced deformation of bovine insulin in Couette flow. J Phys Chem B. 2009 Jun 25;113(25): 8453-7. doi: 10.1021/jp903522e. PMID: 19534559], about three orders of magnitude lower shear stress than encountered in ultra-high pressure homogenization. The shear sensitivity of polypeptides limits the extent to which typical nanoemulsion and solid lipid nanoparticle particle comminution process can be used, limiting lipid nanoparticle synthesis to methods such as the so-called double emulsion technique [Sarmento B, Martins S, Ferreira D, Souto EB. Oral insulin delivery by means of solid lipid nanoparticles. Int J Nanomedicine. 2007;2(4):743-9. PMID: 18203440; PMCID: PMC2676823] which present relatively lower shear processes. Consequently, lipid nanoparticle dispersions that comprise proteins such as insulin generally do not have particle diameters less than 100 nanometers.

This disclosure is directed in part to the preparation of nanoparticle dispersions comprising hydrophilic and amphipathic bioactive agents for oral administration. It is against this background that the current invention is made.

US Patent Application No. 16/748,399, METHOD OF PREPARING NANOPARTICLES BY HOT-MELT EXTRUSION, hereby incorporated by reference herein in its entirety, describes a method of making concentrated lipid nanoparticle dispersions that contain between 25% and 60% of lipid content where "lipid content" means the sum of the concentrations of surfactants, water immiscible oils, hydrophobic drugs, and hydrophobic bioactive agents, where "hydrophobic bioactive ingredient" is defined as a chemical compound or mixture of compounds that have an effect on a living organism, tissue or cell and a logP value (that is, the logarithm of the octanol/water partition coefficient) greater than 1 and where "lipid nanoparticle" means particles with diameter smaller than 150 nm comprising surfactants, water immiscible oils, and one or more hydrophobic bioactive ingredients. Such highly concentrated dispersions of lipid nanoparticles that comprise hydrophobic bioactive agents in water which contain between 25% and 60% lipid content are termed dense nanolipid fluid (DNLF) dispersions. Nanolipid dispersions, that is, dispersions of lipid particles in water with diameters less than 150 nm, that comprise hydrophobic bioactive ingredients are useful to increase the bioavailability of the hydrophobic bioactive ingredients. Nanolipid dispersions that comprise hydrophobic bioactive ingredients can be difficult to prepare, especially those with greater than 25% lipid content, that is, DNLF dispersions. DNLF dispersions offer advantages over nanolipid dispersions with lipid concentrations less than 25%, including capability for administering useful amounts of hydrophobic bioactive agent where administration volumes are low, such as buccal and sublingual administration. DNLF dispersions are also useful to be diluted for administration where the therapeutically effective amount of hydrophobic bioactive agent is low, or the administration volume is high.

DNLF dispersions able to adopt lamellar structures is an important property related to bioavailability. The ability of a DNLF dispersion to adopt a lamellar structure upon heating, evaporation, or contact with other materials can aid in absorption into other lamellar structured materials including skin and cell membranes. Latent lamellar structure, that is, the tendency to form lamellar structures upon physical changes, can be discerned by heating DNLF dispersions and observing for evidence of lamellar structures. Such evidence includes depression of electrical conductivity that appears as a negative peak in a plot of electrical conductivity vs temperature or the appearance of optical birefringence.

In certain aspects, this disclosure is directed to the formulation of stable nanoparticle dispersions comprising bioactive compounds that have conventionally poor oral bioavailability. This disclosure is also directed to methods of treatment that comprise oral administration of nanoparticles comprising drugs such as peptides that are conventionally prone to degradation within the gastrointestinal tract. In certain aspects, DNLF dispersions having improved characteristics, while incorporating peptide compounds of interest are contemplated herein.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a plot of conductivity vs temperature for a mill base composition comprising caffeine compared to a standard (Example 10) Figure 2 is a plot of dasatinib plasma concentration vs. time after administration for Examples 16-19.

Figure 3 is a plot comparing the dasatinib plasma concentration vs time after administration for Examples 17 and 20.

Figure 4 is a plot comparing the dasatinib plasma concentration vs time after administration for Examples 17 and 21.

Figure 5 is a plot of the particle size distribution of a DNLF dispersion before and after lipolysis (Example 22).

Figure 6 is a plot of the particle size of the DNLF of Example 22 before and after lipolysis, expressed as the volume percent of particles having less than a certain diameter.

Figure 7 is a plot showing results of a pH stat titration of Example 23.

Figure 8 is a plot of the particle size of a DNLF before and after lipolysis (Example 23).

Figure 9 is a plot of the particle size of the DNLF of Example 23 before and after lipolysis, expressed as the volume percent of particles having less than a certain diameter.

Figure 10 is a plot comparing the pH stat titration of a DNLF without orlistat to a DNLF with orlistat (Example 28).

Figure 11 is a showing results of a pH stat titration of Example 30.

SUMMARY OF THE INVENTION

The present invention generally provides nanolipid dispersions comprising peptide and hydrophilic bioactive compounds useful for administration to a mammal by oral, sublingual, buccal, cutaneous, subcutaneous and intravascular administration routes. In certain aspects, the nanolipid dispersions can comprise water, a water immiscible oil, a surfactant, and a bioactive agent selected from a peptide (e.g., an amino acid, an oligopeptide, a polypeptide, a protein) a hydrophilic bioactive agent, a hydrophobic bioactive agent, small molecules, or a mixture of bioactive agents.

Nanoparticle dispersions for delivery of a peptide bioactive agent to a mammal are contemplated herein. In certain aspects, nanoparticle dispersions can comprise from 0.01 wt. % to 8.5 wt. % of one or more bioactive agents (e.g., a hydrophilic small molecule, a hydrophilic or amphipathic peptide), from 1.6 wt.% to 11.9 wt.% of one or more high hydrophile-lipophile-balance (HLB) surfactants, from 2.6 wt.% to 12.0 wt.% of one or more low hydrophile-lipophile-balance (HLB) surfactants, from 13.9 wt.% to 41.8 wt.% of one or more water immiscible oils, and from 39.1 wt.% to 58.1 wt.% water. In other aspects, the high hydrophile-lipophile-balance (HLB) surfactant can comprise an ester type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactant, and the low hydrophile-lipophile-balance (HLB) surfactants comprises a phospholipid low hydrophile- lipophile-balance (HLB) surfactant. In other aspects, the high hydrophile-lipophile-balance (HLB) surfactant can comprise an ether type polyethoxylated high hydrophile-lipophile- balance (HLB) surfactant.

In still further aspects, dispersions can comprise an ether type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactants and an ester type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactant. In such aspects, a weight ratio of the ether type polyethoxylated high hydrophile-lipophile-balance HLB surfactant to the ester type polyethoxylated high hydrophile-lipophile-balance HLB surfactant is greater than 1 : 1. Alternatively, dispersions comprising an ether type polyethoxylated high hydrophile- lipophile-balance (HLB) surfactant can further comprise a non-polyethoxylated high hydrophile-lipophile-balance (HLB) surfactants (e.g., sodium laurel sulfate).

In certain aspects, the bioactive agent can be hydrophilic or amphipathic (e g., the bioactive agent is not hydrophobic, and/or the bioactive agent has a logP less than 1). The bioactive agent may be a peptide (e.g., a peptide having a molar mass in a range from 1 kDa to 1,000 kDa), or alternatively, a small molecule. In certain aspects, the bioactive agent can be insulin.

In other aspects, the lipophilic phase (e.g., surfactants, water immiscible oils, hydrophobic therapeutic agents, etc.) of the nanoparticle dispersion is lipolysis-resistant (e.g., less than 25% of ester bonds hydrolyze within one hour in the presence of 500 units/mL of porcine pancreatic lipase; alternatively, indigestible). In certain aspects, the nanoparticle dispersion can comprise a lipase inhibitor (e.g., orlistat). In other aspects, the high HLB surfactant is inert to lipolysis (e.g., indigestible). In still further aspects, the nanoparticles within the nanoparticle dispersion can have a net positive charge. Encapsulated nanoparticle dispersions are also contemplated herein. In certain aspects, the capsule comprises an encapsulating polymer surrounding the nanoparticle dispersion. In other aspects, the encapsulating polymer comprises a carboxylic acid, and the pH of the nanoparticle dispersion is less than (e.g., at least one pH unit less than) a pKa of the encapsulating polymer carboxylic acid group.

Methods for treating a disorder comprising orally administering to a patient in need thereof any of the nanoparticle dispersions or capsules disclosed herein are also contemplated.

DEFINITIONS

As used herein, the term "lipid" refers to fats and fat-derived materials which are insoluble in water except as micellar solutions or dispersions, but which are soluble in organic solvents including surfactants, water immiscible oils, and hydrophobic bioactive ingredients.

As used herein, the term "dense nanolipid fluid (DNLF) dispersion" refers to a dispersion of lipid nanoparticles in water with between 25% and 60% of lipid content where lipophilic content means the sum of the concentrations of surfactants, water immiscible oils, hydrophobic drugs, and hydrophobic therapeutic agents.

As used herein, the term "nanolipid dispersion" refers to a dispersion of lipid particles in water with a volume average particle size less than 150 nm.

As used herein, the term "enteral administration" refers to food or drug administration via the human gastrointestinal tract.

As used herein, the term "therapeutic agent" refers to a chemical compound, complex or composition that exhibits a desirable effect in the biological context, i.e., when administered to a subject.

As used herein, the term "drug" refers to a chemical compound that is regulated as a drug by the US Food and Drug Administration.

As used herein, the term "oral administration" refers to the process by which drugs are delivered by mouth to the alimentary track. As used herein, the term "buccal administration" refers to the process by which therapeutic agents are held or applied in the buccal area and diffuse through the oral mucosa directly into the bloodstream.

As used herein, the term "sublingual administration" refers to the process by which therapeutic agents are held or applied to the area under the tongue and diffuse through the oral mucosa directly into the bloodstream.

As used herein, the term "cutaneous administration" refers to the process by which therapeutic agents are applied to the skin.

As used herein, the term "HLB" refers to Hydrophile-Lipophile Balance, which is an empirical expression for the relationship of the hydrophilic ("water-loving") and hydrophobic ("water-hating") groups of a surfactant.

As used herein, the phrase "low hydrophile-lipophile-balance (HLB) surfactant" refers to a surfactant with an hydrophile-lipophile-balance (HLB) value of less than about 10.

As used herein, the phrase "high hydrophile-lipophile balance (HLB) surfactant" refers to a surfactant with an hydrophile-lipophile-balance (HLB) value of equal to or greater than about 14.

As used herein, the phrase "anionic high hydrophile-lipophile-balance (HLB) surfactant" refers to a surfactant with a hydrophile-lipophile-balance (HLB) value of equal to or greater than about 14 that dissociates in water to give an anion comprising a hydrophobic group covalently bonded to an anionic group such as a sulfate or carboxylate group plus a cation selected from the group of hydrogen ion and alkali metal ions.

As used herein, "cryoprotectant" refers to compounds that are used to retard ice formation upon cooling compositions that contain water.

As used herein, "preservative" refers to a substance or a chemical that is added to products such as food products, beverages, and drugs to prevent decomposition by microbial growth or by undesirable chemical changes.

As used herein, the term "essential oil" refers to a volatile oil derived from the leaves, stem, flower or twigs of plants or synthetically made compounds that have the same chemical attributes. The essential oil usually carries the odor or flavor of the plant. As use herein, the term "immiscible" refers to liquids that will not mix or remain mixed with each other, although at certain conditions, for example, high temperatures, they might mix, but any such mixture will typically be thermodynamically unstable and will typically separate into distinct phases at lower temperatures.

As used herein, the term "water immiscible oil" refers to a compound or mixture of compounds that is not miscible with or soluble in water and is not dispersible in water in the absence of a surfactant, said compound comprising a hydrophobic group such as a water insoluble hydrocarbon chain and not comprising a hydrophilic group such as an ionized group, hydroxyl, amine, carboxylic acid, poly(ethylene oxide).

As used herein, "latent lamellar structure" refers to lamellar structure that is not observable in a dispersion including surfactants, oil and water but becomes observable when the dispersion undergoes heating or evaporation.

As used herein, the phrase "bioactive ingredient" or "bioactive agent" refers to a chemical compound or mixture of compounds that have an effect on a living organism, tissue or cell (e.g., a skin care agent such as hyaluronic acid and derivatives thereof).

As used herein, the phrase "lipid nanoparticle" refers to a particle with a diameter smaller than 150 nm that comprises surfactants, water immiscible oils, and one or more hydrophobic bioactive ingredients.

As used herein, the phrase "vesicular nanoparticle" refers to a nanoparticle with a vesicular structure, that is, comprising a lipid bilayer and enclosing an aqueous core.

As used herein, the phrase "edible" when referring to nanoparticle formulations describes a formulation that is suitable and designed for oral administration. In certain aspects therefore, edible nanoparticle dispersions can comprise, or consist of, ingredients approved for oral consumption by the FDA. However, it will be understood that edible formulations as referred to herein may also comprise additional ingredients suitable for oral ingestion which have not been approved by the FDA for oral consumption, with a vesicular structure, that is, comprising a lipid bilayer and enclosing an aqueous core.

As used herein, the phrases “digestible” and “indigestible” when referring to a nanoparticle dispersion describes a dispersion that maintains its essential structural characteristics under lipolytic conditions. Digestible dispersions will refer to a nanoparticle dispersion comprising a lipid phase with ester-based surfactants. Indigestible dispersions will refer to a nanoparticle dispersion comprising a lipid phase lacking surfactants subject to lipolysis (e.g., ether-based surfactants).

As used herein, agents may be characterized as hydrophilic or hydrophobic, based on the agent having a logP less than 1 , or greater than or equal to 1 , respectively. Separately, agents may be characterized as being amphipathic, i.e., containing both hydrophilic and hydrophobic portions, irrespective of their overall logP value. As an example, peptides generally may be considered to be amphipathic where both hydrophilic and hydrophobic amino acids are included within the amino acid sequence of the peptide. Peptides also can be considered amphipathic when conjugated with hydrophobic groups through covalent bonds. For example, palmitoyl tripeptide 1 (PubChem CID 156595485) is the condensation product of the amine group of the glycine of glycine-histidine-lysine tripeptide with hydrophobic palmitic acid, and semaglutide (PubChem CID 56843331) is the condensation product of a 31-amino acid residue peptide with hydrophobic stearic diacid. Each may be characterized as amphipathic due to the presence of hydrophobic and hydrophilic portions within the compound. Even solitary amino acids comprising a hydrophobic side chain (e.g., phenylalanine, leucine, isoleucine, tyrosine, tryptophan, valine, methionine, proline) may be considered amphipathic, due to the hydrophilic carboxy 1/amino moieties also bound to the alpha carbon.

The logP value for insulin (PubChem Compound Identifier (CID) number 118984375) is reported as -13.1, indicating that insulin is highly hydrophilic. Accordingly, insulin is soluble in water at low pH (e.g., 0.1 N HC1). However, peptides also are amphoteric, and so able to act as an acid or a base depending on conditions such as pH. Insulin, for example, has an isoelectric point of pH 5.4. As the pH of dissolved insulin nears the isoelectric point (beginning around pH 4.3) insulin becomes insoluble and therefore behaves hydrophobically. As pH increases to physiological pH 7.4 insulin again becomes water soluble behaves as hydrophilic.

Despite this behavior, in the context of this disclosure it will be understood that insulin is characterized as hydrophilic according to its logP being less than 1 and characterized as hydrophobic or amphipathic according to the nature of its chemical structure. Notably, the hydrophilicity (logP) of a peptide can depend upon the density of ionized groups, the peptide chain length, and the peptide chain conformation. Peptides can also form a hydrophobic ion pair (HIP). For example, if anionic sodium lauryl sulfate is added to a homogeneous, clear solution of cationic, hydrophilic insulin in 0.1 N HC1, a hydrophobic precipitate forms.

Unless otherwise noted, all percentage-based amounts are weight percentages based on the weight percent of the relevant component to the total weight of the relevant composition.

DETAILED DESCRIPTION OF THE INVENTION

Dense nanolipid dispersions comprising various bioactive compounds including hydrophilic small molecules and amphipathic compounds (e.g., peptides, modified amino acids, palmitoyl tripeptide- 1, and the like), and combinations thereof, are contemplated and described herein. Processes for preparing the dispersions and methods of administering nanoparticle dispersions for the treatment of disease are also disclosed herein. In certain aspects, the nanolipid dispersions can be lipolysis resistant, e.g., by addition of a lipolysis inhibitor, zwitterionic formulation, persistent particle size.

In certain aspects, it is noted that the nanolipid dispersion itself or any components thereof (e.g., the high hydrophile-lipophile balance (HLB) surfactant, the low hydrophile- lipophile-balance (HLB) surfactant, the water immiscible oils, or combinations thereof present in amounts greater than 0.1 % can be listed in the US Food and Drug Administration Code of Federal Regulations Title 21 Part 172 Food additives permitted for direct addition to food for human consumption.

Also disclosed herein are encapsulated nanolipid dispersions. Methods for treating disease using nanolipid dispersions disclosed herein are also contemplated.

NANOLIPID DISPERSIONS

Nanolipid dispersions (e.g., dense nanolipid fluid (DNLF) dispersions) are disclosed herein that may be utilized to incorporate any number of bioactive and non-bioactive substances of interest without disruption of the nature of the dispersion. In certain aspects, dispersions disclosed herein can incorporate oils, water, and surfactants in various amounts as described herein. In certain aspects, dispersions disclosed herein may allow incorporation of a greater amount of any bioactive and non-bioactive substances or combinations thereof, relative to for instance, otherwise identical dispersions wherein the lipid nanoparticles present in the dispersion comprising vesicular lipid nanoparticles (or otherwise identical nanoparticle dispersions wherein the lipid nanoparticle content is greater than 10%, greater than 25%, greater than 50%, or greater than 80% vesicular nanoparticles).

In certain embodiments, the DNLF dispersions can improve the stability and biodelivery of bioactive agents for the customary, typical, and accepted clinical purposes, and with improved efficacy relative to administration forms that suffer from relatively reduced bioavailability. For instance, DNLF dispersions disclosed herein can offer advantages over nanolipid dispersions having lipid concentrations less than 25%, including the capability for administering useful amounts of bioactive agent where administration volumes are low, such as buccal and sublingual administration. DNLF dispersions also may be useful as diluted compositions for administration where the therapeutically effective amount of bioactive agent is low, or the administration volume is high.

Generally, nanolipid dispersions (including DNLF dispersions) disclosed herein can comprise water, a water immiscible oil, a surfactant, and optionally a bioactive agent such as a peptide, biological molecule, or small molecule. In certain aspects, nanoparticle dispersions can further comprise a structure promoting additive that may impart necessary stability in formation of mesophases between the crude mixture of components and formation of the stable nanoparticle dispersions, e.g., during mechanical shearing and kneading conducted within a twin screw extruder. In certain aspects, dispersions contemplated herein can further comprise additives useful for preservation and stability of the dispersions, and other common uses.

Surprisingly, bioactive agents that are not hydrophobic (i.e., either hydrophilic or amphiphilic) were able to be incorporated within the lipid phase of nanoparticle dispersions. For instance, peptides may be hydrophilic, and/or have a net positive charge at physiological pH, the peptide can be paired with an anionic hydrophobic species (e.g., laurel sulfate) such that the peptide ion pair becomes hydrophobic and associated within the lipid phase of the dispersion during preparation. In certain aspects, peptides as incorporated were recovered from the dispersions largely in their natural state, indicating that their incorporation into dispersions does not induce damage to the secondary, tertiary, or quaternary structures of peptides due to unfolding. This is particularly surprising in the case of peptides that comprise external hydrophilic groups for solvation in aqueous media and shift hydrophobic groups to interior portions of the peptide when solvated. Thus, it is unexpected that even hydrophilic and amphipathic peptides may be alternately incorporated and dissociated within the lipid phase, without affecting their overall structures. Incorporation of the peptide to the lipid phase of the dispersion provides a protective effect to the peptide, as the peptides become less available to interact with hydrophilic enzymes that may cleave peptide bonds. Incorporation of the hydrophilic and amphiphilic peptides as disclosed herein also can lead to the unexpected benefit of assisting such compounds in traversing biological membranes as part of the lipid phase dispersion structures, which may allow administration of much larger peptide compounds through skin and other membranes beyond previously accepted limits.

The molecular weight of bioactive agents suitable for the nanoparticle dispersions disclosed herein generally is not limited. For instance, peptides suitable for incorporation into the nanoparticle dispersions described herein generally can be any size, for instance, ranging from monopeptides (amino acids), oligopeptides (generally ranging from 2 to 25 amino acids) including dipeptides, tripeptides, tetrapeptides, and the like, to polypeptides (generally comprising more than about 50 amino acids), to small proteins containing roughly 50 to 500 amino acids, and large proteins containing more than 500 amino acids. Peptide sequences suitable for incorporation into dispersions disclosed herein also can be characterized by their molecular weight, and in certain aspects can be in range from 1 kDa to 2,000 kDa, from 3 kDa to 1,000 kDa, from 5 kDa to 800 kDa, from 10 kDa to 500 kDa, from 25 kDa to 100 kDa. Likewise, the size of small molecules suitable for the dispersions is limited only by practical limits and definition of the class of compounds. Thus, nanoparticle dispersions herein can comprise bioactive agents that are small molecules having a molecular weight less than about 300 g/mol, less than about 500 g/mol, less than about 1000 g/mol, or less than about 1,500 g/mol; alternatively, in a range from about 100 g/mol to about 1,000 g/mol, or from about 250 g/mol to about 750 g/mol.

Bioactive agents suitable for incorporation into the nanoparticle dispersions described herein also can comprise small molecules. In certain aspects, the bioactive agent can be selected from spermadine, caffeine, acetominophen, niacinamide, or combinations thereof. Small molecules for therapies as can be found on the relevant FDA approvals are also contemplated herein.

Peptides and other bioactive agents suitable for incorporation into dispersions disclosed herein can have any number of physiological purposes or classifications. In certain aspects, the bioactive agent can be a peptide selected from a signaling peptide, an antibody, an AA peptide, a hormone, or combinations or derivatives thereof. In certain aspects, peptides suitable for dispersions disclosed herein can comprise lipid derivations, such as may form a lipidized peptide. For instance, in certain aspects dispersions can comprise insulin as the bioactive agent, in any amount or range of amounts as contemplated for the bioactive agent herein. Insulin is a human hormone composed of 51 amino acids, and approximately 5.8 kDa. In other aspects, dispersions can comprise tyrosine kinase inhibitors (e.g., nilotinib, dasatinib), or a skin care agent (e.g., hyaluronic acid).

Contemplated peptides suitable for incorporation into dispersions as described herein can include Abaloparatide, Abarelix, aclerastide, Afamelanotide, Albiglutide, albusomatropin, anamorelin, Angiopeptin, Angiotensin, avexitide, Aviptadil, Bacitracin, Bleomycin, bovine serum albumin, Bremelanotide, Carbetocin, Carfilzomib, Colistin, corticorelin, cyclosporin A, Dalbavancin, Degarelix, Dulaglutide, Enfuvirtide, Etelcalcetide, Exenatide, Goserelin, Icatibant, Lanreotide, lenomorelin, leptin, Linaclotide, Liraglutide, Lixisenatide, Lucinactant, Macimorelin, Mifamurtide, Nesiritide, Octreotide, Oritavancin, Pasireotide, Peginesatide, Plecanatide, Polymyxin B, Pramlintide, Sandostatin, selepressin, Semaglutide, serum albumin, Setmelanotide, somapacitan, somatostatin, Somatuline, Taltirelin, taspoglutide, Teduglutide, Teriparatide, Tesamorelin, thymalfasin, thymosine beta-4, tirzepatide, ularitide, vapreotide, Ziconotide, and combinations thereof.

Peptide-based cosmeceuticals also can be incorporated into the dispersions disclosed herein, and in certain aspects can comprise Acetyl Dipeptide-3 Aminohexanoate, Acetyl Dipeptide- 1 Cetyl Ester, Acetyl Hexapeptide- 1, Acetyl Hexapeptide-3, Acetyl Hexapeptide- 30, Acetyl Hexapeptide-37, Acetyl Hexapeptide-38, Acetyl Hexapeptide-39, Acetyl Hexapeptide-49, Acetyl Hexapeptide-51 Amide, Acetyl Hexapeptide-8, Acetyl Octapeptide-3, Acetyl Tetrapeptide- 11, Acetyl Tetrapeptide-15, Acetyl Tetrapeptide-2, Acetyl Tetrapeptide-22, Acetyl Tetrapeptide-3, Acetyl Tetrapeptide-40, Acetyl Tetrapeptide-5, Acetyl Tetrapeptide-9, Acetyl-Hexapeptide-3, Beta-Glucan, Biotinoyl Tripeptide-1, Caprooyl Tetrapeptide-3, Collagen, Copper Peptide (GHK-Cu), Copper Tripeptide-34, Decapeptide-10, Decapeptide- 12, Decapeptide- 18, Decapeptide-4, Diaminopropionoyl Tripeptide-33, Dipeptide-2, elastin, glutathione, Heptapeptide-6, Heptapeptide-7, Hexapeptide- 10, Hexapeptide- 11, Hexapeptide- 12, Hexapeptide-2, Hexapeptide-3, Hexapeptide-42, Hexapeptide-9, Keratin, Manganese Tripeptide-1, Myristoyl Hexapeptide- 16, Myristoyl Hexapeptide-23, Myristoyl Hexapeptide-4, Myristoyl Pentapeptide- 17, Myristoyl Pentapeptide- 17, Myristoyl Pentapeptide-8, Myristoyl Pentapeptide-8, Myristoyl Tetrapeptide- 12, Myristoyl-Octapeptide-2, Nonapeptide- 1, Octapeptide-2, Oligopeptide- 10, Oligopeptide-20, Oligopeptide-24, Oligopeptide-34, Oligopeptide-51, Oligopeptide-54, Oligopeptide-68, Palmitoyl Dipeptide-5, Palmitoyl Dipeptide-6, Palmitoyl Glutathione, Palmitoyl Hexapeptide, Palmitoyl Hexapeptide- 12, Palmitoyl Pentapeptide-3, Palmitoyl Pentapetide-4, Palmitoyl Tetrapeptide-7, Palmitoyl Tripeptide, Palmitoyl Tripeptide-1, Palmitoyl Tripeptide-3, Palmitoyl Tripeptide-36, Palmitoyl Tripeptide-38, Palmitoyl Tripeptide-5, Palmitoyl Tripeptide-8, Pentapeptide- 18, Pentapeptide- 18, Pentapeptide-25, Pentapeptide-3, Tetrapeptide-21, Tetrapeptide-26, Tetrapeptide-30, Trifluoroacetyl Tripeptide-2, Tripeptide-1, Tripeptide-10 Citrulline, Tripeptide-29, Tripeptide-32, Tripeptide-9 Critulline or combinations thereof.

Suitable bioactive agents also include compounds wherein the pH of the nanodispersion is less than that of an isoelectric point of the bioactive agent. As noted above, insulin has an isoelectric point of at pH 5.4. In aqueous mixtures having a pH similar to the isoelectric point of insulin, beginning around pH 4.3, insulin becomes insoluble and therefore behaves hydrophobically. As the pH drops, insulin again adopts a hydrophilic behavior. Surprisingly, in aspects disclosed herein, it is found that the bioactive agent may be incorporated into the lipid portion of the nanoparticle dispersions, even where the pH of the nanoparticle dispersion is less than the isoelectric point of the bioactive agent. In certain aspects, the nanoparticle dispersion can have a pH less than (0.1 less than, 0.5 less than, 1 less than 1.5 less than, 2.5 less than, 3 less than; alternatively in a range from about 0.5 less than to about 5 less than, from about 1 less than to about 4 less than, from about 2 to about 3 less than) the isoelectric point of the bioactive agent.

In certain aspects, DNLF dispersions comprise lipid nanoparticles that are zwitterionic, lipid nanoparticles have separate positively and negatively charged ions.

Having separated positively and negatively charged ions may, but not necessarily, make lipid nanoparticles and macromolecules such as peptides and proteins hydrophilic. Because association of positively and negatively charged ions may occur in such a way that ion pairs are buried within hydrophobic groups, the presence of charged ions does not always result in the particle or peptide acquiring a hydrophilic, water soluble or water dispersible character. For example, in a hydrophobic ion pair formed from positively charged ions in a peptide associating with negatively charged ions in an anionic surfactant, charged ions are situated so that the complex is hydrophobic. For peptides near their isoelectric point charged ions may also be situated so as to not confer hydrophilicity. Accordingly, peptides near their isoelectric point may be considered to be hydrophobic. In the case that lipid nanoparticle surfaces are not sufficiently hydrophilic for stable dispersibility by virtue of having charged ions, hydrophilicity and dispersion stability can be provided by polyethoxylated chain hydrophilic groups. In the case that peptides are not sufficiently hydrophilic to be soluble, whether as a hydrophobic ion pair or separately, they may be dispersible by encapsulation in a lipid nanoparticle.

In certain aspects, the bioactive agent can be a biological molecule consisting of or comprising a nucleotide, including but not limited to compounds comprising naturally occurring nucleotides (adenosine, guanine, tyrosine, cytosine) and their common synthetic derivatives. Bioactive agents as disclosed herein can include nucleic acid sequences such as DNA and RNA, again, both naturally occurring, synthetic, and fragments thereof.

In certain aspects, nanoparticle dispersions can comprise any suitable amount of water that results in a stable nanoparticle dispersion with appropriate characteristics (e.g., a particle size less than 150 nm, non-vesicular and latent lamellar structure, adequate incorporation of bioactive agents and additives, acceptable ratio of water immiscible oils to bioactive agents, etc.). Without being bound by theory, it is believed that an amount of water suitable for the dispersion may be minimized in certain aspects to provide a dispersion having an excess or large amount of lipid phase, to increase the capacity for the bioactive agents and additives within the dispersions. In certain aspects, nanoparticle dispersions can comprise an amount of water in a range from 10 wt. % to 90 wt. %, from 20 wt. % to 80 wt. %, from 25 wt. % to 75 wt. %, from 35 wt. % to 65 wt. %, from 40 wt. % to 60 wt. %, from 40 wt. % to 55 wt. %, from 45 wt. % to 60 wt. %, or from 50 wt. % to 60 wt. %. In other aspects, nanoparticle dispersions can comprise an amount of water less than 75 wt. %, less than 65 wt. %, less than 55 wt. %, less than 50 wt. %, less than 45 wt. %, less than 40 wt. %, or less than 35 wt. %. Certain aspects can comprise an amount of water in a range from 39.1 to 58.1 wt. %.

Dispersions disclosed herein can further contain water immiscible oils. Without being bound by theory, water immiscible oils may be provided for the purpose of carrying bioactive ingredients within the dispersion and across biological membranes as may be necessary to achieve efficient incorporation of bioactive agents within the nanoparticle dispersion and delivery of the bioactive agents to or within a biological entity for appropriate treatment, respectively. Thus, it may be advantageous to increase the relative amount of water immiscible oil and surfactants in dispersions to maximize the capacity for bioactive agents as discussed above. It may also be advantageous to maximize the amount of lipid phase in dispersions to maximize protection of peptides that are degraded in aqueous settings, as for example, proteolytic enzymatic degradation of insulin in the gastrointestinal tract. In certain aspects, dispersions can comprise an amount of water immiscible oil in a range from 5 wt. % to 65 wt. %, from 10 wt. % to 45 wt. %, from 15 wt. % to 40 wt. %, or from 20 wt. % to 35 wt. %. In certain aspects, the amount of water immiscible oil can be greater than 10 wt. %, greater than 20 wt. %, greater than 30 wt. %, greater than 35 wt. %, greater than 40 wt. %, greater than 45 wt. %, greater than 50 wt. %, or greater than 55 wt. %.

Generally, water immiscible oils are not limited to any particular chemical structure or combination and can be oil or combination of oils that comprises a hydrophobic group such as a water insoluble hydrocarbon. Generally, water immiscible oils will not comprise hydrophilic groups such as an ionized group, a hydroxyl, an amine, a carboxylic acid, or a poly(ethylene oxide), however, exceptions may be made (e.g., in the instance of 2-butyl-l- octanol) based on the overall character of the oil (e.g., the water immiscible oil showing the characteristic of being non-amphipathic in a test).

In certain aspects, the water immiscible oil can comprise cocoyl caprate/caprylate, alkyl alcohols such as Isofol 12 (2-butyl-l -octanol), benzyl alcohol, diisopropyl adipate, capric/caprylic triglyceride oil, isopropyl myristate, limonene, medium chain triglyceride oil, mineral oil, omega 3 fatty acid, oleyl alcohol, isohexadecane, isododecane, a C13 to C15 alkane, or combinations thereof. In other aspects, suitable water immiscible oils useful in the practice of the invention disclosed herein can include medium chain triglyceride oil, coconut oil, isopropyl palmitate, isopropyl myristate, methyl decanoate, ethyl myristate, ethyl oleate, mineral oil, orange essential oil, cyclopentasiloxane, poly(dimethyl siloxane), hexadecane, propylene glycol dicaprylate, isododecane, isoeicosane, isohexadecane, soy biodiesel jojoba oil, cocoyl caprylocaprate, C10 to C13 alkanes, squalane, sunflower seed oil, diacetylated monoglycerides, clove essential oil, and limonene.

Those of skill in the art will understand that water and water immiscible oils as described above alone cannot combine form nanoparticle dispersions as disclosed herein, and readily separate into distinct phases. Surfactants are therefore required to stabilize a dispersion comprising the water and water immiscible oils as a dense nanolipid fluid dispersion and prevent phase separation of the water and water immiscible oil(s). Surfactants as disclosed herein may be characterized by their structure and also their general lipophilicity.

In certain aspects, dispersions disclosed herein can comprise high hydrophile- lipophile-balance (HLB) surfactant, a low hydrophile-lipophile-balance (HLB) surfactant, or both. Generally, the high hydrophile-lipophile-balance (HLB) surfactant can comprise a polyethoxylated high hydrophile-lipophile-balance (HLB) surfactant, an ether type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactant, an ester type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactant, or a nonpolyethoxylated high hydrophile-lipophile-balance (HLB) surfactant. Suitable polyethoxylated high hydrophile-lipophile-balance (HLB) surfactants may include, for example, one or more ether type polyethoxylated high hydrophile-lipophile- balance (HLB) surfactants each independently having a hydrophile-lipophile-balance (HLB) value of equal to or greater than about 14; one or more ester type poly ethoxylated high hydrophile lipophile-balance (HLB) surfactants each independently having a hydrophile- lipophile-balance (HLB) value of equal to or greater than about 14; one or more nonpolyethoxylated high hydrophile-lipophile-balance (HLB) surfactants each independently having a hydrophile lipophile-balance (HLB) value of equal to or greater than about 14; one or more low hydrophile lipophile-balance (HLB) surfactants each independently having a hydrophile-lipophile-balance (HLB) value less than about 10; one or more phospholipid low hydrophile-lipophile-balance (HLB) surfactants each independently having a hydrophile- lipophile-balance (HLB) value less than about 10; and combinations thereof.

Suitable one or more ether type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactants each independently having a hydrophile-lipophile-balance (HLB) value of equal to or greater than about 14 may include, for example, surfactants derived from addition of about 20 to about 100 moles of ethylene oxide to fatty alcohols such as lauryl alcohol, cetyl alcohol, oleyl alcohol, stearyl alcohol, and isotridecyl alcohol which may be referred to as alcohol ethoxylates, polyoxyethylene alkyl ethers, and polyoxyethylated fatty alcohols including laureth-23, ceteth-20, ceteareth-20, ceteareth-25, ceteareth-30, oleth-20, steareth-20, steareth-40, and steareth-100 which are available, for example, as commercial products including Brij® L23, Brij® CS20, Brij ^II! 020, Brij® S20 and Brij® S100. Particularly preferred ether type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactants include, for example; laureth-23, ceteareth-20, ceteareth-30 and steareth-40. Preferably, the one or more ether type polyethoxylated high hydrophile- lipophile balance (HLB) surfactants are selected from the group consisting of laureth-23, laureth-30, steareth-100, steareth-20, steareth-40, ceteareth-20, and ceteareth-30.

Suitable one or more ester type poly ethoxylated high hydrophile-lipophile-balance (HLB) surfactants each independently having a hydrophile-lipophile-balance (HLB) value of equal to or greater than about 14 may include, for example, (1) Ester type poly ethoxylated high hydrophile-lipophile-balance (HLB) surfactants derived from the addition of 20 to 100 moles of ethylene oxide to saturated or unsaturated fatty acids which may be referred to as polyethylene glycol carboxylates, poly(oxyethylene) carboxylates and polyethylene oxide) carboxylate esters including poly(ethylene oxide) laurate esters, poly(ethylene oxide) oleate esters, and poly(ethylene oxide) stearate esters, such as PEG-20 laurate, PEG-20 oleate; PEG-20 stearate, PEG-32 stearate, PEG-40 stearate, and PEG- 100 stearate. Ester type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactants are available, for example, as commercial products including Myij® S40 and Myij® SI 00; (2) Ester type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactants derived from the addition of 20 to 100 moles of ethylene oxide to fatty acid sorbitan esters which may be referred to as polyoxyethylene sorbitan carboxylates, such as polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80 which are available, for example, as commercial products including Tween® 20, Tween® 40, Tween® 60, and Tween® 80; (3) Ester type polyethoxylated high hydrophile lipophile-balance (HLB) surfactants derived from the addition of 20 to 100 moles of ethylene oxide to fatty acid glycerin esters which may be referred to as polyethylene glycol glyceryl carboxylates such as PEG-30 glyceryl cocoate, poly(oxyethylene) glyceryl monolaurate and poly(oxyethylene) glyceryl monostearate, which are available, for example, as commercial products including Jeechem GL-30 and Jeechem GC-30; and (4) Ester type poly ethoxylated high hydrophile-lipophile-balance (HLB) surfactants derived from the addition of 20 to 100 moles of ethylene oxide to castor oil or hydrogenated castor oil such as PEG-25 castor oil ethoxylate, PEG-40 castor oil ethoxylate, PEG-60 castor oil ethoxylate, hydrogenated PEG-25 castor oil ethoxylate, hydrogenated PEG-40 castor oil ethoxylate, and hydrogenated PEG-60 castor oil ethoxylate, which are available, for example, as commercial products including Kolliphor® RH40 and Kolliphor® RH60. Preferably, the one or more ester type polyethoxylated high hydrophile- lipophile-balance (HLB) surfactants are selected from the group consisting of PEG100 stearate; PEG20 stearate; PEG30 glyceryl cocoate; PEG32 stearate; polysorbate 20, and polysorbate 80. Thus, in certain aspects, the ester type polyethoxylated surfactant can comprise at least 40 ethoxylate groups per molecule.

Suitable one or more non-polyethoxylated high hydrophile-lipophile-balance (HLB) surfactants each independently having a hydrophile-lipophile-balance (HLB) value of equal to or greater than about 14 may include, for example, (1) fatty ether mono-, di-and oligoglycosides containing a ether bond between a fatty alcohol and a mono-, di-and oligoglycosides, alkyl polyglucosides, and alkylpolyglycosides such as decyl glucoside, cocoglucoside, poly(D-glucopyranose) ether with (Cs-Cw) linear primary alcohols, and xylityl caprate/caprylate which are available, for example, as commercial products including Plantacare® 2000 UP and Giorbis GiO™ - 103; (2) polyglyceryl fatty acid monoesters with such as triglycerol monolaurate, tetraglycerol monolaurate, triglycerol monooleate, tetraglycerol monooleate, triglycerol monostearate, tetraglycerol monostearate; (3) mono- and di-esters of glycerin with linear or branched long chain (greater than about 8 carbon atoms) fatty acids further esterified with short chain monocarboxylic acids, for example, glycerol monostearate lactate; (4) Saturated or unsaturated, linear or branched aliphatic Cs to C22 alkyl sulfonate and sulfate compounds, for example, octanesulfonic acid, sulfuric acid ester with lauryl alcohol, and salts thereof such as sodium lauryl sulfate; (5) Saturated or unsaturated, linear or branched aliphatic Cs to C22 ethoxylated alkyl sulfonate and sulfate compounds, for example, sulfuric acid ester with the product of addition of four moles of ethylene oxide to lauryl alcohol, and salts thereof such as sodium laureth sulfate; (6) sulfonated succinic acid esters with saturated or unsaturated, linear or branched aliphatic Cs to C22 alcohols, for example, the bis(2-ethylhexyl) ester of sulfosuccinic acid and the lauryl poly(etllylene oxide) ester of sulfosuccinic acid, or a mixture of these surfactants; (7) esters of lactic acid or lactic acid oligomers with fatty acids and salts thereof such as sodium stearoyl-2-lactylate; (8) sulfonates of benzene, cumene, toluene and alkyl substituted aromatic compounds and salts thereof, for example, dodecyl benzene sulfonic acid, or a mixture of these surfactants; (9) carboxylates of alcohol ethoxylates, alcohol propoxylates, alcohol ethoxylate propoxylates and ethoxylated linear and branched alkylpllenol compounds and salts thereof, for example, poly(ethylene oxide) tridecyl alcohol ether carboxylic acid and sodium poly(ethylene oxide) lauryl ether carboxylate, or a mixture of these surfactants; (10) long chain (greater than about 8 carbon atoms) acyl amino acids, for example, acyl glutamates, acyl peptides, acyl sarcosinates, acyl tau-rates, salts thereof, and mixtures of these surfactants; (11) Saturated or unsaturated, linear or branched aliphatic Cs to C22 alkyl amido propyl (dimethyl ammonio) acetate compounds, for example, cocamidopropyl betaine, lauramidopropyl betaine and stearamidopropyl betaine, and mixtures of these surfactants; (12) Sophorolipids, which consist of a hydrophobic fatty acid tail of a hydroxylated 16 or 18 carbon atom fatty acid, which is 13-glycosidically linked to a hydrophilic sophorose head, including free acid (open) and internally esterified (lactonic) forms and acetylated forms (acetylated on the 6'-and/or 6"positions). Sophorolipids useful in the practice of this invention include, for example, product mixtures produced by yeasts, for example, Candida bombicola, Candida apicoia, Starmerella bombicola, and Candida sp. NRRL Y-2720 (as identified by Price et al, Carbohydrate Research, 348 (2012) 33-41) and chemically modified product mixtures; and (13) Rhamnolipids including monorhamnolipids, which consist of one or two 3-(hydroxyalkanoyloxy) alkanoic acid tails and a single rhamnose head and di-rhamnolipids, which consist of one or two 3- (hydroxyalkanoyloxy) alkanoic acid tails and two rhamnose heads, including mixtures of compounds produced by Pseudomonas and Burkholderia bacterial species, for example, Pseudomonas aeruginosa and Burkholderia plantarii. Preferably, the one or more nonpolyethoxylated high hydrophile-lipophile-balance (HLB) surfactants can be selected from the group consisting of cocoglucoside, decyl glucoside, xylityl caprate/caprylate and sodium laureth sulfate.

In general, nanoparticle dispersions disclosed herein can comprise any amount of the high hydrophile-lipophile-balance (HLB) surfactants suitable to maintain the structural characteristics of the dispersion. In certain aspects, the amount of high HLB surfactant can be in a range from about 0.1 wt. % to about 20 wt. %, from about 1 wt. % to about 15 wt. %, from about 1.5 wt. % to about 15 wt. %, or from about 2 wt. % to about 10 wt. %. In certain aspects, the amount of high HLB surfactant can be in a range from 1.6 wt. % to 11.9 wt. %. Similarly, nanoparticle dispersions can comprise an amount of low HLB surfactant in a range from about 0.1 wt. % to about 20 wt. %, from about 1 wt. % to about 15 wt. %, from about 1.5 wt. % to about 15 wt. %, or from about 2 wt. % to about 10 wt. %. In certain aspects, the amount of high HLB surfactant can be in a range from 2.6 wt. % to 12.0 wt. %.

Suitable one or more low hydrophile-lipophile-balance (HLB) surfactants each independently having a hydrophile-lipophile-balance (HLB) value less than about 10 may include, for example, (1) Fatty acid esters including saccharide residues including sorbitan monolaurate, sorbitan monopalmitate, sorbitan stearate, sorbitan oleate, sorbitan isostearate, sorbitan sesquioleate, sorbitan trioleate, and sorbitan tristearate, which are available, for example, as commercial products including Span" 120, Span® 20, Span¹® 60, Span®’ 80, Span® 83, and Span® 85; (2) fatty acid glycerides, for example, glycerol monooleate, glyceryl monostearate, glycerol dioleate, glycerol distearate, which are available, for example, as commercial products including Jeechem GMS-0 and Jeechem GMIS; (3) fatty alcohol ethoxylates, fatty alcohol propoxylates, and fatty alcohol ethoxylate propoxylates for example, oleth-2, ceteareth-2, and lauryl alcohol 3 mole ethoxylate/6 mole propoxylate, which are available, for example, as commercial products including Brij® L4, Brij®05, Brij® S2, and Alkomol®L 306; and (4) Saturated or unsaturated, linear or branched aliphatic Cs to C22 carboxylic acid functional compounds including fatty acids derived from the saponification of vegetable and animal fats and oils, for example, octanoic acid, coconut fatty acid, oleic acid, ricinoleic acid, stearic acid, and carboxylic acid terminated short chain (e.g., n=4) polymers of ricinoleic acid and mixtures of such surfactants. Preferably, the one or more low hydrophile-lipophile-balance (HLB) surfactants selected from the group consisting of stearic acid, octanoic acid, glyceryl monostearate, sorbitan oleate, and sorbitan stearate.

Suitable one or more phospholipid low hydrophile-lipophile-balance (HLB) surfactants each independently having a hydrophile-lipophile-balance (HLB) value less than about 10 may include, for example, phosphatidyl choline, phosphatidylethanolamine, and phosphatidylinositol and compositions which include mixtures of these, for example, lecithins. Phospholipid products are available, for example, as commercial products including Phospholipon® 90G, Phospholipon®⁾ 90H, Alcolec® XTRA-A, Alcolec® PC 75 and Sunlipon® 65. Preferably, the one or more phospholipid low hydrophile-lipophile-balance (HLB) surfactants selected from the group consisting of phosphatidylcholine and lecithin.

In certain aspects, it is noted that the nanolipid dispersion itself or any components thereof (e g., the high hydrophile-lipophile balance (HLB) surfactant, the low hydrophile- lipophile-balance (HLB) surfactant, the water immiscible oils, or combinations thereof present in amounts greater than 0.1 % can be listed in the US Food and Drug Administration Code of Federal Regulations Title 21 Part 172 Food additives permitted for direct addition to food for human consumption.

Use of relatively longer chain poly(ethylene oxide) surfactants in a process of phase inversion resulting from a change in temperature is described in U.S. Patent No. 6,221,370. Typically phase inversion processes require polyethoxylated surfactants which have the property of decreasing hydrophilicity with increasing temperature. According to U.S. Patent No. 6,221,370. Examples F1-F3, compositions including an ester type polyethoxylated high hydrophile lipophile balance (HLB) surfactant (palmitic/stearic acid + 30 moles of ethylene oxide) were processed using a phase inversion temperature (PIT) emulsification method to give dispersions that were stable when stored for 4 weeks at 40°C. If the ester type poly ethoxylated surfactant was replaced with an ether type poly ethoxylated high hydrophile- lipophile-balance (HLB) surfactant (cetearyl alcohol + 30 moles of ethylene oxide, Example F4) the processed dispersions are not stable.

Previously, it was found that, through a process of phase inversion, a composition with ibuprofen which included an ether type polyethoxylated high hydrophile-lipophile- balance (HLB) surfactant could give concentrated nanoparticle dispersions that are stable at 40 °C but compositions with ibuprofen and ester type polyethoxylated high hydrophile- lipophile-balance (HLB) surfactants gave large particle size, non-nanoparticle dispersions that are unstable.

These results are the opposite of those disclosed in U.S. Patent No. 6,221,370. It appeared that including ibuprofen, an organic compound with a carboxylic acid group, negated the need for the ester group, a carboxylic acid group residue, in the polyethoxylated high hydrophile lipophile- balance (HLB) surfactant. Subsequently, it has been discovered that the requirement for ether type and not ester type poly ethoxylated surfactants in order to obtain stable dispersions is not limited to compositions that contain ibuprofen, it also applies to ibuprofen free compositions with other hydrophobic drugs that have carboxylic acid groups and carboxylic acid group residues including diclofenac (carboxylic acid), aspirin (carboxylic acid and ester), and lidocaine (ester), and to ibuprofen-free compositions that include, for example, drugs with no carboxylic acid group residues (hydrocortisone). As disclosed herein, nanoparticle dispersions having advantageous characteristics can be prepared without hydrophobic drugs as well. In preferred embodiments of the present invention concentrated nanoparticles are prepared that are free of ibuprofen and S-ibuprofen. Surprisingly, a modified twin screw extruder can be used to prepare stable, highly concentrated nanoparticle dispersions through reactions characterized by phase inversion where the compositions contain ether type polyethoxylated high hydrophile-lipophile- balance (HLB) surfactants but not ester type polyethoxylated high hydrophile-lipophile- balance (HLB) surfactants, the opposite of what is useful in preparing stable PIT emulsions according to U.S. Patent No. 6,221,370. By using ether type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactants and a modified twin screw extruder, it is possible to prepare highly concentrated nanoparticle dispersions with volume average particle size less than 150 nm which may include peptide bioactive agents such as those disclosed herein, as well as hydrophobic drugs, oil soluble vitamins and provitamins, and botanical extracts. In certain aspects, highly concentrated dispersions are possible whether or not the compositions include, for example, ibuprofen or other compounds with carboxylic acid group residues.

Owing to their commercial importance, the phase behavior of ether type poly ethoxylated surfactants has been studied in detail, and a key feature of these surfactants is their self-assembling and structuring behavior in aqueous compositions. The structuring behavior gives rise to many different phases depending upon concentration, temperature and the presence or absence of water immiscible oils. At higher concentrations, ether type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactants form phases including liquid crystalline (LC), hexagonal, and cubic phases which exhibit marked gel-like properties and extraordinarily high viscosity (see, e.g., Kunieda et al., Highly Concentrated Cubic-Phase Emulsions: Basic Study on D-Phase Emulsification using Isotropic Gels. J Oleo Sci 50(8):633- 639 • January 2001). The tendency towards highly structured phases and high viscosity can be remarkably reduced if a carbonyl (C=O) group is inserted into ether type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactants (see, e.g., Spiering et al., Changes in Phase Behavior from the Substitution of Ethylene Oxide with Carbon Dioxide in the Head Group of Nonionic Surfactants. ChemSnsChem. 2019, Nov 25. doi: 10.1002/cssc.201902855.), however the presence of a carbonyl group (from a carboxylic acid residue) in ester type polyethoxylated high hydrophile-lipopllile-balance (HLB) surfactants is apparently the reason that compositions with ester type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactants fail to give nanoparticle dispersions when processed with a modified twin screw extruder (compare examples disclosed in U.S. Application No. 16/748,399, e.g., Examples 10 -13 with Examples 5 - 9 and Example 18 with Example 17).

Although important for nanostructure and nanoparticle development, the rich phase behavior of ether type polyethoxylated surfactants can be problematic for processing because of the formation of highly viscous phases. Because the surfactant - oil - water mixtures pass through complex; high viscosity intermediate phases, there is a need for constant positive mechanical provided by kneading or mastication during the process. It is for this reason that simply cooling surfactant - oil - water mixtures with a conventional plate type or concentric tube type heat exchanger gives coagulated, large particle size emulsions rather than nanoparticle dispersions.

In certain aspects a total amount of surfactants (e.g., high and low HLB surfactants) within the dispersions disclosed herein can be in a range from 2 wt. % to 30 wt. %, from 5 wt. % to 28 wt. %, or from 15 wt. % to 25 wt. %. In other aspects, the total amount of surfactants can be less than about 40 wt. %, less than about 35 wt. %, less than about 30 wt. %, less than about 28 wt. %, less than about 25 wt. %, less than about 22 wt. %, less than about 20 wt. %, less than about 15 wt. %, or less than about 10 wt. %.

However, it was also found that certain combination of high and low HLB surfactants can lead to stable nanolipid dispersions with exceptional and advantageous characteristics, as disclosed herein. In certain aspects, the dispersion can comprise an ester-type polyethoxylated high HLB surfactant (e.g., PEG100 stearate, PEG20 stearate, PEG30 glyceryl cocoate, PEG32 stearate, polysorbate 20, polysorbate 80) and a phospholipid low HLB surfactant (e.g., phosphatidylcholine and lecithin). In other aspects, dispersions contemplated herein can comprise an ether type polyethoxylated high HLB surfactant (e.g., laureth-23, laureth-30, steareth-100, steareth-20, steareth-40, ceteareth-20, and ceteareth-30) and an ester type polyethoxylated high HLB surfactant (e.g., PEG100 stearate, PEG20 stearate, PEG30 glyceryl cocoate, PEG32 stearate, polysorbate 20, polysorbate 80). In such aspects the weight ratio of the ether type polyethoxylated high HLB surfactant to the ester- type poly ethoxylated high HLB surfactant can be greater than 1 : 1, greater than 2 : 1 , or greater than 4:1, for example, in a range from 1: 1 to 4: 1, or from 1: 1 to 2: 1. Alternatively, dispersions can comprise an ether type polyethoxylated high HLB surfactant can further comprise a non-polyethoxylated high HLB surfactant (e.g., sodium laurel sulfate).

Dispersions disclosed herein also can comprise one or water soluble polymers or gums, including: (1) Polysaccharides such as dextrins, gums, including maltodextrin, cyclodextrin, hyaluronic acid, xanthan gum, guar gum, and water dispersible or water- soluble starches; (2) Water soluble cellulose derivatives including cellulose ethers such as methyl cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, and carboxyethyl cellulose; (3) Poly(acrylic) acid and salts thereof including Carbomer 940, and sodium carbomer such as Neutragel DA (product of 3V Sigma USA, Georgetown SC); (4) Acrylates/Vinyl Crosspolymers such as Rapidgel EZ1 (product of 3V Sigma USA); and (5) Poly(vinyl pyrrolidone). Preferably, the one or more water soluble polymers or gums are selected from the group consisting of methyl cellulose, sodium carbomer, and acrylates/vinyl crosspolymers.

The nanoparticle dispersion may also include, for example, one or more cryoprotectants that prevent the freezing of nanoparticle dispersions, or prevents decomposition of nanoparticle dispersions during freezing. Preferred cryoprotectants are water miscible or water-soluble compounds including glycerin, propylene glycol, ethylene glycol, diethylene glycol, diethylene glycol ethyl ether, sucrose, sorbitol, trehalose, and dimethyl sulfoxide (DMSO). Preferably, the one or more cryoprotectants are selected from the group consisting of diethylene glycol, dimethyl sulfoxide, ethylene glycol, glycerin, propylene glycol, sorbitan, and trehalose.

The nanoparticle dispersion may also include, for example, one or more additives such as antioxidants, chelating agents, acidulants, and antimicrobial agents. Useful antioxidants include, for example, butylated hydroxyl toluene (BHT) and mixed tocopherols. Useful chelating agents include, for example, phosphates, ethylenediaminetetriacetic acid and salts thereof, sodium phytate, and nitrilotri acetic. Useful antimicrobial agents include, for example, phenoxy ethanol and caprylyl glycol such as Optiphen, a product of Ashland Chemicals and Jeechem CAP-4, a product of Jeen. Preferably, the one or more additives are selected from the group consisting of tetrasodium ethylenediaminetetraacetic acid, citric acid, butylated hydroxy toluene, and sodium chloride.

Support for the conclusion that structure formation during phase changes is responsible for fine particle size in extruded dispersions is provided by the observation that adding structure promoting additives allows the use of ester type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactants in the practice of the current invention. It has been found to be possible to compensate for the inadequate structure forming properties of ester type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactants by adding structure forming low hydrophile-lipophile-balance (HLB) surfactants as additives. Particularly useful structure forming low hydrophile-lipophile-balance (HLB) surfactants are phospholipids. Examples 19-23 of U.S. Patent No. 11,504,327 show that by adding phospholipids to compositions with ester type polyethoxylated high hydrophile-lipophile- balance (HLB) surfactants that alone do not give nanoparticle dispersion. It is possible to obtain useful and stable nanoparticle dispersions. Useful phospholipids include, for example, dioleyl phosphatidyl choline, distearoyl phosphatidyl choline, and lecithin.

Compounds such as ibuprofen also can be considered a structure promoting additive within the context of this disclosure and may be exemplified by ibuprofen. Without being bound by theory, examining the structure of ibuprofen suggests that ibuprofen may interact with surfactants in the mixture as phases of the mixtures are converted from a crude mixture to a nanolipid dispersion having particle size less than 150 nm. Specifically, ibuprofen includes a hydrophobic alkyl group positioned in a phenyl ring opposite a carboxylic group that is mildly hydrophilic at pH 5 or less and more hydrophilic at higher pH where the carboxylic group is neutralized at physiological pH. These opposing groups may allow ibuprofen to modulate and facilitate the interaction between surfactants, water, and water immiscible oils within the mixture. Thus, in certain aspects, dispersions contemplated herein can comprise an amount of ibuprofen (or acetaminophen, or other structure promoting additives) in a range from about 0.01 wt. % to 10 wt. %, from 0.05 wt. % to 5 wt. %, or from 0.1 wt. % to 3 wt. %. Of course, as ibuprofen (and other structure promoting additives) may also have biological activity and can be considered bioactive agents independently as defined herein, for the purposes of this disclosure it will be understood that ibuprofen and/or acetaminophen will be considered structure promoting additives in dispersions where other bioactive agents are also present. Alternatively, in certain aspects dispersions can be substantially free of ibuprofen (or free of lidocaine, or free of acetaminophen) where the combination of surfactants remains stable upon formulation and maintain desirable characteristics as discussed below.

Dispersions disclosed herein may advantageously maintain their stability and other desirable characteristics while accommodating an increased loading of the bioactive compounds disclosed herein, specifically relative to dispersions comprising vesicular nanoparticles. Without being bound by theory, it is believed that by identifying stable dispersions with an increased amount of the hydrophobic lipid components allows the dispersion to incorporate an increased amount of the bioactive substance within the lipid phase of the dispersion. Moreover, the nature of the lipid phase dispersion as non-vesicular lipid particles allows the dispersions to incorporate the bioactive agents within the entire volume of the particulate lipid phase, instead of limited within lipid layers present throughout vesicles and other lipid membrane structures (e.g., lamellar, etc.) Again, without being bound by theory, it is contemplated that nanolipid dispersions disclosed herein can be formed entirely, or in substantial part, by lipid particulates dispersed within an aqueous medium comprising hydrophilic components.

Given the above discussion, certain aspects disclosed herein can comprise an amount of bioactive agent that may drastically exceed otherwise identical nanoparticulate compositions comprising otherwise identical components but adopting another nanoparticulate form. In certain aspects, it is contemplated that the dispersions can comprise as much as 2 wt. %, 5 wt. %, 10 wt. %, 20 wt. %, 30 wt. % or 40 wt. % of bioactive agent relative to the total weight of the dispersion. Alternatively, dispersions can comprise an amount of the bioactive agent in a range from about 0.01 wt. % to about 40 wt. % of the bioactive agent, from about 0.01 wt. % to about 20 wt. %, or from about 0.1 wt. % to about 10 wt. %, from about 1 wt. % to about 10 wt. % or from about 3 wt. % to about 8 wt. %. The amount of bioactive agent may also be expressed relative to the amount of water immiscible oil in the dispersion. Particularly, a relatively high weight ratio of water immiscible oil to bioactive agent can have a demonstrated effect of encapsulating the bioactive compound within protective nanoparticles and away from lipophilic surfaces that are adjacent hydrophilic components, where interactions between the bioactive agents and hydrophilic components of the environment may occur. Dispersions having a relatively high weight ratio of water immiscible oil to the bioactive ingredients therefore may exhibit a protective effect to the bioactive agents, preventing degradation of the bioactives due to environmental factors and enzymes that may become present in the hydrophilic portion (such as may occur upon ingestion, or application to the skin). It is also contemplated that significant excess of water immiscible oils relative to the bioactive ingredient may improve biodelivery of the bioactives, for instance by increased dermal permeation of the nanolipid particles. As discussed above, the non-vesicular, non-lamellar structure of the nanolipid dispersion also may contribute to the protective effect. In certain aspects, the weight ratio of water immiscible oils to the bioactive agent can be in a range from 10,000: 1 to 1 :1, from 1,000: 1 to 10: 1, from 800: 1 to 25:1, from 500: 1 to 50: 1, from 500: 1 to 10: 1, from 200:1 to 10: 1, or from 100:1 to 10: 1.

The ability of nanoparticles to permeate skin is known to be related to particle size, with generally better permeation for smaller paliicles. The appropriate quantity for paliicle size in a distribution of particles as it relates to dermal permeation is the weight average particle diameter because it defines the average on the basis of mass fractions of nanoparticles with a particular diameter and what matters is the mass fraction of nanoparticles that have sufficiently small diameters so as to effectively permeate skin. By comparison, an unspecified majority by mass fraction of particles can have diameters much greater than the number average particle diameter, making number average particle diameter a poor metric for permeability. In certain aspects, nanoparticle dispersions disclosed herein can have volume average particle diameter less than 150 nm, less than 100 nm, less than 60 nm, or less than 50 nm; alternatively, in a range from about 10 to about 150 nm, from about 20 nm to about 120 nm, from about 30 nm to about 100 nm, or from about 30 nm to about 80 nm.

Dispersions disclosed herein may also be characterized by their lipid content. Without being bound by theory, it is believed that an increased lipid content can provide additional capacity to load and protect bioactive agents as disclosed herein. In certain aspects, the dispersions disclosed herein can comprise greater than 25 wt. %, greater than wt. 30%, greater than 35 wt. %, greater than 40 wt. %, greater than 45 wt. %, greater than 50 wt. %, greater than 55 wt. %, or greater than about 60 wt. %. In other aspects, dispersions can comprise a lipid content in a range from 25 wt. % to 55 wt. %, or from 35 wt. % to 50 wt. %. In certain aspects, dispersions can comprise a lipid content greater than 25 wt. % and a particle size less than 100 nm. In alternative aspects, dispersions can comprise a lipid content in a range from 35 wt. % to 50 wt. %, and a particle size less than 60 nm.

Another important attribute of highly permeating nanoparticles is poly dispersity in hydrophile-lipophile-balance (HLB) surfactant values. Hydrophile-lipophile-balance (HLB) poly dispersity promotes formation of lamellar structures for nanoparticles which otherwise have no layer structures as prepared when they are applied to skin. Layered structures have been shown to be important for promoting dermal permeation of nanoparticles, for example, in liposomes and niosomes. Hydrophile-lipophile-balance (HLB) poly dispersity can be calculated as the hydrophile-lipophile-balance (HLB) weight mean square deviation, WMSDHLB which is the sum of the products of the weight fraction of the z-th surfactant species times the square of the deviation of the hydrophile-lipophile-balance (HLB) of the z-th surfactant species from the weight average hydrophile-lipophile-balance (HLB) divided by the weight average hydrophile lipophile-balance (HLB) hydrophile-lipophile-balance (HLB) poly dispersity with WMSDHLB greater than about 1.5 is required to support development of lamellar structures subsequent to skin application for dispersions of nanoparticles with no layered structure as prepared. At high values of WMSDHLB, nanoparticles with aqueous cores may form including multi-layered structures which are undesirable. In certain nanoparticle dispersions, nanoparticles can have oily cores and WMSDHLB is between about 1.5 and about 4.5, between about 1.75 and about 3.5, and between about 2 and about 3. In certain aspects, DNLF dispersions disclosed herein can have latent lamellar structure, that is, the propensity for nanoparticle dispersions without lamellar structure to develop lamellar structure. In certain aspects, dispersions disclosed herein can adopt lamellar structure by evaporation or heating. The phase transition from non-lamellar dispersions to a lamellar dispersion that characterizes latent lamellar character can be observed by the development of optical birefringence or increased electrical impedance in a sample under appropriate conditions (e.g., evaporation, heating, or both). Optical birefringence can be observed by viewing through crossed polarized films. Electrical impedance can be observed as a negative peak in a plot of conductivity versus temperature and a corresponding positive peak in the first derivative plot of conductivity vs temperature for heated and stirred nanoparticle dispersions when heating at a rate of between 1 and 4 °C per minute and measuring conductivity with an open cell geometry electrode such as a 013005MD 4-cell conductivity electrode available from ThermoScientific.

In certain aspects, the transition between non-lamellar structure and lamellar structure can be characterized by the temperature at which the phase transition begins, i.e., a phase transition onset temperature, and the temperature at which the phase transition is complete, i.e., a phase transition finish temperature. The phase inversion onset temperature is the temperature at which the electrical conductivity dropped to less than 80% of the maximum conductivity while heating. The phase inversion finish temperature was determined as the highest temperature at which the conductivity dropped to less than 10% of the maximum conductivity. In certain aspects, latent lamellar structure can be characterized by the nanoparticle dispersion adopting a lamellar structure upon heating to a temperature (e.g., a phase inversion onset temperature) in a range from 40 °C to 95 °C, 45 °C to 90 °C, 50 °C to 85 °C, 60 °C to 80 °C, or 65 °C to 75 °C. In certain aspects, evaporation may also induce a phase transition, either in combination with gentle heating, or without heating altogether.

Alternatively, nanoparticle dispersions disclosed and comprising latent lamellar structure can be characterized by the dispersion exhibiting a positive peak in the first derivative plot of normalized conductivity versus temperature, wherein the positive peak has a peak amplitude greater than about 0.05 °C'¹, greater than about 0.1 °C'¹, or greater than about 0.3 °C'¹, at a temperature in a range between about 45 °C and 80 °C (or any phase inversion onset temperature noted above).

Further still, nanoparticle dispersions disclosed herein and comprising latent lamellar structure can be characterized by the dispersion exhibiting an optical birefringence when observing the dispersion through cross polarized films upon heating to temperatures in a range from 60 °C to 95 °C (or any phase inversion onset temperature noted above).

In certain aspects, dispersions may become relatively unstable after phase inversion, and phase separation may be observed in a scale of many hours to several days. However, this does not pose an issue for use as prepared as ready-for-use treatments. Non-lamellar dispersions (e.g., nanolipid dispersions with latent lamellar character, prior to phase inversion) disclosed herein have demonstrated an excellent stability under storage conditions. For instance, in certain aspects, peptide compound DNLF dispersions retain a high kinetic stability such that they do not phase separate within 28 days when stored at temperatures between 18 °C and 23 °C and do not phase separate within 7 days when stored at 40 °C. In another aspect, nanolipid dispersions can be stable with respect to phase separation for 5 days at 40 °C.

Separately, nanoparticle dispersions disclosed herein can demonstrate stability with respect to retaining the bioactive agent within the nanoparticle dispersion. In certain aspects, the bioactive agent does not form crystals within the nanoparticle dispersion at a temperature from about 18 °C to about 22 °C for greater than about 1 month, greater than about 2 months, greater than about 3 months, 8 months, greater than about 12 months, greater than about 18 months, or greater than about 24 months; alternatively, in a range from 1 month to 24 months, in a range from 3 months to 12 months. The nanoparticle dispersions disclosed herein may remain free from crystals even where the concentration of the bioactive agent is greater than the solubility of the bioactive agent in the aqueous phase of the dispersion, even outside the context of the nanoparticle. Surprisingly, the characteristic of super solubility remains applicable even where the bioactive agent has a logP less than 1, and thereby demonstrating a significant solubility in aqueous solutions.

METHODS FOR PREPARING NANOPARTICLE DISPERSIONS DNLF dispersions do not form spontaneously within 2 weeks after oil phase and aqueous phase after low shear mixing of an oil phase with an aqueous phase. Low shear mixing includes mixing using a magnetic stir bar and magnetic stirrer and stirring using a stirring element including a blade, a paddle, or a spiral paint mixer such as a Red Devil® 5 Gallon Drywall Mud/Paint Mixer Model Number 4041 or equivalent. Surprisingly, it was found that a modified twin screw extruder in which the majority of screw elements are conveying elements with a minor fraction of mixing or kneading screw elements is capable to provide sufficient mechanical kneading and mastication during phase transformations so as to provide concentrated nanoparticles in the extrudate.

We further found that highly concentrated dispersions with up to 60 weight percent dispersed nanoparticles and volume average particle size as low as about 25 nm can be made by a single step process using a twin screw extruder modified so as to provide a temperature gradient of greater than about 35°C for surfactant-oil-water composition conveyed through it. Processing times for the conversion of a coarse, non-nanoparticle dispersion to a final concentrated nanoparticle dispersion can be less than 2 minutes which is remarkable considering that time and temperature cycling have been shown to be important for making nanoparticle dispersions by phase inversion methods (Heurtault et al., Pharm. Res. 19, 875, 2002 and Anton et al., Int J Pharm. 2007 Nov 1 ;344(l-2):44-52).

Certain aspects can comprise forming a crude mill base to be processed within the extruder, by preparation of an aqueous mixture and a lipid mixture, each comprising hydrophilic and hydrophobic components as described above, respectively. The crude mill base can be formed by addition of the aqueous and lipid mixtures to produce a single mixture, which may then be processed within an extruder.

In certain aspects, processing the crude mill base can be conducted at rates up to about 380 grams per minute using pilot scale twin screw extruders with 24 mm or 27 mm screw diameters. Preferably, the crude mill base can be processed within the extruder with temperature gradients that include, for example, the temperature at which the composition is a microemulsion. The temperature gradient during processing can be provided by preheating the composition prior to introduction to the extruder or by heating in one or more of the first temperature control zones of the extruder, or both plus cooling the composition in one or more of the latter temperature zones of the extruder. In preferred embodiments, the process deltaT defined as the difference between the maximum of the composition temperature as introduced to the extruder and the extruder zone temperature minus the minimum temperature of the cooled extruder zones is greater than about 35 °C, greater than about 50 °C, and greater than about 60 °C.

Surprisingly, a modified twin screw extruder can be used to prepare stable, highly concentrated nanoparticle dispersions through reactions characterized by phase inversion where the compositions contain ether type polyethoxylated high hydrophile-lipophile- balance (HLB) surfactants but not ester type polyethoxylated high hydrophile-lipophile- balance (HLB) surfactants, the opposite of what is useful in preparing stable PIT emulsions according to U.S. Patent No. 6,221,370. By using ether type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactants and a modified twin screw extruder, it is possible to prepare highly concentrated nanoparticle dispersions with volume average particle size less than 150 nm which include many active ingredients such as hydrophobic drugs, oil soluble vitamins and provitamins, and botanical extracts whether or not the compositions include structure promoting additives such as ibuprofen or other compounds with carboxylic acid group residues.

NANOPARTICLE DISPERSIONS COMPRISING BIO ACTIVE AGENTS AS ORAL FORMULATIONS WITH IMPROVED BIO AVAILABILITY

Where practicable, drugs and therapeutic agents are often administered in oral dosage forms because such forms are easy for patients to administer themselves (for example, compared to injectable medication). Orally administered drugs, however, encounter several challenges before they arrive at the desired location within the body. Such challenges may include extreme pH environments, poor absorption, and significant quantities of the drug being processed by the body as waste. Consequently, some drugs have poor bioavailability when administered in an oral dosage form.

US Patent 11,504,327 entitled METHOD OF PREPARING NANOPARTICLES BY HOT-MELT EXTRUSION, which is hereby incorporated in its entirety herein by reference, describes a method of making concentrated lipid nanoparticle dispersions that contain between 25% and 60% of lipid content. It is disclosed and demonstrated herein that such highly concentrated dispersions of lipid nanoparticles can also comprise hydrophilic drugs and bioactive agents as described in US Patent Application 63/384,585, filed November 21, 2022 or peptides as described in in US Patent Application 63/384,584, filed November 21, 2022 each of which are also incorporated in their entirety herein by reference.

Surprisingly, oral administration of lipid nanoparticle dispersions disclosed herein can result in excellent Tmax and Cmax characteristics that are more typical of an injection. In certain aspects, of a peak plasma concentration that occurs at less than one hour after administration by oral gavage to a Jackson Labs C57BL/6J mice. In one embodiment, the lipid nanoparticle dispersion provides a plot of plasma concentration vs time in which d(plasma concentration)/dt is negative at every time greater than one hour after administration by oral gavage to a Jackson Labs C57BL/6J mice.

In certain aspects, oral administration of the nanoparticle dispersion results in a peak plasma concentration in the patient at less than one hour (or from 10 minutes to 1 hour, or less than 30 minutes, or less than 15 minutes, or from 5 to 30 minutes) after administering the nanoparticle or capsule to the patient. In other aspects, a plot of plasma concentration vs time in which d(plasma concentration)/dt is negative at each time point after administering the nanoparticle or capsule greater than one hour (e.g., from 1 to 24 hours).

Those of ordinary skill in the art will appreciate that the nanoparticle dispersions disclosed herein are suitable for known methods of treatment for myriad well-known bioactive agents and their known applications. For instance, the use of insulin in the treatment of diabetes mellitus type 1 and type 2 is well known, despite no previously known effective oral formulation. It is contemplated that the nanoparticle dispersions disclosed herein containing insulin as a bioactive agent will be suitable for methods of treating diabetes mellitus type 1 and type 2 based on the ability to achieve suitable Cmax and Tmax values following oral administration. Similarly, methods of treatment for other bioactive compounds contemplated herein, in reliance on treatment methods recognized in the art that may be improved by increased oral bioavailability, based on the ability to achieve Cmax and Tmax values that are comparable to existing methods of treatment. It is also observed that nanoparticle dispersions may be absorbed intact from the GI tract, and therefore may provide a protective effect on the bioactive agents through oral administration of the dispersions comprising the bioactive agents. In this manner, it is contemplated that dispersions disclosed herein may demonstrate a rate of drug absorbance is greater than (e.g., more than 1.5x greater than, more than 2x greater than; alternatively, in a range from 1 to 5 times greater than) a rate of drug release from the lipid phase of the nanoparticle dispersion in the GI tract. Ultimately, the amount of drug released from the nanoparticle dispersions by digestion is less than that absorbed from the GI tract. Therefore, the majority of the bioactive agent may be absorbed into the body under milder physiological conditions.

Formulations comprising the nanoparticle dispersions disclosed hereinabove may further comprise inactive ingredients that have been used in a US Food and Drug Administration (FDA) approved drug product for a particular route of administration. When inactive ingredients have been approved for a route of administration, the inactive ingredient is not considered new and may require a less extensive review when seeking approval for DNLF dispersion drug products.

In one aspect, all of the inert ingredients in a topical DNLF dispersion drug product appear in the FDA Inert Ingredients Guide (IIG) for one or more drugs approved for topical administration.

In one aspect, all of the inert ingredients in a topical DNLF dispersion drug product appear in the FDA Inert Ingredients Guide (IIG) for one or more drugs approved for oral administration.

In certain aspects, DNLF dispersions for oral administration comprise an ether type polyethoxylated high HLB surfactant. Ether type polyethoxylated high HLB surfactants lack ester bonds and are therefore not susceptible to hydrolytic cleavage of a bond between the hydrophobic and hydrophilic surfactant groups which would cause the surfactant properties to be lost, particle size to increase, and phase separation to occur. Several ether type polyethoxylated high HLB surfactant have been approved for use in drugs for oral administration including ceteareth-20 (CAS number 68439-49-6), steareth-40 (CAS number 9005-00-9), poloxamer 124, poloxamer 188, poloxamer 331, and poloxamer 407. Ether type polyethoxylated high HLB surfactants with polyethoxylated chain lengths greater than about 20 ethoxylate units and molecular weight less than 2500 Daltons such as ceteareth-20 and steareth-40 are particularly useful for extruding DNLF dispersions.

In one aspect, DNLF dispersions include an ether type polyethoxylated high HLB surfactant that appears in the FDA Inert Ingredients Guide (IIG) for one or more drugs approved for oral administration.

In one aspect, DNLF dispersions include an ether type polyethoxylated high HLB surfactant that appears in the FDA Inert Ingredients Guide (IIG) for one or more drugs approved for oral administration wherein the ether type polyethoxylated high HLB surfactant has polyethoxylated chain length greater than about 20 ethoxylate units and molecular weight less than 2500 Daltons.

In one aspect, DNLF dispersions for oral administration include an ether type polyethoxylated high HLB surfactant selected from ceteareth-20 and steareth-40.

Lipolysis-resistant nanolipid dispersions

Further improvements to the bioavailability of bioactive agents beyond those discussed above are also contemplated herein.

Biopharmaceutics Classification Class II and Class IV drugs which are poorly soluble in the aqueous environment of the GI tract are mostly eliminated by excretion rather than being absorbed. The conventional strategy to improving the absorption and bioavailability of Class II and Class IV drugs is to improve the effective solubility by creating super saturated solutions. For example, the solubility of Class II or Class IV drug may be increased by providing the compound in the form of very fine particles or as an amorphous solid dispersion (ASD) in which the drug is molecularly dissolved in a water-soluble polymer.

Another approach to generating supersaturated solutions of drugs in the GI tract is digestion of lipids in a lipid-based drug delivery (LBDD) system. Formulating the drug as a lipid-based formulation such as oil solution or suspensions, emulsions, self-emulsifying drug delivery systems (SEDDS), self-micro emulsifying drug delivery system (SMEDDS) or self-nano emulsifying drug delivery system (SNEDDS) can increase the solubility and dissolution of lipophilic drugs and facilitate the formation of solubilized species, from which absorption occurs. There is little evidence to suggest the potential for direct absorption of the solubilized construct (for example, a micelle or an emulsion droplet), and the prevailing view is that drug absorption is facilitated by drug movement from the solubilized lipid reservoir to free drug as an intermicellar solution followed by absorption of free drug from the intermicellar phase. Digestion of lipids forces drugs out of the oil phase and into intermicellar solution and is believed to be critical for absorption of drugs from lipid formulations.

The dearth of evidence for direct absorption of the dispersed lipid particles and nanoparticles is likely the result of inability to prepare particles sufficiently small enough to be easily absorbed directly while also being sufficiently resistant to digestion to exist long enough in the intestinal lumen to be absorbed. Generally, particles that are small enough to undergo rapid absorption, for example by caveolin mediated endocytosis, are so rapidly digested that the absorption result is essentially the same as other methods that create supersaturated aqueous drug solutions.

Tmax, the time required to reach the maximum plasma drug concentration (Cmax) for drugs that are administered in LBDD systems is generally one hour or more. It is reasonable to presume an hour or more is required for digestion of LBDD lipids plus subsequent solubilization of released drug in micelles by bile salts. Plots of plasma concentration of drugs administered orally as LBDD formulations generally show increasing drug concentrations during the first 1 to 10 hours following by a decline in concentrations thereafter due as the drug is metabolized and eliminated. By contrast, intravenous bolus injection of drugs gives plots with ever decreasing plasma concentrations.

The present invention provides drugs encapsulated in lipid nanoparticles which are directly absorbed from the intestinal lumen, exhibiting drug plasma Tmax values of 30 minutes or less when the lipid nanoemulsion is administered orally. The present invention provides lipid nanoemulsions that exhibit monotonically decreasing drug plasma concentrations upon oral administration that monotonically decrease beginning 30 minutes or less after oral administration. Inventive lipid nanoemulsions may comprise lipids that lack ester bonds and are therefore not subject to enzymatic decomposition from lipase, or they may comprise compounds that are inhibitors for lipases, or they may comprise surfactants that block access of lipases across oil water interfaces. Inventive lipid nanoemulsions have volume average particle diameters sufficiently small to allow direct absorption via endocytosis, particularly caveolin and clathrin mediated endocytosis. In caveolin and clathrin mediated endocytosis, about 60 nm to 100 nm diameter pits or invaginations form in cell walls which yield endocytic vesicles that become increasingly effective vehicles for particle absorption as absorbate particle diameters decrease below about 80 nm.

The invention provides a lipid nanoparticle dispersion comprising a hydrophobic therapeutic agent encapsulated in a nanoparticle composed of lipophilic materials wherein the nanoparticles have volume average particle size less than 100 nm wherein lipophilic materials are lipolysis resistant meaning that less than 25% of ester bonds in the lipophilic materials hydrolyze within one hour in the presence of a concentration 50 units per milliliter of porcine pancreatic lipase wherein “lipophilic materials” include all surfactants, water immiscible oils, hydrophobic drugs, and hydrophobic therapeutic agents.

In one embodiment the lipid nanoparticle dispersion includes a high HLB polyethoxylated surfactant that is inert to lipolysis. In one embodiment the lipid nanoparticle dispersion includes a high HLB polyethoxylated surfactant that lacks an ester group. In one embodiment the lipid nanoparticle dispersion includes an ether type high HLB polyethoxylated surfactant.

In one embodiment the lipid nanoparticle dispersion includes a lipase inhibitor. In one embodiment the lipase inhibitor can be selected from the group consisting of anandamide, diacylglycerol lipase, orlistat, 2-arachidonoylglycerol, cannabinoid, endocannabinoid, fatty acid amidase, acylglycerol lipase, and combinations thereof. In certain aspects, the lipase inhibitor is orlistat. In one embodiment the lipid nanoparticle dispersion comprises greater than 25 weight percent lipophilic material content.

Nanoparticle dispersions disclosed herein also may comprises lipid nanoparticles with a positive charge. In one embodiment the lipid nanoparticle dispersion comprises lipid nanoparticles with a zeta potential greater than 1.0 millivolt; alternatively, in a range from 0.5 to 5 mV, or from 1 mV to 3 mv). Thus, in certain aspects, nanoparticle dispersions disclosed herein can comprise zwitterionic surfactants. Zwitterionic surfactants comprise separated positively and negatively charged ions which are not conformationally allowed to associate in such a way as to become hydrophobic. The presence of charged ions on lipid nanoparticle surfaces may be effective to reduce bending forces in cell membranes and allow more facile puckering so as to promote endocytosis; alternatively, zwitterionic groups on lipid nanoparticle surfaces may mimic improved attachment, cellular membrane penetration and absorption observed for capsid viruses.

Accordingly, in certain aspects, nanoparticle dispersions comprise amphipathic compounds with a betaine group. In one aspect, nanoparticle dispersions comprise cocamidopropyl betaine. In one aspect, nanoparticle dispersions comprise amphipathic compounds with a choline phosphate group. In one aspect, DNLF dispersions comprise a phosphatidyl choline compound.

Lipolysis resistance may be characterized by changes in the particle size, as ester bonds are hydrolyzed within the dispersions. In certain aspects, the nanoparticle dispersion can have greater than 80% (or greater than 60%, or greater than 70%, or greater than 90%) of particles in the dispersion on a volume basis have a diameter less than 100 nm after 60 minutes in a lipolysis solution with 2.6% DNLF dispersion, pH about 6.8 containing calcium, one or more bile salts, and 0.4% lipase. In still further aspects, less than 10% (e.g., less than 5%, less than 2%; alternatively, in a range from 1 to 10 %) of the lipid ester bonds are lipolyzed after 60 minutes in a lipolysis solution with pH about 6.8 containing calcium, one or more bile salts, and 0.4% lipase.

Still, even where lipolysis occurs, it is observed that nanoparticle dispersions as disclosed herein may retain a reduced particle size. For instance, it is seen that greater than 80% of particles on a volume basis can have a diameter less than 100 nm after 30% or more of lipid nanoparticle ester bonds have been hydrolyzed by lipase.

Encapsulated nanolipid dispersions The high water activity of nanolipid dispersions can cause deterioration of gelatin capsules. Left overnight at temperatures between 4 °C and 50 °C, gelatin capsules filled with DNLFs will become too soft to easily swallow. One solution is to fill the gelatin capsule with DNLF shortly before swallowing, but such a method is inconvenient and creates risk of incorrectly measuring the required dosage.

A solution to poor systemic absorption is provided by providing drugs and therapeutic agents in dense nanolipid fluid (DNLF) dispersions which are highly concentrated lipid nanoemulsions in a continuous aqueous phase.

It has been discovered that DNLF dispersions can be encapsulated in so called enteric polymers that have carboxylic acid functional groups if the pH of the DNLF dispersion is below the pKa value of the polymer carboxylic acid groups, even in the case that such polymers may be plasticized by absorption of some of the water in the DNLF dispersion. Furthermore, DNLF dispersions are stable to encapsulation in carboxylic acid functional enteric polymers, even in the case that some of the water in the DNLF dispersion is lost to absorption in the capsule wall.

The present invention generally provides capsules comprising DNLF dispersions. In certain aspects, the DNLF dispersion can comprise hydrophobic active compounds, hydrophilic active compounds, and or peptides encapsulated in capsules comprising carboxylic acid functional polymers. In certain aspects, the capsules can comprise a carboxylic acid functional polymer and an encapsulated DNLF dispersion.

In another aspect, the DNLF dispersion can be encapsulated in a carboxylic acid functional polymer. In one embodiment the pH of the DNLF dispersion is less than the pKa value of the encapsulating polymer carboxylic acid groups. In one embodiment the pH of the DNLF dispersion is more than one pH unit less than the pKa value of the encapsulating polymer carboxylic acid groups. In one embodiment, the encapsulating polymer is the product of vinyl polymerization.

In one embodiment, the encapsulating polymer comprises acrylate monomer units. In one embodiment, the encapsulating polymer comprises methacrylate monomer units. In one embodiment, the encapsulating polymer comprises vinyl acetate monomer units. In one embodiment, the encapsulating polymer comprises acrylic acid monomer units. In one embodiment, the encapsulating polymer comprises methacrylic acid monomer units. In one embodiment, the encapsulating polymer comprises vinyl 4-hydroxyl phthalate monomer units. In one embodiment, the encapsulating polymer comprises modified cellulose. In one embodiment, the encapsulating polymer comprises cellulose esterified with acetic acid and phthalic acid. In one embodiment, the encapsulating polymer comprises cellulose esterified with acetic acid and succinic acid. In one embodiment, the encapsulating polymer is methyl acrylate - methacrylic acid copolymer. In one embodiment, the encapsulating polymer is methyl methacrylate - methacrylic acid copolymer. In one embodiment, the encapsulating polymer is ethyl acrylate - methacrylic acid copolymer. In one embodiment, the encapsulating polymer is vinyl acetate - vinyl 4-hydroxyl phthalate copolymer.

In one embodiment, the encapsulating polymer is hydroxypropyl methyl cellulose acetate succinate. In one embodiment, the encapsulating polymer is cellulose acetate phthalate. In one embodiment, the encapsulating polymer is Eudragit L 30 D-55.

In one embodiment, the capsule comprises a surface layer comprising carnauba wax, dimethicone and organo-modified silicone. In one embodiment, the capsule comprises a surface layer comprising carnauba wax.

In one embodiment, the capsule comprises an innermost layer consisting of a carboxylic acid functional polymer, an intermediate layer of gelatin, and a surface layer comprising carnauba wax.

EXAMPLES

Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

Table 1. Abbreviations, Descriptions, and Sources of Materials

The particle size distributions of extruded DNLF dispersions prepared in Examples below were measured within two days of preparation on samples diluted approximately 50 times with deionized water using a NanoFlex dynamic light scattering (DLS) instrument (Microtrac Instruments, York PA). Particle polydispersity was estimated as the ratio of volume average particle size (D_v) to number average particle size (D_n).

GENERAL PROCEDURES

The following Examples were conducted under the following general procedures, modified as noted throughout the Examples.

Preparation of crude emulsion mill bases.

Lipid components including surfactants and water immiscible oils, were combined and warmed until solid components melted or dissolved to give liquids ranging in clarity from clear to moderately hazy. Water soluble peptides were converted to lipophilic, water insoluble hydrophobic ion pairs by dissolving the peptide in 0.1 N HC1 and then adding an aqueous solution of sodium lauryl sulfate to give a suspension of hydrophobic precipitate which was then mixed with the previously formed mill base of the remaining components. Water soluble components were dissolved in water in a separate vessel. The aqueous solution was added to the warm mixture of lipid components and stirred to give the mill base as an opaque emulsion.

Preparation of dense nanolipid fluid (DNLF) dispersions

DNLF dispersions were prepared in either of a 27 mm twin screw extruder or a 11 mm twin screw extruder.

In examples using a 27 mm twin screw extruder, a 50 g sample of the crude emulsion base was heated to between 90 and 98 °C in a 2-quart pyrex pitcher using a microwave. The heated emulsion base was then poured by hand into the inlet of a Leistritz ZSE 27/GL-36D extruder, modified as described in Example 57 of US Patent Appl. Ser. No. 16/748,399, which is hereby incorporated by reference herein in its entirety. The temperature of the extruded DNLF dispersion was maintained below 10 °C throughout the processing.

In examples using an 11 mm twin screw extruder, a 50 g sample of the crude emulsion mill base was first pre-heated in bulk in a 250 mL pyrex beaker using a microwave oven to about 60 °C and then to between 80 and 90 °C using a handheld torch. The heated sample was then introduced to the inlet port of a modified Thermo Fisher Process 11 extruder. Generally, the extruder carried zone temperatures of zone 2 = approximately 50 °C, zone 3 = 35 °C, zone 4 = 9 °C, zone 5 = 9 °C, zone 6 = 7 °C, zone 7 = 9 °C, and zone 8 = 8 °C and screw rotation rate = 200 rpm.

Alternatively, the crude emulsion mill base was heated using a heat exchanger consisting of a 50.0 cm length of 3 mm ID x 4 mm OD copper tubing coiled to an inside diameter of 13 mm situated in a 25 mm radius times 75 mm length cylindrical cavity about

8 mm from the end of a 225 mm long, 38 mm square beam of aluminum. The aluminum beam was heated by placing the end opposite of the cylindrical cavity on a hot plate stirrer. The extruder was modified to provide cooling of the barrel by removing port plugs from the top of the barrel and adding a copper heat exchanger consisting of a 2-inch wide by 0.75 inch thick by 12-inch4ong copper bar bolted on top of final 12 inches of the barrel (exit end). The copper block was cooled by a water / propylene glycol circulating transversely through

9 horizontal channels in zones of 3 channels. Cold coolant passed through the first three channels, returned back through the next three channels, and passed again to the side opposite of where it had first entered through the final three channels before returning to the chiller. The coolant temperature was maintained at 5 °C.

Determination of latent lamellar character by observed birefringence.

DNLF dispersion samples were tested for latent lamellar structure by heating while measuring conductivity and observing for birefringence. Approximately 10 g of a sample DNLF dispersion was added to a 30 mL beaker with a magnetic stirrer and warmed at a rate of 2 to 3 °C per minute with stirring using a hotplate stirrer. The sample was observed through two orthogonally oriented pieces of polarizing film. Latent lamellar structure was evident upon observing birefringence (i.e., a pattern of alternating darker and lighter regions or colored regions in a stirring sample) within the stirring sample when observed through the polarized film, while the sample is simultaneously transparent, isotropic and featureless when observed without polarizing film.

Lipid nanoparticle dispersions were checked for latent lamellar structure by heating on a hot plate / magnetic stirrer at a rate of about 10 °C per minute while recording temperature and conductivity. In examples where samples were too viscous be stirred, they were warmed carefully using a microwave oven in defrost mode until the viscosity was sufficiently low to allow stirring. Samples that formed transparent microemulsion when heated were checked for birefringence.

The change in electrical impedance also can be observed as a negative peak in a plot of conductivity versus temperature and a corresponding positive peak in the first derivative plot of conductivity vs temperature for heated and stirred nanoparticle dispersions when heating at a rate of between 1 and 4 °C per minute and measuring conductivity with an open cell geometry electrode such as a O13OO5MD 4-cell conductivity electrode available from ThermoScientific.

Test for oral bioavailability.

Lipid nanoparticle dispersions were tested for oral bioavailability in male Jackson Labs C57BL 6J mice by administering 10 mg/kg dasatinib by oral gavage and measuring plasma concentrations as a function of time after administration using high-performance liquid chromatography-mass spectrometry (HPLC-MS). Before administration, the concentration of dasatinib was brought to 1.25 mg/L by diluting with deionized water. For a 20 g mouse the administration volume was 160 pL. Plasma samples were collected and pooled from three mice for HPLC-MS analysis for each time point.

Examples 1-12. Preparation of extruded DNLF dispersions for comprising hydrophilic and/or amphipathic bioactive agents for topical administration.

Previous disclosures have shown the ability to incorporate hydrophobic compounds within the lipid phase of a nanolipid dispersion, in form stable and suitable for topical applications. Surprisingly, as demonstrated by at least Examples 1-12 below, stable DNLF dispersions also may be formed with appreciable concentrations of a hydrophilic or amphipathic agents. Even more surprisingly, the heating and extrusion processes employed to prepare the nanolipid dispersions are mild enough to include peptides and other small molecules that are sensitive to harsh conditions including high shear, temperature, and pH. As shown in Example 4, a dispersion comprising insulin was processed by a twin screw extruder to produce a DNLF dispersion with a loss of only 30% the initial insulin concentration. As shown, DNLF dispersions can be adapted to formulations comprising hydrophilic and/or amphipathic bioactive agents suitable for topical administration.

Example 1. Insulin DNLF for topical administration.

A crude emulsion mill base was prepared by warming a mixture of oil phase components consisting of 7.2% laureth-23, 5.5% PEG32 stearate, 3.6% sorbitan oleate, 1.5% dioleyl phosphatidyl choline (Phospholipon 90G, product of Lipoid USA), 2.0% lauric acid, 25.0% isopropyl myristate, 6.0% limonene, 3.0% medium chain triglyceride oil, and 6.5% ibuprofen and adding 0.2% sodium citrate dihydrate and 39.6% deionized water. In a separate beaker, 106.0 mg of recombinant human insulin (available from Sigma Aldrich, catalog number 91077C) was dissolved in 20.2 g of 0.1 N HC1. To the resulting clear insulin solution, 0.69 g of 10% sodium lauryl sulfate was added with stirring to give a white suspension. To the white suspension, 182.0 g of crude emulsion was added to give a crude extrudable emulsion premix. The crude extrudable emulsion premix contained 0.0475% insulin, 5.8% laureth-23, 4.5% PEG32 stearate, 2.9% sorbitan oleate, 1.2% dioleyl phosphatidyl choline, 1.7% lauric acid, 20.4% isopropyl myristate, 4.9% limonene, 2.4% medium chain triglyceride oil, 5.3% ibuprofen, 0.033% HC1, 0.2% sodium citrate dihydrate and 50.7% deionized water. The ratio of water immiscible oil to insulin was 583 to 1.

Prior to extrusion, the crude emulsion (150 g) was placed in a 250 mL pyrex beaker and heated in a microwave oven for 60 seconds to a temperature of about 65 °C. It was then heated over about 2 minutes with a handheld torch while stirring with a stick thermometer until it reached a temperature of 85 °C. The hot crude emulsion was poured into the inlet of a modified Process 11 extruder with zone temperatures zone 2 = approximately 50 °C, zone 3 = 35 °C, zone 4 = 9 °C, zone 5 = 9 °C, zone 6 = 7 °C, zone 7 = 9 °C, and zone 8 = 8 °C and screw rotation rate = 200 rpm. The processing rate was about 40 g per minute and the entire batch was extruded in less than 4 minutes to give an insulin DNLF dispersion as a translucent beige liquid with a viscosity of approximately 10,000 cSt (determined as relative to the consistency of honey). The volume and number average particle diameters and the poly dispersity index were 53.1 nm, 42.4 nm and 0.1151, respectively and the lipid concentration was 49.2%. The pH was 4.6.

A sample (23.4g) was placed in a 30 mb beaker and heated on a hot plate while monitoring conductivity and temperature. When heated to 71 °C, the sample was transparent and birefringent, indicating the DNLF dispersion has latent lamellar structure. The plot of d(normalized conduct! vity)/dT showed positive peak of magnitude 0.05 °C’¹ at 68 °C. The phase inversion onset temperature and finish temperature were 56 and 69 °C, respectively.

A sample of the DNLF dispersion (0.41 g) was diluted with 3.63 g of 4% bovine serum albumin PBS buffer and the diluted sample was diluted 1 to 100 twice using 4% bovine serum albumin PBS buffer for an overall dilution of 1 to 100,000. The concentration of insulin based on formulation data was 5.3 parts per billion by weight. The insulin concentration was measured using a human insulin ELISA kit from Crystal Chem (catalog number 90095) using a standard curve prepared from 1.0, 2.0, 3.0, 4.0 and 5.0 ppb standards prepared from the same Sigma Aldrich, catalog number 91077C human recombinant insulin. According to this measurement, the concentration of insulin was 1.2 ppb, equivalent to a recovered yield of insulin of 23%.

In a separate measurement, a sample of the DNLF dispersion (1.00 g) was diluted with 10.0 g of 10% Triton X-100 (4-tert-octylphenol ether with 9 mole poly(ethylene glycol), product of Union Carbide) to give a clear solution. The clarity of the solution compared to the hazy appearance of DNLF dispersion diluted with deionized water or with phosphate buffered saline is an indication that Triton X-100 was disrupting lipid nanoparticles and facilitating release of insulin. The DNLF plus 10% Triton X-100 solution was diluted 1 to 100 twice using 4% bovine serum albumin PBS buffer for an overall dilution of 1 to 110,000. The concentration of insulin based on formulation data was 4.7 ppb. When measured using a human insulin ELISA test as described above, the concentration of insulin was 1.1 ppb, equivalent to a recovered yield of insulin of 23%. This experiment indicates that the amount of insulin in the DNLF dispersion surviving the extrusion process was 23% and that insulin is effectively released from DNLF lipid nanoparticles simply by dilution with physiological pH 7.4 bovine serum albumin phosphate buffered saline. Example 2. Bovine Serum albumin DNLF for topical administration.

A crude emulsion was prepared by warming a mixture of oil phase components consisting of 7.2% laureth-23, 5.5% PEG32 stearate, 3.6% sorbitan oleate, 1.5% dioleyl phosphatidyl choline (Phospholipon 90G, product of Lipoid USA), 2.0% lauric acid, 25.0% isopropyl myristate, 6.0% limonene, 3.0% medium chain triglyceride oil, and 6.5% ibuprofen and adding 0.2% sodium citrate dihydrate and 39.6% deionized water. In a separate beaker, 0.50 g of bovine serum albumin (available from Sigma Aldrich, catalog number A9647) was dissolved in 20.0 g of 0.1 N HC1. To the resulting clear bovine serum albumin solution, 0.68 g of 10% sodium lauryl sulfate was added with stirring to give a white suspension. To the white suspension, 183.4 g of crude emulsion was added to give a crude extrudable emulsion premix. The crude extrudable emulsion premix contained 0.0223% bovine serum albumin, 5.8% laureth-23, 4.5% PEG32 stearate, 2.9% sorbitan oleate, 1.2% dioleyl phosphatidyl choline, 1.7% lauric acid, 20.4% isopropyl myristate, 4.9% limonene, 2.4% medium chain triglyceride oil, 5.3% ibuprofen, 0.033% HC1, 0.2% sodium citrate dihydrate and 50.4% deionized water. The ratio of water immiscible oil to bovine serum albumin was 124 to 1. The crude emulsion (150 g) was placed in a 250 mb pyrex beaker and in a microwave oven for 60 seconds to a temperature of about 65 °C. It was then heated over about 2 minutes with a handheld torch while stirring with a stick thermometer until it reached a temperature of 85 °C. The hot crude emulsion was poured into the inlet of a modified Process 11 extruder with zone temperatures zone 2 = approximately 50 °C, zone 3 = 35 °C, zone 4 = 9 °C, zone 5 = 9 °C, zone 6 = 7 °C, zone 7 = °9 C, and zone 8 = °8 C and screw rotation rate = 200 rpm. The processing rate was about 40 g per minute and the entire batch was processed in less than 4 minutes to give an insulin DNLF dispersion as a translucent beige liquid with approximately honey viscosity.

The volume and number average particle diameters and the poly dispersity index were 58.7 nm, 44.3 nm and 0.0623, respectively and the lipid concentration was 49.2%. The pH was 4.2.

A sample (22.6 g) was placed in a 30 mL beaker and heated on a hot plate while measuring conductivity and temperature. When heated to 72 °C, the sample was transparent and birefringent, and the plot of d(normalized conduct! vity)/dT showed positive peak of magnitude 0.17 °C'¹ at 70.5 °C, indicating the DNLF dispersion has latent lamellar structure. The phase inversion onset temperature and finish temperature were 64 and 73 °C, respectively.

A sample of the DNLF dispersion (1.00 g) was diluted with 10.0 g of 10% Triton X- 100 (4-tert-octylphenol ether with 9 mole poly(ethylene glycol), product of Union Carbide) to give a translucent dispersion. The diluted dispersion was diluted 1 to 100 twice using 4% bovine serum albumin PBS buffer for an overall dilution of 1 to 110,000. The insulin concentration was measured using a human insulin ELISA kit from Crystal Chem (catalog number 90095) using a standard curve prepared from 1.0, 2.0, 3.0, 4.0 and 5.0 ppb standards prepared from the same Sigma Aldrich, catalog number 91077C human recombinant insulin. According to this measurement, the concentration of insulin in the DNLF dispersion was 0.27 ppb (baseline value - empty lipid nanoparticles encapsulating bovine serum albumin).

Example 3. Cyclosporin A DNLF for topical administration.

A crude emulsion was prepared by warming a mixture of oil phase components consisting of 0.8% cyclosporin A, 6.0% laureth-23, 4.7% PEG40 stearate, 3.1% sorbitan oleate, 1.5% soy lecithin, 11.5% isopropyl myristate, 8.7% acetic acid esters of mono- and di-glycerides, 5.2% limonene, and 5.0% ibuprofen and adding 0.2% sodium chloride and 53.4% deionized water. The ratio of water immiscible oil to cyclosporin A was 33 to 1. The millbase was pumped at a rate of 12.5 mL per minute through a heat exchanger maintained at a temperature between 86 and 91 °C into the inlet of the modified Thermo Fisher Process 11 extruder with screws rotating at 300 rpm to give a translucent ivory liquid with viscosity similar to vegetable oil. The volume and number average particle diameters and the poly dispersity index were 94.4 nm, 43.1 nm and 0.396, respectively.

A sample (23.2 g) was placed in a 30 mL beaker and heated on a hot plate while measuring conductivity and temperature. The plot of d(normalized conductivity )/dT showed positive peak of magnitude 0.12 °C’¹ at 84.8 °C, indicating the DNLF dispersion has latent lamellar structure. The phase inversion onset temperature and finish temperature were 73 and 78 °C, respectively. Example 4. Nonapeptide- 1 DNLF dispersion for topical administration, comprising nonapeptide- 1, laureth-23, PEG40 stearate, sodium lauryl sulfate, sorbitan oleate, soy lecithin, lauric acid, isopropyl myristate, cold pressed orange oil, medium chain triglyceride oil, ibuprofen, and water.

A crude emulsion was prepared by warming a mixture of oil phase components consisting of 6.2% laureth-23, 4.8% PEG40 stearate, 3.1% sorbitan oleate, 5.5% soy lecithin (Alcolec XTRA-A, product of American Lecithin), 1.8% lauric acid, 14.1% isopropyl myristate, 5.2% cold pressed orange oil, 10.1% medium chain triglyceride oil, 5.7% ibuprofen, 0.8% Jeecide CAP-4, 0.2% sodium citrate dihydrate and 34.5% deionized water. In a separate beaker, 0.072% of nonapeptide-1 (available from Active Peptide Company) was dissolved in 10.8% of 0.1 N HC1. To the resulting clear nonapeptide-1 solution, 0.04% of sodium lauryl sulfate as a 30% aqueous solution was added with stirring to give a white suspension and the oil phase added to give a crude emulsion millbase. The ratio of water immiscible oil to nonapeptide-1 was 412 to 1. The crude extrudable emulsion millbase was placed in a 60 mb polyethylene syringe and heated for 7 seconds in a microwave oven to increase the temperature to 31 °C and pumped at a rate of 12.5 mL per minute through a heat exchanger maintained at a temperature between 86 and 94 °C into the inlet of the modified Thermo Fisher Process 11 extruder with screws rotating at 300 rpm to give a translucent ivory liquid with viscosity similar to honey. The volume and number average particle diameters and the poly dispersity index were 67.6 nm, 29.5 nm and 0.0979, respectively.

Example 5. Nonapeptide-1 DNLF dispersion for topical administration comprising nonapeptide-1, ceteareth-20, sorbitan oleate, soy lecithin, isopropyl myristate, niacinamide, d-panthenol, kojic acid, and water.

A crude emulsion was prepared by warming a mixture of oil phase components consisting of 8.6% ceteareth-20, 8.6% sorbitan oleate, 0.5% soy lecithin, and 25.9% isopropyl myristate. In a separate beaker, 0.06% nonapeptide - 1 was dissolved in 3.5% 0.1 N HC1 and 0.30% sodium lauryl sulfate was added, giving a white precipitate. The oil phase and the nonapeptide-1 hydrophobic ion pair suspension were mixed and 0.52% Jeecide CAP-4, 51.1% water, 0.19% niacinamide, 0.6% -panthenol, 0.5% kojic acid and 0.15% sodium chloride were added to give a crude emulsion mill base. The ratio of water immiscible oil to nonapeptide-1 was 452 to 1. The crude extrudable emulsion millbase (1000 g) was placed in a 2 L Pyrex measuring cup and heated in a microwave to 95°C and poured into the inlet of the modified Leistritz ZSE 27/GL-36D extruder with screws rotating at 200 rpm to give a translucent ivory liquid with viscosity similar to honey. The volume and number average particle diameters and the poly dispersity index were 46.7 nm, 38.0 nm and 0.1 186, respectively.

A sample (60.4 g) was placed in a 100 mL beaker was warmed gently to 40 °C in a microwave oven using the defrost setting to reduce the viscosity and subsequently heated and stirred on a hot plate while measuring conductivity and temperature. The plot of d(normalized conduct! vity)/dT showed positive peak of magnitude 0.51 °C’¹ at 89.4 °C, indicating the DNLF dispersion has latent lamellar structure. The phase inversion onset temperature and finish temperature were 84 and 92 °C, respectively.

Example 6. Copper tripeptide DNLF dispersion for topical administration comprising copper tripeptide, ceteareth-20, sorbitan oleate, soy lecithin, isopropyl myristate, niacinamide, d-panthenol, kojic acid, and water.

A crude emulsion was prepared by adding an oil phase consisting of 9.5% ceteareth- 20, 9.5% sorbitan oleate, 0.6% soy lecithin, 28.6% isopropyl myristate and 0.6% Jeecide CAP-4 to an aqueous phase consisting of 0.16% copper tripeptide, 0.17% sodium chloride and 51.0% water. The ratio of water immiscible oil to copper tripeptide was 183 to 1. The crude extrudable emulsion millbase (1000 g) was placed in a 2 L Pyrex measuring cup and heated in a microwave to 95°C and poured into the inlet of the modified Leistritz ZSE 27/GL-36D extruder with screws rotating at 200 rpm to give a translucent ivory liquid with viscosity similar to honey. The volume and number average particle diameters and the poly dispersity index were 46.9 nm, 40.2 nm and 0.0600, respectively.

A sample (56.3 g) was placed in a 100 mL beaker was warmed gently to 60 °C in a microwave oven using the defrost setting to reduce the viscosity and subsequently heated and stirred on a hot plate while measuring conductivity and temperature. The plot of d normalized conductivity)/dT showed positive peak of magnitude 0.12 °C'¹ at 87 °C, indicating the DNLF dispersion has latent lamellar structure. The phase inversion onset temperature and finish temperature were 79 and 87 °C, respectively.

Example 7. Palmitoyl tripeptide- 1 DNLF dispersion for topical administration comprising hydrolyzed collagen, palmitoyl tripeptide- 1, palmitoyl tetrapeptide-7 , spermidine, ceteareth-20, ceteareth-30, sorbitan oleate, cetyl alcohol, isopropyl myristate, medium chain triglyceride oil, mineral oil and water.

A crude emulsion was prepared by adding an oil phase consisting of 550 ppm palmitoyl tripeptide- 1, 540 ppm palmitoyl tetrapeptide-7, 900 ppm spermidine, 6.2% ceteareth-20, 3.1% ceteareth-30, 9.3% sorbitan oleate, 0.5% cetyl alcohol, 9.8% isopropyl myristate, 8.9% medium chain triglyceride oil, 9.4% light mineral oil, and 0.6% Jeecide CAP-4 to an aqueous phase consisting of 0.76% hydrolyzed collagen, 0.2% sodium chloride and 51.1% water. The ratio of water immiscible oil to hydrolyzed collagen, palmitoyl tripeptide-1, and palmitoyl tetrapeptide-7 was 32 to 1. The crude extrudable emulsion millbase (1000 g) was placed in a 2 L Pyrex measuring cup and heated in a microwave to 95°C and poured into the inlet of the modified Leistritz ZSE 27/GL-36D extruder with screws rotating at 200 rpm to give a translucent ivory liquid with viscosity similar to honey. The volume and number average particle diameters and the poly dispersity index were 55.8 nm, 43.4 nm and 0.0866, respectively.

A sample (56.6 g) was placed in a 100 mL beaker was warmed gently to 60 °C in a microwave oven using the defrost setting to reduce the viscosity and subsequently heated and stirred on a hot plate while measuring conductivity and temperature. The plot of d(normalized conductivity)/dT showed positive peak of magnitude 0.32 °C'¹ at 94 °C, indicating the DNLF dispersion has latent lamellar structure. The phase inversion onset temperature and finish temperature were 92 and 99 °C, respectively.

Example 8. DNLF dispersion for topical administration comprising undecylenoyl phenylalanine, glutathione, arbutin, nicotinamide mononucleotide, ceteareth-20, ceteareth-30, sorbitan oleate, cetyl alcohol, isopropyl myristate, medium chain triglyceride oil, mineral oil and water.

A crude emulsion was prepared by adding an oil phase consisting of 0.55% undecylenoyl phenylalanine, 5.9% ceteareth-20, 3.0% ceteareth-30, 9.4% sorbitan oleate, 0.5% cetyl alcohol, 9.4% isopropyl myristate, 8.5% medium chain triglyceride oil, 9.0% light mineral oil, and 0.5% Jeecide CAP-4 to an aqueous phase consisting of 1.1% glutathione, 0.5% arbutin, 0.5% nicotinamide mononucleotide 0.2% sodium chloride and 51.4% water. The ratio of water immiscible oil to undecylenoyl phenylalanine plus glutathione was 16 to 1. The crude extrudable emulsion millbase (1000 g) was placed in a 2 L Pyrex measuring cup and heated in a microwave to 95°C and poured into the inlet of the modified Leistritz ZSE 27/GL-36D extruder with screws rotating at 200 rpm to give a translucent ivory liquid with viscosity similar to honey. The volume and number average particle diameters and the poly dispersity index were 54.9 nm, 43.9 nm and 0.0724, respectively.

A sample (63.3 g) was placed in a 100 mb beaker was warmed gently to 50 °C in a microwave oven using the defrost setting to reduce the viscosity and subsequently heated and stirred on a hot plate while measuring conductivity and temperature. The plot of d(normalized conduct! vity)/dT showed positive peak of magnitude 1.1 °C'¹ at 90 °C, indicating the DNLF dispersion has latent lamellar structure. The phase inversion onset temperature and finish temperature were 87 and 93 °C, respectively.

Example 9. DNLF dispersion for topical administration comprising acetaminophen, ceteareth-20, sorbitan oleate, isopropyl myristate, medium chain triglyceride oil, and water.

A crude emulsion was prepared by warming a mixture of oil phase components consisting of 8.9% ceteareth-20, 13.8% sorbitan oleate, 7.9% isopropyl myristate, and 15.7% medium chain triglyceride oil to give transparent solution. To this was added 0.16% sodium chloride, 1.5% acetaminophen and 52.1% water to give a crude emulsion millbase. This is the same millbase as Example 1 except for the addition of acetaminophen. The crude extrudable emulsion millbase (1000 g) was placed in a 2 L Pyrex measuring cup and heated in a microwave to 95° C and poured into the inlet of the modified Leistritz ZSE 27/GL-36D extruder with screws rotating at 200 rpm to give a translucent ivory liquid with viscosity similar to vegetable oil. The volume and number average particle diameters and the poly dispersity index were 58.7 nm, 43.2 nm and 0.1421, respectively.

A sample (23.4g) was placed in a 30 mL beaker and heated on a hot plate while measuring conductivity and temperature. When heated to 87⁰ C, the sample was transparent and birefringent, indicating the DNLF dispersion has latent lamellar structure. The phase inversion onset temperature and finish temperature were 88 and 95 ⁰ C, respectively.

After storing at between 18 and 21 ⁰ C for 5 days, there were no crystals of acetaminophen visible in the DNLF dispersion. In a separate experiment, 1.50 g of acetaminophen was heated in 53.0 g of deionized water (this is the same composition as the extruded DNLF except omitting lipids) to give a clear solution. After standing for 3 hours at room temperature, acetaminophen formed crystals.

What this example shows is that adding acetaminophen, a hydrophilic drug can to a millbase composition of ceteareth-20, sorbitan oleate, isopropyl myristate, medium chain triglyceride oil, and water is effective to allow the millbase to be extruded to give a DNLF dispersion with volume average particle size less than 100 nm wherein the DNLF dispersion has latent lamellar structure. This example also demonstrate that the lipid nanoparticles are effective to solubilize acetaminophen in an aqueous solution relative to a solution that lacks lipid nanoparticles.

Example 10. DNLF dispersion for topical administration comprising caffeine , ceteareth- 20, sorbitan oleate, isopropyl myristate, medium chain triglyceride oil, and water.

A crude emulsion was prepared by warming a mixture of oil phase components consisting of 8.9% ceteareth-20, 13.8% sorbitan oleate, 7.9% isopropyl myristate, and 15.7% medium chain triglyceride oil to give transparent solution. To this was added 0.16% sodium chloride, 1.5% caffeine and 52.1% water to give a crude emulsion millbase. This is the same millbase as Example 1 except for the addition of caffeine. The crude extrudable emulsion millbase (1000 g) was placed in a 2 L Pyrex measuring cup and heated in a microwave to 95° C and poured into the inlet of the modified Leistritz ZSE 27/GL-36D extruder with screws rotating at 200 rpm to give a translucent ivory liquid with viscosity similar to vegetable oil. The volume and number average particle diameters and the polydispersity index were 51.2 nm, 40.3 nm and 0.0789, respectively.

A sample (25 g) was placed in a 30 mL beaker and heated on a hot plate while measuring conductivity and temperature. When heated to 87⁰ C, the sample was transparent and birefringent, and the indicating the DNLF dispersion has latent lamellar structure. The phase inversion onset temperature and finish temperature were 88 and 95 ⁰ C, respectively.

In a separate experiment, 1.50 g of caffeine was heated in 53.0 g of deionized water (this is the same composition as the extruded DNLF except omitting lipids) to give a clear solution. After standing for overnight at room temperature, caffeine crystallized as long needle shaped crystals.

What this example shows is that adding caffeine, a hydrophilic drug to a millbase composition of ceteareth-20, sorbitan oleate, isopropyl myristate, medium chain triglyceride oil, and water is effective to allow the millbase to be extruded to give a DNLF dispersion with volume average particle size less than 100 nm wherein the DNLF dispersion has latent lamellar structure. This example also demonstrate that the lipid nanoparticles are effective to solubilize caffeine in an aqueous solution relative to a solution that lacks lipid nanoparticles.

In an additional separate experiment, conductivity vs temperature was measured for a base composition consisting of 9.0% ceteareth-20, 14.0% sorbitan oleate, 15.9% medium chain triglyceride oil, 10% isopropyl myristate, 0.2% sodium chloride, and 50.9% water. Then 1.0% caffeine was added to the composition and conductivity vs temperature measured again. Plots of conductivity vs temperature are shown in Figure 1. Adding caffeine caused the phase inversion temperature to increase by 5 °C and shrunk the negative peak attributable to attenuation of conductivity by lamellar structure (weakened lamellar structure). The shifting of the phase inversion temperature and the weakening of the lamellar structure is evidence that caffeine is encapsulated in or is otherwise associated with the lipid phase (water immiscible oil plus surfactants).

What this example shows is that adding caffeine, a hydrophilic drug to a mill base composition of ceteareth-20, sorbitan oleate, isopropyl myristate, medium chain triglyceride oil, and water is effective to allow the mill base to be extruded to give a DNLF dispersion with volume average particle size less than 100 nm wherein the DNLF dispersion has latent lamellar structure. This example also demonstrate that the lipid nanoparticles are effective to solubilize caffeine in an aqueous solution relative to a solution that lacks lipid nanoparticles and the example provides additional evidence that caffeine is encapsulated in or is otherwise associated with lipids in DNLF dispersions.

Example 11. DNLF dispersion for topical administration comprising 5-fluorouracil, ceteareth-20, sorbitan oleate, isopropyl myristate, medium chain triglyceride oil, and water.

A crude emulsion was prepared by warming a mixture of oil phase components consisting of 8.7% ceteareth-20, 13.4% sorbitan oleate, 11.5% isopropyl myristate, and 15.3% medium chain triglyceride oil to give transparent solution. To this was added 0.15% sodium chloride, 0.5% 5-fluorouracil and 50.6% water to give a crude emulsion millbase. This is the same millbase as Example 1 except for the addition of 5-fluorouracil and isopropyl myristate. The crude extrudable emulsion millbase (1000 g) was placed in a 2 L Pyrex measuring cup and heated in a microwave to 95° C and poured into the inlet of the modified Leistritz ZSE 27/GL-36D extruder with screws rotating at 200 rpm to give a translucent ivory liquid with viscosity similar to vegetable oil. The volume and number average particle diameters and the polydispersity index were 53.2 nm, 38.4 nm and 0.0821 , respectively.

A sample (25g) was placed in a 30 mL beaker and heated on a hot plate while measuring conductivity and temperature. The plot of d(normalized conductivity )/dT showed positive peak of magnitude 1 ⁰ C'¹ at 88 ⁰ C, indicating the DNLF dispersion has latent lamellar structure. The phase inversion onset temperature and finish temperature were 85 and 91 ⁰ C, respectively.

What this example shows is the preparation of an extruded DNLF dispersion comprising 5-fluorouracil, ceteareth-20, sorbitan oleate, isopropyl myristate, medium chain triglyceride oil, and water with a volume average particle size less than 100 nm. Example 12. DNLF dispersion for topical administration comprising gemcitabine, ceteareth-20, sorbitan oleate, isopropyl myristate, medium chain triglyceride oil, and water.

A crude emulsion was prepared by warming a mixture of oil phase components consisting of 8.5% ceteareth-20, 13.3% sorbitan oleate, 11.5% isopropyl myristate, and 15.4% medium chain triglyceride oil to give transparent solution. To this was added 0.17% sodium chloride, 0.5% gemcitabine and 50.6% water to give a crude emulsion millbase. This is the same millbase as Example 1 except for the addition of gemcitabine and isopropyl myristate. The crude extrudable emulsion millbase (500 g) was placed in a 2 L Pyrex measuring cup and heated in a microwave to 95° C and poured into the inlet of the modified Leistritz ZSE 27/GL-36D extruder with screws rotating at 200 rpm to give a translucent ivory liquid with viscosity similar to vegetable oil. The volume and number average particle diameters and the poly dispersity index were 53.2 nm, 38.4 nm and 0.0821, respectively.

A sample (25g) was placed in a 30 mL beaker and heated on a hot plate while measuring conductivity and temperature. The plot of d(normalized conductivity )/dT showed positive peak of magnitude 1 ⁰ C'¹ at 88 ⁰ C, indicating the DNLF dispersion has latent lamellar structure. The phase inversion onset temperature and finish temperature were 85 and 91 ⁰ C, respectively.

What this example shows is the preparation of an extruded DNLF dispersion comprising gemcitabine, ceteareth-20, sorbitan oleate, isopropyl myristate, medium chain triglyceride oil, and water with a volume average particle size less than 100 nm.

Examples 13-15. Preparation of extruded DNLF dispersions for comprising hydrophilic and/or amphipathic bioactive agents for oral administration.

Surprisingly, as shown by Examples 13-15, DNLF dispersions also were able to be prepared as formulations comprising hydrophilic and/or amphipathic bioactive agents and suitable for oral administration.

Example 13. Insulin DNLF for oral administration. A crude emulsion was prepared by warming a mixture of oil phase components consisting of 12.9% polysorbate 80, 3.1% sorbitan stearate, 2.5% soy lecithin, 2.6% stearic acid, 27.8% ethyl oleate, and 11.5% medium chain triglyceride oil and adding 0.2% sodium chloride and 39.4% deionized water. In a separate beaker, 93.9 mg of recombinant human insulin (available from Sigma Aldrich, catalog number 91077C) was dissolved in 19.7 g of 0.1 N HC1. To the resulting clear insulin solution, 0.68 g of 10% sodium lauryl sulfate was added with stirring to give a white suspension. To the white suspension, 180.0 g of crude emulsion and 2.27 g of 10% sodium citrate dihydrate were added to give a crude extrudable emulsion premix. The crude extrudable emulsion premix contained 0.0463% insulin, 11.4% polysorbate 80, 0.033% sodium lauryl sulfate, 2.7% sorbitan stearate, 2.2% soy lecithin, 2.3% stearic acid, 24.6% ethyl oleate, 10.2% medium chain triglyceride oil, 0.036% HC1, 0.11% sodium citrate dihydrate, 0.16% sodium chloride and 46.2% water. The ratio of water immiscible oil to insulin was 753 to 1. The crude emulsion (150 g) in a 250 mL pyrex beaker was heated in a microwave oven for 60 seconds to a temperature of about 65 C. It was then heated over about 2 minutes with a handheld torch while stirring with a stick thermometer until it reached a temperature of 85 C. The hot crude emulsion was poured into the inlet of a modified Process 11 extruder with zone temperatures zone 2 = approximately 50 C, zone 3 = 35 C, zone 4 = 9 C, zone 5 = 9 C, zone 6 = 7 C, zone 7 = 9 C, and zone 8 = 8 C and screw rotation rate = 200 rpm. The processing rate was about 40 g per minute and the entire batch was processed in less than 4 minutes to give an insulin DNLF dispersion as a translucent beige viscous liquid.

The volume and number average particle diameters and the poly dispersity index were 68.7 nm, 54.5 nm and 0.0698, respectively and the lipid concentration was 53.5%. The pH was 4.9.

A sample (22.2 g) was placed in a 30 mL beaker and heated on a hot plate while measuring conductivity and temperature. At 66 °C and at 77 °C, the sample was transparent and birefringent, and the plot of d(normalized conductivity)/dT showed positive peak of magnitude 0.19 °C'¹ at 76.5 °C, indicating the DNLF dispersion has latent lamellar structure. The phase inversion onset temperature and finish temperature were 66 and 78 °C, respectively. A sample of the DNLF dispersion (0.41 g) was diluted with 3.60 g of 4% bovine serum albumin PBS buffer and the diluted sample was diluted 1 to 100 twice using 4% bovine serum albumin PBS buffer for an overall dilution of 1 to 100,000. The concentration of insulin based on formulation data was 4.7 parts per billion by weight. The insulin concentration was measured using a human insulin ELISA kit from Crystal Chem (catalog number 90095) using a standard curve prepared from 1.0, 2.0, 3.0, 4.0 and 5.0 ppb standards prepared from the same Sigma Aldrich, catalog number 91077C human recombinant insulin. According to this measurement, the concentration of insulin was 1.2 ppb, equivalent to a recovered yield of insulin of 26%.

In a separate measurement, a sample of the DNLF dispersion (1.00 g) was diluted with 10.0 g of 10% Triton X-100 (4-tert-octylphenol ether with 9 mole poly(ethylene glycol), product of Union Carbide) to give a translucent dispersion. The diluted dispersion was diluted 1 to 100 twice using 4% bovine serum albumin PBS buffer for an overall dilution of 1 to 110,000. The concentration of insulin based on formulation data was 4.2 ppb. When measured using a human insulin ELISA test as described above, the concentration of insulin was 1.1 ppb, equivalent to a recovered yield of insulin of 26%.

An empty extrudable emulsion premix was prepared as described above but omitting insulin. The empty extrudable emulsion premix contained 11.4% polysorbate 80, 0.031% sodium lauryl sulfate, 2.7% sorbitan stearate, 2.2% soy lecithin, 2.3% stearic acid, 24.6% ethyl oleate, 11.4% medium chain triglyceride oil, 0.033% HC1, 0.10% sodium citrate dihydrate, 0.16% sodium chloride and 45.0% water. The crude emulsion (150 g) in a 250 mL pyrex beaker was heated in a microwave oven for 60 seconds to a temperature of about 65 C. It was then heated over about 2 minutes with a handheld torch while stirring with a stick thermometer until it reached a temperature of 85 C. The hot crude emulsion was poured into the inlet of a modified Process 11 extruder with zone temperatures zone 2 = approximately 50 C, zone 3 = 35 C, zone 4 = 9 C, zone 5 = 9 C, zone 6 = 7 C, zone 7 = 9 C, and zone 8 = 8 C and screw rotation rate = 200 rpm. The processing rate was about 40 g per minute and the entire batch was processed in less than 4 minutes to give a DNLF dispersion as a translucent beige viscous liquid. The volume and number average particle diameters and the poly dispersity index were 67.6 nm, 53.7 nm and 0. 1038, respectively and the lipid concentration was 54.7%. The pH was 4.8.

A sample of the DNLF dispersion (0.39 g) was diluted with 3.58 g of 4% bovine serum albumin PBS buffer and the diluted sample was diluted 1 to 100 twice using 4% bovine serum albumin PBS buffer for an overall dilution of 1 to 100,000. The insulin concentration was measured using a human insulin ELISA kit from Crystal Chem (catalog number 90095) using a standard curve prepared from 1.0, 2.0, 3.0, 4.0 and 5.0 ppb standards prepared from the same Sigma Aldrich, catalog number 91077C human recombinant insulin. According to this measurement, the concentration of insulin was 0.26 ppb in the DNLF dispersion (baseline value - empty lipid nanoparticles).

Example 14. Insulin DNL for oral administration

A crude emulsion was prepared by warming a mixture of oil phase components consisting of 11.7% polysorbate 80, 2.8% sorbitan stearate, 2.2% soy lecithin, 2.3% stearic acid, 14.0% ethyl oleate, 10.9% isopropyl myristate, and 10.3% medium chain triglyceride oil and adding 0.16% sodium citrate dihydrate and 35.4% deionized water. The ratio of water immiscible oil to insulin was 682 to 1. In a separate beaker, 520 ppm of recombinant human insulin (available from Sigma Aldrich, catalog number 91077C) was dissolved in 10.1% g of 0.1 N HC1. To the resulting clear insulin solution, 300 ppm of sodium lauryl sulfate was added with stirring to give a white suspension. The oil phase components were added to the suspension of insulin to give a crude extrudable emulsion. The millbase was pumped at a rate of 12.5 mL per minute through a heat exchanger maintained at a temperature between 85 and 90 °C into the inlet of the modified Thermo Fisher Process 11 extruder with screws rotating at 300 rpm to give a translucent ivory liquid with viscosity similar to vegetable oil. The volume and number average particle diameters and the poly dispersity index were 77.6 nm, 55.9 nm and 0.0714, respectively. At a flow rate of 12.5 mL per minute, the residence time of the millbase in the hot heat exchanger (path length = 50.0 cm, cross sectional area = 0.071 cm², and volume = 3.5 mL) was 17 seconds. The pH of the extruded sample was 5.5. A sample of the DNLF dispersion (0.56 g) was diluted with 5.67 g of 4% bovine serum albumin PBS buffer and the diluted sample was diluted 1 to 100 twice using 4% bovine serum albumin PBS buffer for an overall dilution of 1 to 100,000. The concentration of insulin based on formulation data was 5.2 parts per billion by weight. The insulin concentration was measured using a human insulin ELISA kit from Crystal Chem (catalog number 90095) using a standard curve prepared from 1.0, 2.0, 3.0, 4.0 and 5.0 ppb standards prepared from the same Sigma Aldrich, catalog number 91077C human recombinant insulin. According to this measurement, the concentration of insulin was 3.6 ppb, equivalent to a recovered yield of insulin of 70%. In this example, heating a crude emulsion to 85 to 90 °C in 17 seconds was sufficient to form an intermediate structure capable of being processed in a twin screw extruder to produce a DNLF dispersion with a loss of only 30% the initial insulin concentration.

Example 15. Cyclosporin A DNLF for oral administration.

A crude emulsion was prepared by warming a mixture of oil phase components consisting of 0.5% cyclosporin A, 11.5% polysorbate 80, 2.7% sorbitan stearate, 2.2% soy lecithin, 2.2% stearic acid, 18.6% isopropyl myristate, 8.7% acetic acid esters of mono- and di-glycerides, and 7.4% medium chain triglyceride oil and adding 0.2% sodium chloride and 46.0% deionized water. The ratio of water immiscible oil to cyclosporin A was 68 to 1. The millbase was pumped at a rate of 12.5 mL per minute through a heat exchanger maintained at a temperature between 87 and 89 °C into the inlet of the modified Thermo Fisher Process 11 extruder with screws rotating at 300 rpm to give a translucent ivory liquid with viscosity similar to vegetable oil. The volume and number average particle diameters and the poly dispersity index were 67.1 nm, 46.4 nm and 0.0852, respectively.

Examples 16-21. Improvements to oral bioavailability of DNLF formulations.

With the understanding that DNLF dispersions comprising hydrophilic and amphipathic compounds may be suitable for oral administration (e.g., insulin, as demonstrated in Examples 13-14), the oral bioavailability of DNLFs was examined against a suspension. As shown in Examples 16-21 below, DNLFs comprising dasatinib were, quite surprisingly, able to deliver a maximum concentration of the active compound in the blood stream (Tmax) on the order of minutes, as opposed to more than an hour. A maximum dasatinib concentration in the bloodstream (Cmax) was also relatively high, attributable to the quick absorption from the GI system. Notably, Examples 20-21 show further improvement to the bioavailability by imparting a lipolysis resistance to the DNLFs.

Comparative Example 16. Oral administration of dasatinib suspension.

A suspension of dasatinib powder (1.4%, product of ChemScene, Monmouth Junction, NJ) in a solution of methyl cellulose HV (0.75%, product of Modernist Pantry, Eliot ME) and phosphate buffered saline (pH 7.4) was prepared. The suspension was diluted to 0.125% dasatinib (1.25 mg/mL) using a solution of 0.75% methyl cellulose HV in phosphate buffered saline (pH 7.4). The suspension was administered to three Jackson Labs C57BL/6J mice at a dosage of 10 mg/Kg, and blood samples withdrawn periodically. Blood samples were pooled for the three mice and combined plasma was collected by centrifugation and dasatinib quantified by HPLC-MS. Plasma concentrations vs time are shown in Table 2.

Example 17. Oral administration of a DNLF dispersion susceptible to lipolysis comprising polysorbate 80, sorbitan stearate, soy lecithin, oleic acid, isopropyl myristate, medium chain triglyceride oil, dasatinib, and water.

A crude emulsion was prepared which comprised 14.6 percent polysorbate 80, 1.5 percent sorbitan stearate, 1.8 percent soy lecithin, 5.3 percent oleic acid, 26.6 percent isopropyl myristate, 6.5 percent medium chain triglyceride oil, 0.3 percent sodium benzoate, 0.1 percent citric acid, 42.2 percent water and 1.2 percent dasatinib. The crude emulsion was processed in a Process 11 extruder which had been modified with a heat exchanger for cooling the barrel and a heat exchanger for heating the crude emulsion before introducing to the barrel to give a DNLF dispersion with volume average particle size 54.7 nm, number average particle size 35.0 nm and zeta potential 5.6 mV. In this DNLF, polysorbate 80, sorbitan stearate, isopropyl myristate, and medium chain triglyceride oil comprise ester bonds and are susceptible to lipolysis. The DNLF was diluted to 0.125% dasatinib (1.25 mg/mL) with deionized water and administered to three Jackson Labs C57BL/6J mice at a dosage of 10 mg/Kg. Blood samples were withdrawn periodically, pooled for the three mice and combined plasma was collected by centrifugation and dasatinib quantified by HPLC-MS. Plasma concentrations vs time are shown in Table 2.

Example 18. Oral administration of a DNLF dispersion susceptible to lipolysis comprising polysorbate 80, PEG40 stearate, sorbitan stearate, soy lecithin, oleic acid, isopropyl myristate, medium chain triglyceride oil, trilinolein, dasatinib, and water DNLF dispersion comprising polysorbate 80, sorbitan stearate, soy lecithin, oleic acid, isopropyl myristate, medium chain triglyceride oil, dasatinib, and water

A crude emulsion was prepared which comprised 6.7 percent polysorbate 80, 6.8 percent PEG40 stearate, 2.0 percent sorbitan stearate, 0.9 percent soy lecithin, 6.2 percent oleic acid, 21.4 percent isopropyl myristate, 7.9 percent medium chain triglyceride oil, 5.3 percent trilinolein (Maisine CC, product of Gattefosse), 0.3 percent sodium benzoate, 0.1 percent citric acid, percent water and 1.4 percent dasatinib. The crude emulsion was processed in a Process 11 extruder which had been modified with a heat exchanger for cooling the barrel and a heat exchanger for heating the crude emulsion before introducing to the barrel to give a DNLF dispersion with volume average particle size 62.1 nm, number average particle size 46.9 nm, and zeta potential -0.8 mV. In this DNLF, polysorbate 80, EG40 stearate, sorbitan stearate, isopropyl myristate, medium chain triglyceride oil, and trilinolein comprise ester bonds and are susceptible to lipolysis. The DNLF was diluted to 0.125% dasatinib (1.25 mg/mL) with deionized water and administered to three Jackson Labs C57BL/6J mice at a dosage of 10 mg/Kg. Blood samples were withdrawn periodically, pooled for the three mice and combined plasma was collected by centrifugation and dasatinib quantified by HPLC-MS. Plasma concentrations vs time are shown in Table 2.

Example 19. Oral administration of a DNLF dispersion susceptible to lipolysis comprising polysorbate 80, PEG40 stearate, sorbitan stearate, soy lecithin, oleic acid, isopropyl myristate, medium chain triglyceride oil, trilinolein, sesame seed oil, sodium benzoate, citric acid, dasatinib, and water.

A crude emulsion was prepared which comprised 6.6 percent polysorbate 80, 6.6 percent PEG40 stearate, 2.0 percent sorbitan stearate, 0.8 percent soy lecithin, 6.1 percent oleic acid, 13.7 percent isopropyl myristate, 5.9 percent medium chain triglyceride oil, 7.9 percent trilinolein (Maisine CC, product of Gattefosse), 7.9 percent sesame seed oil, 0.2 percent sodium benzoate, 0.1 percent citric acid, 40.8 percent water and 1.4 percent dasatinib.

The crude emulsion was processed in a Process 11 extruder which had been modified with a heat exchanger for cooling the barrel and a heat exchanger for heating the crude emulsion before introducing to the barrel to give a DNLF dispersion with volume average particle size 66.6 nm, number average particle size 42.5 nm and zeta potential 7.4 mV.

In this DNLF, polysorbate 80, EG40 stearate, sorbitan stearate, isopropyl myristate, medium chain triglyceride oil, trilinolein and sesame seed oil comprise ester bonds and are susceptible to lipolysis.

The DNLF was diluted to 0.125% dasatinib (1.25 mg/mL) with deionized water and administered to three Jackson Labs C57BL/6J mice at a dosage of 10 mg/Kg. Blood samples were withdrawn periodically, pooled for the three mice and combined plasma was collected by centrifugation and dasatinib quantified by HPLC-MS. Plasma concentrations vs time are shown in Table 2.

Example 20. Oral administration of a DNLF dispersion not susceptible to lipolysis comprising polysorbate 80, sorbitan stearate, soy lecithin, oleic acid, isopropyl myristate, medium chain triglyceride oil, orlistat, dasatinib, and water

A crude emulsion was prepared which comprised 14.4 percent polysorbate 80, 1.5 percent sorbitan stearate, 1.8 percent soy lecithin, 5.2 percent oleic acid, 26.4 percent isopropyl myristate, 6.4 percent medium chain triglyceride oil, 1.0 percent orlistat, 0.2 percent sodium benzoate, 0.1 percent citric acid, 41.8 percent water and 1.2 percent dasatinib. The crude emulsion was processed in a Process 11 extruder which had been modified with a heat exchanger for cooling the barrel and a heat exchanger for heating the crude emulsion before introducing to the barrel to give a DNLF dispersion with volume average particle size 110.3 nm, number average particle size 46.4 nm and zeta potential -0.1 mV.

In this DNLF, polysorbate 80, sorbitan stearate, isopropyl myristate, and medium chain triglyceride oil comprise ester bonds and are susceptible to lipolysis and orlistat is an inhibitor of lipase enzymes.

The DNLF was diluted to 0.125% dasatinib (1.25 mg/mL) with deionized water and administered to three Jackson Labs C57BL/6J mice at a dosage of 10 mg/Kg. Blood samples were withdrawn periodically, pooled for the three mice and combined plasma was collected by centrifugation and dasatinib quantified by HPLC-MS. Plasma concentrations vs time are shown in Table 2.

Example 21. Oral administration of a DNLF dispersion not susceptible to lipolysis comprising ceteareth-30, sorbitan stearat , oleic acid, isopropyl myristate, medium chain triglyceride oil, mineral oil, dasatinib, and water.

A crude emulsion was prepared which comprised 9.5 percent ceteareth-30, 3.8 percent sorbitan stearate, 8.3 percent oleic acid, 9.8 percent isopropyl myristate, 8.2 percent medium chain triglyceride oil, 8.9 percent mineral oil, 0.3 percent sodium benzoate, 0.1 percent citric acid, 49.7 percent water and 1.5 percent dasatinib. The crude emulsion was processed in a Process 11 extruder which had been modified with a heat exchanger for cooling the barrel and a heat exchanger for heating the crude emulsion before introducing to the barrel to give a DNLF dispersion with volume average particle size 66.0 nm, number average particle size 47.1 nm, and zeta potential 3.9 mV.

In this DNLF, sorbitan stearate, isopropyl myristate, and medium chain triglyceride oil comprise ester bonds and are susceptible to lipolysis and ceteareth-30 and mineral oil have no ester bonds and are not susceptible to lipolysis.

The DNLF was diluted to 0.125% dasatinib (1.25 mg/mL) with deionized water and administered to three Jackson Labs C57BL/6J mice at a dosage of 10 mg/Kg. Blood samples were withdrawn periodically, pooled for the three mice and combined plasma was collected by centrifugation and dasatinib quantified by HPLC-MS. Plasma concentrations vs time, and AUC4, are shown in Table 2. Local maxima in plots of concentration vs time are indicated by underlining.

Table 2. Plasma concentrations vs time

Discussion of Examples 16-21

Three dasatinib DNLFs based on lipolysis susceptible polysorbate 80 were prepared and tested for oral bioavailability (Examples 2 - 4) and compared to dasatinib suspension (Example 1). The DNLFs showed enhanced absorption compared to the suspended powder (Figure 2). They also showed a local maximum in the dasatinib plasma concentration vs time curve occurring at the earliest sampling time point in addition to a local maximum at 2 hours. The early local maximum is attributed to rapid absorption of already formed nanoemulsion droplets and the peak at 2 hours is attributed to absorption of micelles formed from bile salts and the lipolysis degradation products of the nanoemulsion droplets.

The conclusion that the second local maximum in the plasma concentration vs time curve is attributable to micellization and absorption of lipolytic degradation products of lipid nanoemulsion droplets is supported by the observation that including orlistat (a lipase inhibitor) in the DNLF causes the second peak to disappear (Example 20 in Figure 3). The truly remarkable feature of this plot is the monotonically decreasing plasma concentration. While it is not unusual and actually quite typical to find improvements of up to 4 times and even greater for AUCs in academic literature, the observation of Cmax at the first time point is never seen for oral dosage forms. In fact, it is a characteristic of intravenously administered drugs. The AUC was not improved compared to DNLFs without orlistat because the diameter of the nanoemulsion droplets doubled due to including orlistat.

Attributing the second local maximum to micellization and absorption of lipolytic degradation products is further supported by the observation that replacing lipolysis susceptible polysorbate 80 with ceteareth-30 (not susceptible to lipolysis) also causes the second peak to disappear (Example 21 in Figure 4). In this case the AUC was greatly improved compared to DNLFs with polysorbate 80 in the absence of orlistat because drug is not lost to precipitation when lipids and surfactant hydrolyze.

Examples 22-24. DNLF dispersions maintain particle size distributions under lipolytic conditions.

The performance of DNLFs under lipolytic conditions was also examined and shown that DNLFs formulated as disclosed herein may retain characteristics (e.g., particle size less than 100 nm) for a significant amount of time. Given the time to traverse the portions of the GI tract with lipolytic conditions is on the scale of 1 hour, the unexpected ability of DNLFs to largely retain a particle size suitable for absorption beyond this time frame suggests the DNLFs of Examples 1-15 may be able to be administered in certain aspects without further considerations to lipase inhibitors, encapsulation, and the like.

Comparative Example 22. Preparation of a digestible DNLF dispersion comprising polysorbate 80, sorbitan stearate, soy lecithin, isopropyl myristate, medium chain triglyceride oil, glycerin, sodium chloride, and water.

A DNLF dispersion was prepared consisting of 14.6% polysorbate 80, 7.2% sorbitan stearate, 0.9% soy lecithin, 25.5% isopropyl myristate, 10.9% medium chain triglyceride oil, 2.3% glycerin, 0.17% sodium chloride, and 40.2% water. The dispersion was a hazy transparent light yellow liquid with viscosity similar to light syrup. The volume and number average particle diameters and the poly dispersity index were 70.5 nm, 57.5 nm and 0.0733, respectively. The sample has 1.86 mmol of ester bonds per gram. A pH stat experiment for susceptibility to ester bond hydrolysis by lipase was done using 1.52 g of DNLF dispersion in 60.06 g of lipolysis media at 40 °C. The experiment was started by adding 0.25 g of Carolina Biologicals Laboratory Grade Lipase (catalog number 872500). The particle size distribution was measured at the start of the experiment and after one hour using a NanoFlex dynamic light scattering (DLS) instrument (Microtrac Instruments, York PA). The particle size distributions are shown in Figure 5 and the cumulative volume percent of particles with diameters less than (% passing through a screen of diameter) vs diameter is shown in Figure 6. Carolina Biologicals Laboratory Grade Lipase is useful for the determination of particle size vs time using DLS because it dissolves in water to give a clear, homogeneous solution which does not interfere with the particle size measurement.

What this example shows is that after 60 minutes of lipolysis using Carolina Biologicals Laboratory Grade Lipase, none of the particles have a diameter less than 100 nm.

Example 23. Preparation of a DNLF dispersion in which greater than 80 volume % of nanoparticles have diameter less than lOOnm after 1 hour of lipolysis comprising ceteareth-20, sorbitan stearate, isopropyl myristate, medium chain triglyceride oil, mineral oil, quinine, sodium chloride, and water.

A DNLF dispersion was prepared consisting of 9.9% ceteareth-20, 9.9% sorbitan stearate, 9.8% isopropyl myristate, 9.8% medium chain triglyceride oil, 9.9% mineral oil, 1.1% quinine, 0.2% sodium chloride, and 49.3% water. The sample has 1.17 mmol of ester bonds per gram.

A pH stat experiment for susceptibility to ester bond hydrolysis by lipase was done using 1.83 g of DNLF dispersion in 60.11 g of lipolysis media at 40 °C. Once the pH was stable at 6.80 the experiment was started by adding 0.24 g of Carolina Biologicals Laboratory Grade Lipase (catalog number 872500) was added and pH logged. The pH dropped to 6.90 upon addition of lipase, and then remained constant at 6.90 for 40 minutes. Plots of the pH and % ester bond hydrolysis as a function of time are shown in Figure 7. The particle size distribution was measured at the start of the experiment and after one hour using a NanoFlex dynamic light scattering (DLS) instrument (Microtrac Instruments, York PA). The particle size distributions are shown in Figure 8 and the cumulative volume percent of particles with diameters less than (% passing through a screen of diameter) vs diameter is shown in Figure 9.

What this example shows is that after 60 minutes of lipolysis using Carolina Biologicals Laboratory Grade Lipase, 84 volume % of the particles have a diameter less than 100 nm even though greater than 20% of the ester groups had been hydrolytically cleaved by lipase.

Example 24. Preparation of a DNLF dispersion with zwitterionic particles comprising ceteareth-20, sorbitan stearate, medium chain triglyceride oil, isopropyl myristate, mineral oil, cocamidopropyl betaine, palmitoyl tripeptide 1, palmitoyl tetrapeptide 7, sodium chloride, and water.

A DNLF dispersion was prepared consisting of 9.8% ceteareth-20, 9.7% sorbitan stearate, 9.7% medium chain triglyceride oil, 9.8% isopropyl myristate, 10.0% mineral oil, 1.5% cocamidopropyl betaine, 0.13% palmitoyl tripeptide 1, 0.12% palmitoyl tetrapeptide 7, 0.19% sodium chloride, and 49.1% water. The DNLF dispersion had volume average particle size 54.7 nm and number average particle size 37.1 nm.

Examples 25-27. Preparation of encapsulated DNLF dispersions.

In addition to improvements resulting from lipolysis-resistance and maintenance of particle size distribution, it is contemplated herein that DNLFs comprising bioactive agents may be formulated within a capsule for delivery to the particular segment of the GI tract where conditions for absorption without damaging the bioactive agents are most favorable. For instance, delivering the DNLFs to the small intestine by a capsule may avoid exposure of the DNLFs to digestion by lipase present elsewhere in the GI tract. Examples 25-27 provide encapsulated DNLFs in stable form for storage and oral administration. Example 25. Encapsulation of an oral DNLF dispersion comprising polysorbate 80, sorbitan stearate, soy lecithin, isopropyl myristate, light mineral oil, clove essential oil, and water in a capsule comprised ofEudragit FL 30 D-55 polymer.

A DNLF dispersion was prepared which comprised 14.7 % polysorbate 80, 5.2 % sorbitan stearate, 0.7 % soy lecithin, 26.0 % isopropyl myristate, 11.2% light mineral oil, 0.3% clove essential oil, 0.1% sodium benzoate, 0.3% sodium citrate dihydrate, 0.4% citric acid, 2.3 % glycerin, and 39.0% water. The volume and number average particle diameters and the poly dispersity index were 58.7 nm, 44.7 nm and 0.0766, respectively and the lipid concentration was 58.0%. The pH was 4.1.

A film of Eudragit FL 30 D-55 polymer was prepared by drying a puddle on a silicone baking sheet. The areal weight of film was 0.048 g per cm². The film was wrapped around a rod with diameter 7 mm and heat sealed to give a tube weighing 0.89 g. The tube was heat sealed on one end, partially filled with 1.46 g of the DNLF dispersion and heat sealed on the other tube end. After storing at room temperature for 4 days, the weight of the capsule and contents decreased from 2.36 g to 2.16 g and the capsule remained intact and firm, and not noticeably softer than before filling. The capsule was cut open and the DNLF contents squeezed out, leaving an empty capsule weighing 0.95 g (6 % increase) and contents weighing 1.21 g (17% decrease). The volume and number average particle diameters and the poly dispersity index of the stored DNLF dispersion were 62.4 nm, 50.1 nm and 0.0549, respectively. What this example shows is that when a DNLF dispersion with pH = 4.1 is stored in a capsule of Eudragit FL 30 D-55, both the DNLF dispersion and the capsule are stable and that about 35% of the water initially present in the DNLF dispersion was lost to evaporation. In a separate experiment, when two similar capsules were stored at 40 °C for four days, they became stuck together, indicating that filled Eudragit FL 30 D-55 capsules become tacky under such storage conditions.

Example 26. Encapsulation of an oral DNLF dispersion comprising polysorbate 80, sorbitan stearate, soy lecithin, isopropyl myristate, light mineral oil, clove essential oil, and water in a capsule comprised ofEudragit FL 30 D-55 polymer with an exterior surface layer comprising carnauba wax, dimethicone and organo-modified silicone. A polymer film was prepared by drying a puddle of Eudragit FL 30 D-55 polymer weighing 7.5 g contained in a 6 cm by 8 cm rectangular silicone soap mold. The areal weight of film was 0.047 g per cm². While still in the mold, the film was sprayed with 0.20 g of Megiuar brand Gold Class Carnauba Plus premium quick wax spray (available from Meguiar, Irvine CA). The as applied beaded spray wet film was manually smoothed to give a continuous film and dried at 50 C for 18 hours. The film was wrapped with the coated side outward around a rod with diameter 7 mm and heat sealed to give a tube weighing 0.99 g. The tube was heat sealed on one end, partially filled with 1.90 g of the DNLF dispersion from Example and heat sealed on the other tube end. The capsule plus a second similar capsule were stored at 40 °C for two days. The capsules remained intact and firm, and not noticeably softer than before filling and were very slightly stuck together and easily separated. What this example shows is that Eudragit FL 30 D-55 capsules that are coated with carnauba wax, dimethicone and organo-modified silicone do not become tacky when filled with DNLF dispersion with pH 4.1 and stored at 40 °C.

Example 27. Encapsulation of an oral DNLF dispersion comprising polysorbate 80, sorbitan stearate, soy lecithin, isopropyl myristate, light mineral oil, clove essential oil, and water in a capsule comprised of Eudragit FL 30 D-55 polymer with an exterior surface layer comprising carnauba wax.

A polymer film was prepared by drying a puddle of Eudragit FL 30 D-55 polymer weighing 5.0 g contained in a 6 cm by 8 cm rectangular silicone soap mold. The areal weight of film was 0.032 g per cm². While still in the mold, 0.5 g of a 1.0 weight percent solution of carnauba wax (food grade flakes, available from Better Shea Butter Company, Cedar Park TX) in trichloroethylene was spread on top of the film and dried at 50 °C for 4 hours. The film was wrapped with the coated side outward around a rod with diameter 7 mm and heat sealed to give a tube weighing 0.55 g. The tube was heat sealed on one end, partially filled with 1.08 g of the DNLF dispersion from Example and heat sealed on the other tube end. The capsule plus a second similar capsule were stored at 40 °C overnight whereupon the weight of the capsule and contents decreased from 1.63 g to 1.45 g. The capsules remained intact and firm, and not noticeably softer than before filling and were not stuck together. When placed into 500 mL of slowly stirring PBS 7.4 buffer at 40 °C, the capsule became opaque in 5 minutes and after 90 minutes had the consistency of melted cheese. Very gentle squeezing caused it to rupture. What this example shows is that Eudragit FL 30 D-55 capsules that are coated with carnauba wax do not become tacky when filled with DNLF dispersion with pH 4.1 and stored at 40 °C, but becomes very soft and physically weak when stirred in 90 minutes in pH 4 buffer at 40 °C.

Examples 28-30. Preparation of extruded DNLF dispersions comprising insulin for oral administration with high bioavailability.

Given the prior Examples, it is contemplated herein that bioactive compounds previously unavailable by oral administration may be provided in an oral formulation with high bioavailability. A formulation for the oral administration of insulin has long been sought without success. Examples 28-29 present a lipolysis resistant DNLF comprising insulin that, according to the results of Examples 20-21, is able to deliver insulin through an oral administration route to the blood stream with acceptable Tmax and Cmax similar to less desirable alternative administration pathways (e.g., injection).

Example 28. Insulin. Preparation of an oral DNLF dispersion which is not susceptible to degradation by lipolysis comprising insulin, polysorbate 80, sodium lauryl sulfate, sorbitan stearate, soy lecithin, stearic acid, ethyl oleate, isopropyl myristate, medium chain triglyceride oil, orlistat, and water.

A DNFL dispersion was prepared comprising an oil phase consisting of 540 ppm insulin as a hydrophobic ion pair with 310 ppm sodium lauryl sulfate, 11.7% polysorbate 80, 2.8% sorbitan stearate, 2.1% soy lecithin, 2.3% stearic acid, 13.9% ethyl oleate, 10.7% isopropyl myristate, 10.2% medium chain triglyceride oil, plus 0.23% orlistat as a lipase inhibitor and an aqueous phase consisting or 0.2% sodium citrate dihydrate, 410 ppm HC1, and 45.7% water as a hazy transparent yellow liquid with viscosity similar to light syrup. The volume and number average particle diameters and the polydispersity index were 61.7 nm, 31.7 nm and 0.0652, respectively and the pH was 4.93. The sample has 1.66 mmol of ester bonds per gram. This is the same formulation as Example 14 except it includes orlistat. A pH stat experiment for susceptibility to ester bond hydrolysis by lipase was done using 1.61 g of DNLF dispersion in 59.68 g of lipolysis media at 40 °C. Once the pH value was stable at 6.93, 0.28 g of Sigma-Aldrich catalog number L-3126 pancreatic porcine lipase (>125 units/mg protein using olive oil and a 30-minute incubation) was added as a solid giving a lipolysis mixture with >560 units/mL and pH logged. The pH dropped to 6.90 upon addition of lipase, and then remained stable at 6.90 for 40 minutes. No NaOH was required to be added to neutralize acid produced by lipolysis. By comparison, 35% of the ester bonds were hydrolyzed in 25 minutes for the same DNLF dispersion without orlistat. Plots of pH vs time and % hydrolysis vs time are shown in Figures 10 and 11, respectively. What this example shows is that a lipid nanoparticle comprising insulin, polysorbate 80, sodium lauryl sulfate, sorbitan stearate, soy lecithin, stearic acid, ethyl oleate, isopropyl myristate, medium chain triglyceride oil and orlistat is not degraded by pancreatic lipase at pH 6.93.

Example 29. Insulin. Preparation of an indigestible oral DNLF dispersion with lipid nanoparticles comprising insulin, polysorbate 80, PEG 100 stearate, sodium lauryl sulfate, sorbitan stearate, soy lecithin, stearic acid, isopropyl myristate, mineral oil, sodium citrate dihydrate, and water.

A DNFL dispersion was comprising an oil phase consisting of 485 ppm insulin as a hydrophobic ion pair with 360 ppm sodium lauryl sulfate, 11.5% polysorbate 80, 3.3% PEG 100 stearate, 3.9% sorbitan stearate, 0.4% soy lecithin, 7.2% stearic acid, 28.4% isopropyl myristate, 3.7% light mineral oil, and an aqueous phase consisting or 0.2% sodium citrate dihydrate, 329 ppm HC1, and 41.4% water as a hazy transparent light yellow liquid with viscosity similar to honey. The volume and number average particle diameters and the poly dispersity index were 87.3 nm, 63.3 nm and 0.0834. The sample has 1.25 mmol of ester bonds per gram.

A pH stat experiment for susceptibility to ester bond hydrolysis by lipase was done using 1.58 g of DNLF dispersion in 59.68 g of lipolysis media at 40 °C. Once the pH value was stable at 6.71, 0.26 g of Sigma-Aldrich catalog number L-3126 pancreatic porcine lipase was added as a solid giving a lipolysis mixture with >540 units/mL and pH logged. The pH rose to 6.74 upon addition of lipase, and then slowly increased further to 6.78 in 13 minutes where it remained for the duration of the 60-minute experiment. No NaOH was required to be added to neutralize acid produced by lipolysis. What this example shows is that a lipid nanoparticle comprising insulin, polysorbate 80, PEG 100 stearate, sodium lauryl sulfate, sorbitan stearate, soy lecithin, stearic acid, isopropyl myristate, mineral oil is not degraded by pancreatic lipase at pH 6.78.

Example 30. Cyclosporin A. Preparation of an indigestible oral DNLF dispersion with cationic lipid nanoparticles comprising cyclosporin A, polysorbate 80, sorbitan stearate, soy lecithin, acetic acid esters of mono- and di-glycerides, orlistat, dimethyl lauryl amine, sodium chloride, and water.

A DNLF dispersion was prepared consisting of 0.43% cyclosporin A, 12.0% polysorbate 80, 3.1% sorbitan stearate, 2.2% soy lecithin, 4.0% dimethyl lauryl amine, 9.1% acetic acid esters of mono- and di-glycerides (Kerry Myvacet 9-45K), 20.3% isopropyl myristate, 0.25% sodium chloride, 0.12% orlistat, and 48.5% water. The dispersion was a hazy transparent yellow liquid with viscosity similar to light syrup. The volume and number average particle diameters and the poly dispersity index were 59.5 nm, 43.3 nm and 0.0703, respectively and the pH was 8.59. The sample has 1.61 mmol of ester bonds per gram.

A pH stat experiment for susceptibility to ester bond hydrolysis by lipase was done using 1.59 g of DNLF dispersion in 59.42 g of lipolysis media at 40 °C. Once the pH value was stable at 6.93, 0.28 g of Sigma-Aldrich catalog number L-3126 pancreatic porcine lipase was added as a solid giving a lipolysis mixture with >560 units/mL and pH logged. The pH dropped to 6.90 upon addition of lipase, and then remained constant at 6.90 for 40 minutes. No NaOH was required to be added to neutralize acid produced by lipolysis. A plot of pH and % hydrolysis vs time is shown in Figure 11 .

The zeta potential of the sample was measured using a Malvern Zetasizer. DNLF dispersion (0.82 g) was dispersed in 17.9 g of 0.1 M potassium dihydrogen phosphate / dipotassium hydrogen phosphate buffer. The zeta potential was + 42.4 at pH 6.4.

What this sample shows is preparation of a cationic, small particle size DNLF dispersion which is stable to degradation by pancreatic lipase. ASPECTS

Aspect 1. A nanoparticle dispersion comprising; from 0.01 wt. % to 20 wt. % of one or more bioactive agents; from 0.1 wt.% to 20 wt.% of one or more high hydrophile-lipophile-balance (HLB) surfactants; from 0.1 wt.% to 20 wt.% of one or more low hydrophile-lipophile-balance (HLB) surfactants; from 10 wt.% to 45 wt.% of one or more water immiscible oils; and from 25 wt.% to 75 wt.% water; wherein the bioactive agent is hydrophilic or amphipathic (e.g., the bioactive agent is not hydrophobic, and/or the bioactive agent has a logP less than 1).

Aspect 2. A nanoparticle dispersion comprising: from 0.01 wt. % to 20 wt. % of one or more bioactive agents; from 0.1 wt.% to 20 wt.% of one or more high hydrophile-lipophile-balance (HLB) surfactants; from 0.1 wt.% to 20 wt.% of one or more low hydrophile-lipophile-balance (HLB) surfactants; from 10 wt.% to 45 wt.% of one or more water immiscible oils; and from 25 wt.% to 75 wt.% water; wherein the lipophilic phase (e.g., surfactants, water immiscible oils, hydrophobic therapeutic agents, etc.) of the nanoparticle dispersion is lipolysis-resistant (e.g., less than 25% of ester bonds hydrolyze within one hour in the presence of 500 units /mb porcine pancreatic lipase; alternatively, indigestible).

Aspect 3. The nanoparticle dispersion of aspect 1 or 2, wherein: the one or more high hydrophile-lipophile-balance (HLB) surfactants comprises an ester type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactant; and the one or more low hydrophile-lipophile-balance (HLB) surfactants comprises a phospholipid low hydrophile-lipophile-balance (HLB) surfactant. Aspect 4. The nanoparticle dispersion of any one of aspects 1-3, wherein: the ester type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactant is selected from the group consisting of PEG100 stearate, PEG20 stearate, PEG30 glyceryl cocoate, PEG32 stearate, polysorbate 20, and polysorbate 80; and the phospholipid low hydrophile-lipophile-balance (HLB) surfactant is selected from the group consisting of phosphatidylcholine and lecithin.

Aspect 5. The nanoparticle dispersion of any one of aspects 1-4, wherein: the one or more high hydrophile-lipophile-balance (HLB) surfactants comprises one or more ether type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactants.

Aspect 6. The nanoparticle dispersion of aspect 5, further comprising one or more ester type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactants (e.g., an ester type polyethoxylated surfactant comprising at least 40 ethoxylate groups per molecule).

Aspect 7. The nanoparticle dispersion of aspect 6, wherein a weight ratio of the ether type poly ethoxylated high hydrophile-lipophile-balance HLB surfactant to the ester type polyethoxylated high hydrophile-lipophile-balance HLB surfactant is greater than 1 :1.

Aspect 8. The nanoparticle dispersion of aspect 6, wherein: the ether type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactant is selected from the group consisting of laureth-23, laureth-30, steareth-100, steareth-20, steareth-40, ceteareth-20, and ceteareth-30; and the ester type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactant is selected from the group consisting of PEG100 stearate, PEG20 stearate, PEG30 glyceryl cocoate, PEG32 stearate, and polysorbate 20. Aspect 9. The nanoparticle dispersion of any one of aspects 1-4, further comprising one or more non -poly ethoxylated high hydrophile-lipophile-balance (HLB) surfactants.

Aspect 10. The nanoparticle dispersion of aspect 9, wherein the nonpolyethoxylated high hydrophile-lipophile-balance (HLB) surfactant is sodium laurel sulfate.

Aspect 11. The nanoparticle dispersion of any one of aspects 1-10, wherein the bioactive agent is a skin care agent.

Aspect 12. The nanoparticle dispersion of any one of aspects 1-11, wherein the bioactive agent is a tyrosinase inhibitor.

Aspect 13. The nanoparticle dispersion of any one of aspects 1-12, wherein the bioactive agent is a peptide.

Aspect 14. The nanoparticle dispersion of aspect 13, wherein the peptide has a molar mass in a range from 1 kDa to 1,000 kDa.

Aspect 15. The nanoparticle dispersion of aspect 13 or 14, wherein the peptide is a signaling peptide, an AApeptide, a hormone or derivative thereof.

Aspect 16. The nanoparticle dispersion of aspect 13, wherein the peptide is insulin.

Aspect 17. The nanoparticle dispersion of aspect 13, wherein the peptide is bovine serum albumin. Aspect 18. The nanoparticle dispersion of any one of aspects 1-12, wherein the bioactive agent is a hydrophilic bioactive agent.

Aspect 19. The nanoparticle dispersion of aspect 18, wherein the hydrophilic bioactive agent is a nucleotide or nucleic acid.

Aspect 20. The nanoparticle dispersion of aspect 18 or 19, wherein the hydrophilic bioactive agent has a MW less than 1,000 g/mol.

Aspect 21. The nanoparticle dispersion of aspect 18, wherein the hydrophilic bioactive agent is selected from the group consisting of spermadine, caffeine, acetominophen, and niacinamide.

Aspect 22. The nanoparticle dispersion of any one of aspects 1-21, comprising a latent lamellar structure characterized by the nanoparticle dispersion adopting a lamellar structure upon heating to a temperature in a range from 40 °C to 95 °C.

Aspect 23. The nanoparticle dispersion of any one of aspects 1-22, comprising a latent lamellar structure characterized by the dispersion exhibiting a positive peak between about 45 °C and 80 °C in the first derivative plot of normalized conductivity versus temperature, the positive peak having a peak amplitude greater than about 0.1 °C'¹.

Aspect 24. The nanoparticle dispersion of any one of aspects 1-23, comprising a latent lamellar structure characterized by the dispersion exhibiting an optical birefringence when observing the dispersion through cross polarized films upon heating to temperatures in a range from 60 °C to 95 °C.

Aspect 25. The nanoparticle dispersion of any one of aspects 1-24, wherein the bioactive agent does not form crystals within the nanoparticle dispersion (e.g., for greater than about 1 month, greater than about 2 months, greater than about 3 months, greater than about 8 months) at a temperature from about 18 °C to about 22 °C.

Aspect 26. The nanoparticle dispersion of any one of aspects 1-25, wherein: the nanoparticle dispersion is free from crystals and the concentration of the bioactive agent is greater than the solubility limit in the aqueous phase of the dispersion outside the context of the nanoparticle; and the bioactive agent has a logP less than 1 .

Aspect 27. The nanoparticle dispersion of any one of aspects 1-26, further comprising a structure promoting additive.

Aspect 28. The nanoparticle dispersion of any one of aspects 1-27, wherein the nanoparticle dispersion has a pH less than an isoelectric point of the peptide.

Aspect 29. The nanoparticle dispersion of any one of aspects 1-28, comprising a zwitterionic nanoparticle.

Aspect 30. The nanoparticle dispersion of any one of aspects 1-29, wherein the bioactive agent is present as a component of a hydrophobic ion pair, the hydrophobic ion pair further comprising an anionic surfactant.

Aspect 31. The nanoparticle dispersion of any one of aspects 1-30, wherein a volume average particle diameter of the dispersion is less than 80 nm (e.g., in a range from 30 nm to 80 nm).

Aspect 32. The nanoparticle dispersion of any one of aspects 1-31, wherein the lipophilic phase is greater than 25 weight percent of the nanoparticle dispersion. Aspect 33. The nanoparticle dispersion of aspect 2, wherein the high HLB surfactant is inert to lipolysis (e.g., indigestible).

Aspect 34. The nanoparticle dispersion of aspect 33, wherein the high HLB surfactant lacks an ester group.

Aspect 35. The nanoparticle dispersion of aspect 32 or 33, wherein the high HLB surfactant is an ether-type high HLB polyethoxylated surfactant.

Aspect 36. The nanoparticle dispersion of any one of aspects 1-35, further comprising a lipase inhibitor.

Aspect 37. The nanoparticle dispersion of aspect 36, wherein the lipase inhibitor comprises orlistat.

Aspect 38. The nanoparticle dispersion of any one of aspects 1-37, wherein the nanoparticles within the nanoparticle dispersion have a net positive charge.

Aspect 39. The nanoparticle dispersion of any one of aspects 1-38, wherein the nanoparticles within the nanoparticle dispersion comprise a zeta potential greater than 1.0 millivolt.

Aspect 40. The nanoparticle dispersion of any one of aspects 2 and 33-39, wherein oral administration of the nanoparticle dispersion to a Jackson Labs C57BL/6J mouse results in a peak plasma concentration at less than one hour after administration.

Aspect 41. The nanoparticle dispersion of aspect 40, wherein a plot of plasma concentration vs time in which d(plasma concentration)/dt is negative for each time after administration by oral gavage to a Jackson Labs C57BL/6J mouse greater than one hour (e.g., from 1 to 24 hours). Aspect 42. The nanoparticle dispersion of any one of aspects 1-41, wherein greater than 80% of particles in the DNLF on a volume basis have a diameter less than 100 nm after 60 minutes in a lipolysis solution with 2.6% DNLF dispersion, pH about 6.8 containing calcium, one or more bile salts, and 0.4% lipase.

Aspect 43. The nanoparticle dispersion of any one of aspects 1-42, wherein greater than 80% of particles on a volume basis have diameter less than 100 nm after greater than 30% of lipid nanoparticle ester bonds have been hydrolyzed by lipase.

Aspect 44. The nanoparticle dispersion of any one of aspects 1-43, wherein less than 10% of the lipid ester bonds are lipolyzed after 60 minutes in a lipolysis solution with pH about 6.8 containing calcium, one or more bile salts, and 0.4% lipase.

Aspect 45. The nanoparticle dispersion of any one of aspects 1-44, wherein in the gastrointestinal tract a rate of drug absorbance is greater than a rate of drug release from the lipid phase of the nanoparticle dispersion.

Aspect 46. The nanoparticle dispersion of any one of aspects 1-45, wherein in the gastrointestinal tract a rate of drug absorbance is greater than a rate of ester bond hydrolysis in the lipid phase of the nanoparticle dispersion.

Aspect 47. A capsule comprising the nanoparticle dispersion of any one of aspects 1-46.

Aspect 48. The capsule of aspect 47, the capsule comprising an encapsulating polymer surrounding the nanoparticle dispersion.

Aspect 49. The capsule of aspect 48, wherein the encapsulating polymer comprises a carboxylic acid. Aspect 50. The capsule of aspect 48 or 49, wherein a pH of the nanoparticle dispersion is less than (e.g., at least one pH unit less than) a pKa of the encapsulating polymer carboxylic acid group.

Aspect 51. The capsule of any one of aspects 48-50, wherein the encapsulating polymer comprises a plurality of acrylate monomer units, a plurality of methacrylate monomer units, a plurality of methacrylate monomer units, a plurality of vinyl acetate monomer units, a plurality of acrylic acid monomer units, a plurality of methacrylic acid monomer units, a plurality of vinyl 4-hydroxyl phthalate monomer units, a vinyl polymer, modified cellulose, or combinations thereof.

Aspect 52. The capsule of any one of aspects 48-51, wherein the encapsulating polymer comprises modified cellulose, the modified cellulose comprising cellulose esterified with acetic acid and/or phthalic acid, or cellulose esterified with acetic acid and/or succinic acid.

Aspect 53. The capsule of any one of aspects 48-52, wherein the encapsulating polymer is selected from the group consisting of a methyl acrylate - methacrylic acid copolymer, a methyl methacrylate - methacrylic acid copolymer, a ethyl acrylate - methacrylic acid copolymer, a vinyl acetate - vinyl 4-hydroxyl phthalate copolymer, a hydroxypropyl methyl cellulose acetate succinate, a cellulose acetate phthalate, Eudragit L 30 D-55, or combinations thereof.

Aspect 54. The capsule of any one of aspects 48-53, wherein the capsule further comprises a surface layer external to the encapsulating polymer.

Aspect 55. The capsule of aspect 48-54, wherein the surface layer comprises carnauba wax, dimethicone, organo-modified silicone or any combination thereof. Aspect 56. The capsule of any one of aspects 48-55, wherein the capsule further comprises an intermediate layer between the encapsulating polymer and the surface layer.

Aspect 57. The capsule of aspect 56, wherein the capsule comprises an innermost layer consisting of the encapsulating polymer, an intermediate layer comprising a gelatin, and a surface layer comprising a carnauba wax.

Aspect 58. The capsule of any one of aspects 47-57, wherein the capsule is not digested within 30 minutes by stomach acid (e.g.,) at pH less than 2.

Aspect 59. A method for treating a disorder selected from the group consisting of diabetes mellitus type-1 and diabetes mellitus type-2 comprising: the oral administration of insulin, the method comprising orally administering to a patient in need thereof: a nanoparticle dispersion of any one of aspects 1-46; or a capsule of any one of aspects 47-58.

Aspect 60. The method of aspect 59, wherein oral administration of the nanoparticle dispersion results in a peak plasma concentration in the patient at less than one hour (or from 10 minutes to 1 hour, or less than 30 minutes, or less than 15 minutes, or from 5 to 30 minutes) after administering the nanoparticle or capsule to the patient.

Aspect 61. The method of aspect 59 or 60, wherein a plot of plasma concentration vs time in which d(plasma concentration)/dt is negative at each time point after administering the nanoparticle or capsule greater than one hour (e.g., from 1 to 24 hours).

Claims

CLAIMS I claim:

1. A nanoparticle dispersion comprising; from 0.01 wt. % to 20 wt. % of one or more bioactive agents; from 0.1 wt.% to 20 wt.% of one or more high hydrophile-lipophile-balance (HLB) surfactants; from 0.1 wt.% to 20wt.% of one or more low hydrophile-lipophile-balance (HLB) surfactants; from 10 wt.% to 45 wt.% of one or more water immiscible oils; and from 25 wt.% to 75 wt.% water; wherein the bioactive agent is hydrophilic or amphipathic.

2. The nanoparticle dispersion of claim 1, wherein: the one or more high hydrophile-lipophile-balance (HLB) surfactants comprises an ester type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactant; and the one or more low hydrophile-lipophile-balance (HLB) surfactants comprises a phospholipid low hydrophile-lipophile-balance (HLB) surfactant.

3. The nanoparticle dispersion of claim 1, wherein: the ester type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactant is selected from the group consisting of PEG100 stearate, PEG20 stearate, PEG30 glyceryl cocoate, PEG32 stearate, polysorbate 20, and polysorbate 80; and the phospholipid low hydrophile-lipophile-balance (HLB) surfactant is selected from the group consisting of phosphatidylcholine and lecithin.

4. The nanoparticle dispersion of claim 1, wherein: the one or more high hydrophile-lipophile-balance (HLB) surfactants comprises one or more ether type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactants.

5. The nanoparticle dispersion of claim 4, further comprising one or more ester type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactants.

6. The nanoparticle dispersion of claim 5, wherein a weight ratio of the ether type polyethoxylated high hydrophile-lipophile-balance HLB surfactant to the ester type poly ethoxylated high hydrophile-lipophile-balance HLB surfactant is greater than 1: 1.

7. The nanoparticle dispersion of claim 5, wherein: the ether type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactant is selected from the group consisting of laureth-23, laureth-30, steareth- 100, steareth-20, steareth-40, ceteareth-20, and ceteareth-30; and the ester type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactant is selected from the group consisting of PEG100 stearate, PEG20 stearate, PEG30 glyceryl cocoate, PEG32 stearate, and polysorbate 20.

8. The nanoparticle dispersion of claim 4, further comprising one or more nonpolyethoxylated high hydrophile-lipophile-balance (HLB) surfactants.

9. The nanoparticle dispersion of claim 8, wherein the non-polyethoxylated high hydrophile-lipophile-balance (HLB) surfactant is sodium laurel sulfate.

10. The nanoparticle dispersion of claim 17, wherein the hydrophilic bioactive agent selected from the group consisting of spermadine, caffeine, acetominophen, and niacinamide.

11. The nanoparticle dispersion of claim 1, wherein the bioactive agent is a peptide.

12. The nanoparticle dispersion of claim 11, wherein the peptide is insulin.

13. The nanoparticle dispersion of claim 11, wherein the peptide is bovine serum albumin.

14. The nanoparticle dispersion of claim 1, comprising a latent lamellar structure.

15. The nanoparticle dispersion of claim 1, wherein the bioactive agent does not form crystals within the nanoparticle dispersion for greater than about 8 months at a temperature from about 18 °C to about 22 °C.

16. The nanoparticle dispersion of claim 1, wherein the nanoparticle dispersion has a pH less than an isoelectric point of the peptide.

17. A lipolysis-resistant nanoparticle dispersion comprising: from 0.01 wt. % to 20 wt. % of one or more bioactive agents; from 0.1 wt.% to 20 wt.% of one or more high hydrophile-lipophile-balance (HLB) surfactants; from 0.1 wt.% to 20 wt.% of one or more low hydrophile-lipophile-balance (HLB) surfactants; from 10 wt.% to 45 wt.% of one or more water immiscible oils; and from 25 wt.% to 75 wt.% water; wherein: the one or more high HLB polyethoxylated surfactants consists of an ether-type high HLB polyethoxylated surfactant; the nanoparticle dispersion further comprises a lipase inhibitor; the nanoparticle dispersion comprises a zeta potential greater than 1.0 millivolt; or any combination thereof.

18. A capsule comprising: a nanoparticle dispersion of claim 1 or a nanoparticle dispersion of claim 17, an encapsulating polymer comprising a carboxylic acid and selected from the group consisting of a methyl acrylate - methacrylic acid copolymer, a methyl methacrylate - methacrylic acid copolymer, a ethyl acrylate - methacrylic acid copolymer, a vinyl acetate - vinyl 4-hydroxyl phthalate copolymer, a hydroxypropyl methyl cellulose acetate succinate, a cellulose acetate phthalate, Eudragit L 30 D-55, or combinations thereof; optionally, a surface layer external to the encapsulating polymer, the surface layer comprising carnauba wax, dimethicone, organo-modified silicone or any combination thereof; and optionally, an intermediate layer between the encapsulating polymer and the surface layer; wherein: a pH of the nanoparticle dispersion is less than a pKa of the encapsulating polymer carboxylic acid group; and the capsule remains undigested after 30 minutes in an aqueous mixture at a pH of less than 2.

19. The capsule of claim 18, wherein the capsule comprises an innermost layer consisting of the encapsulating polymer, an intermediate layer comprising a gelatin, and a surface layer comprising a carnauba wax.

20. A method for treating a disorder selected from the group consisting of diabetes mellitus type 1 and diabetes mellitus type 2 comprising orally administering to a patient in need thereof: the nanoparticle dispersion of claim 1; the nanoparticle dispersion of claim 17; or the capsule of claim 18; wherein: a time to reach the peak plasma concentration in the patient (Tmax) is in a range from 2 minutes to 1 hour; and d(plasma concentration)/dt is negative in a plot of plasma concentration vs time for each time point in a range from 1 to 24 hours after oral administration.