Cell wall binding proteins specifically targeting propionibacterium acnes

Abstract

The present invention relates to recombinant targeting peptides, compositions comprising said targeting peptides and methods of targeted delivery of therapeutic agents suitable for the control, amelioration and / or treatment of acne. More particularly, the present invention provides an engineered targeting peptide that specifically binds to the cell wall of Propionibacterium acnes, the engineered targeting peptide being capable of providing a homing mechanism for transporting nanoparticles comprising a therapeutic agent to Propionibacterium acnes. Accordingly, the invention also provides methods and compositions comprising the recombinant targeting peptides for topical delivery of therapeutic agents to Propionibacterium acnes.

Description

Technical Field

[0001] The present invention relates to a recombinant targeting peptide, a composition comprising the targeting peptide, and a method for targeted delivery of a therapeutic agent suitable for controlling, improving and / or treating acne. More particularly, the present invention provides an engineered targeting peptide that specifically binds to the cell wall of Propionibacterium acnes, and the engineered targeting peptide can provide a homing mechanism for transporting nanoparticles containing a therapeutic agent to Propionibacterium acnes. Therefore, the present invention also provides a method and composition comprising the recombinant targeting peptide for topical delivery of a therapeutic agent to Propionibacterium acnes. Background Art

[0002] Acne, also known as acne vulgaris, is a chronic inflammatory disorder of the pilosebaceous unit. It is one of the most common skin diseases in the world, and almost all adolescents and adults may experience it at some point in their lives. In addition to causing long-term physical effects (such as scarring), acne can also have a serious impact on the patient's mental, social and emotional well-being. For example, acne can reduce a patient's self-esteem and confidence, which may affect his / her social interactions, school and work performance. In some cases, the distress caused by acne may even lead to depression and suicidal thoughts.

[0003] One of the causes of acne is closely related to the bacterium Propionibacterium acnes, which is a rod-shaped anaerobic Gram-positive commensal bacterium that lives on the skin of most healthy adults. The bacteria are primarily found deep within hair follicles and pores and feed on sebum and byproducts of the surrounding skin tissue. The role of P. acnes in causing acne is complex and is likely to involve multiple pathological processes and contributing factors. For example, when pores become clogged with cellular debris and sebum, this can cause an overgrowth of P. acnes, which can irritate the pore lining and induce inflammation, which in turn leads to acne.

[0004] Bacteria control is a core part of acne treatment. Active ingredients such as benzoyl peroxide and antibiotics are commonly used to reduce the bacterial population on human skin. While these actives are effective, they have several undesirable side effects. A common side effect of benzoyl peroxide is dry skin, as it is a harsh, bleach-like chemical. Over-reliance on antibiotics has created antibiotic-resistant bacteria on the skin. For example, Propionibacterium acnes has become increasingly resistant to antibiotic therapy. Additionally, benzoyl peroxide and antibiotics indiscriminately kill bacteria, including the beneficial bacteria that make up our skin microbiome. Long-term use of these actives will permanently disrupt the microbiome, leading to more acne, rashes, and other skin complications.

[0005] Therefore, there is a need to provide improved medicaments, compositions and methods of producing the same for the management and treatment of acne that overcome or at least ameliorate one or more of the above-mentioned disadvantages. Summary of the invention

[0006] Disclosed herein are isolated recombinant targeting peptides that are suitable for managing, treating or preventing acne specifically targeting Propionibacterium acnes, recombinant DNA molecules and expression vectors encoding the targeting peptides, nanoparticles and compositions comprising the targeting peptides, and methods for producing the targeting peptides, nanoparticles and compositions thereof. It should be understood that the present invention can be used for cosmetic treatment or therapeutic treatment, depending on the active agent loaded in the nanoparticles and the situation or nature of acne.

[0007] In a first aspect of the present invention, a composition is provided, comprising: (i) nanoparticles; (ii) a targeting peptide that binds to Propionibacterium acnes; and (iii) a cargo comprising one or more antimicrobial agents and / or anti-acne active agents, wherein the nanoparticles encapsulate the cargo and the targeting peptide is a component of the surface of the nanoparticles.

[0008] In the second aspect of the present invention, there is provided use of the composition of the first aspect in the manufacture of a medicament for treating or preventing acne.

[0009] In a third aspect of the present invention, there is provided a method of treating or preventing acne, the method comprising administering an effective amount of the composition of the first aspect to a subject in need of such treatment.

[0010] In a fourth aspect of the present invention, an isolated recombinant DNA molecule is provided, wherein the isolated recombinant DNA molecule comprises a DNA sequence encoding the Propionibacterium acnes targeting peptide disclosed in the first aspect.

[0011] In the fifth aspect of the present invention, an expression vector comprising the recombinant DNA molecule of the fourth aspect is provided.

[0012] In the sixth aspect of the present invention, there is provided use of the expression vector of the fifth aspect for recombinantly producing a Propionibacterium acnes targeting peptide.

[0013] In the seventh aspect of the present invention, an isolated recombinant Propionibacterium acnes targeting peptide comprising the following amino acid sequence and having Propionibacterium acnes cell wall binding activity is provided, wherein the amino acid sequence is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:5.

[0014] In the eighth aspect of the present invention, a method for producing a recombinant Propionibacterium acnes targeting peptide as disclosed in the first aspect is provided, the method comprising the following steps: (i) culturing eukaryotic or prokaryotic cells transfected with the recombinant DNA molecule of the fourth aspect or the expression vector of the fifth aspect in a culture medium, and (ii) recovering the expressed recombinant Propionibacterium acnes targeting peptide from the cells or the culture medium.

[0015] In the ninth aspect of the present invention, a method for producing Propionibacterium acnes targeting nanoparticles is provided, the method comprising mixing a cargo comprising one or more antimicrobial agents and / or anti-acne active agents with poly(D,L-lactide-co-glycolide) (PLGA), and then combining the mixture with polyvinyl alcohol (PVA) and ultrasonically treating to form anionic nanoparticles; extracting the PLGA and the cargo nanoparticles; mixing the Propionibacterium acnes targeting peptide as defined in the first aspect with the PLGA and the cargo nanoparticles until the nanoparticles are coated with the targeting peptide.

[0016] Alternatively, the P. acnes targeting lipid nanoparticles can be produced by the following method, the method comprising mixing a cargo comprising one or more antimicrobial agents and / or anti-acne active agents with a lipid to form anionic liposomes + cargo nanoparticles, mixing a P. acnes targeting peptide as defined in the first aspect with the liposomes and the cargo nanoparticles until the nanoparticles are coated with the targeting peptide. Preferably, the lipids are dipalmitoylphosphatidylcholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

[0017] Advantageously, the nanoparticles and compositions of the present disclosure help protect the human skin microbiome by selectively targeting and killing only Propionibacterium acnes. More advantageously, the specificity of the engineered nanoparticles and compositions of the present disclosure also reduces the effective concentration and amount of the antimicrobial agent and / or the anti-acne active agent required, thereby minimizing the adverse side effects that may be associated with the agent. These and other advantages of the present disclosure will become clear to those skilled in the art based on the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings illustrate the disclosed embodiments and serve to explain the principles of the disclosed embodiments. However, it should be understood that the accompanying drawings are designed for illustration purposes only and not as a definition of the limits of the present invention.

[0019] Figure 1Shown is the binding spectrum of SmartNova C. Confocal microscopy images of SmartNova C with P. acnes 6919 (A1-3), P. acnes 11828 (B1-3), P. acnes 11827 (C1-3), P. acnes HC038-PAI (D1-3), Enterococcus faecalis OG1RF (E1-3), Pseudomonas aeruginosa (F1-3), and Staphylococcus epidermidis (G1-3).

[0020] Figure 2 Confocal microscopy images of NovaC with P. acnes 6919 (A1-3) and P. acnes 11828 (B1-3); DukeC2Rap with P. acnes 6919 (C1-3) and P. acnes 11828 (D1-3) are shown. White bars indicate 10 μm.

[0021] Figure 3 A conceptual schematic diagram of SmartArrow is shown. SmartArrow consists of lipid- or polymer-based nanoparticles encapsulating / loading anti-acne active ingredients, and the surface of the nanoparticles is coated with SmartNovaC.

[0022] Figure 4 Shown are the minimum inhibitory concentration (MIC) values ​​of benzoyl peroxide (BPO) against Propionibacterium acnes. An OTC product containing 10% BPO was used in this experiment.

[0023] Figure 5 A schematic diagram of the single emulsion technique is shown.

[0024] Figure 6 Depicted is a direct correlation between the input amount of BPO used experimentally and the amount of BPO loaded into the PLGA nanoparticles as measured by mass spectrometry.The input amount of PLGA was 5 mg.

[0025] Figure 7 SmartNovaC coated on liposomes and polymer nanoparticles are shown. (A, C) Positively charged SmartNovaC effectively reduces the net charge of the nanoparticles when coated on the surface of the nanoparticles, as reflected by the zeta potential value. (B, D) Coating of nanoparticles with GFP-SmartNovaC can be quantified by measuring the fluorescence signal.

[0026] Figure 8Confocal microscopy images depicting the binding of SmartArrow nanoparticles loaded with Nile Red fluorescent dye and coated with SmartNovaC on P. acnes cells. Treated cells were imaged in (A) green channel, (B) red channel, (C) bright field. The image in (D) was generated by combining images from (A)-(C).

[0027] Fig. 9 The efficacy of SmartArrow nanoparticles loaded with BPO and coated with SmartNovaC is shown. The number of colony forming units (CFU) in each sample was determined by plating 10-fold serial dilutions and compared with buffer-treated controls by two-tailed Student's t-test with Welch correction. *P<0.05; **P<0.01.

[0028] Fig.10 Results for one or more secondary structure positions in supernova predicted using JPRED are shown. The secondary structure prediction web server JPRED suggests that residue 171 is a good starting residue for defining the CBD of supernova.

[0029] Fig.11 A photograph of NovaC visualized using the software VMD is shown. A black helix consisting of 17 residues (white arrow) was removed to generate SmartNovaC. DETAILED DESCRIPTION

[0030] For convenience, the references mentioned in this specification are listed in the form of a reference list and attached at the end of the examples. The entire contents of such bibliographic references are incorporated herein by reference, but mentioning them in this specification does not mean that they constitute part of the common general knowledge.

[0031] definition

[0032] For convenience, certain terms used in the specification, examples, and appended claims are collected here.

[0033] Generally, the technical, scientific and medical terms used herein have the same meaning as those understood by those skilled in the art to which the present invention belongs. In addition, the following technical comments and definitions are provided. These definitions should not in any way limit the scope of the present invention to only those terms, but are proposed for a better understanding of the following description.

[0034] As used herein, "a" or "an" may mean one or more than one, unless the context indicates otherwise or there is another clear meaning.

[0035] As used herein, "antimicrobial agent" refers to a natural or synthetic substance that kills or inhibits the growth of microorganisms such as bacteria, fungi and algae. "Antimicrobial agent" used in this context preferably kills or inhibits the growth of the bacterium Propionibacterium acnes.

[0036] As used herein, "anti-acne active agent" refers to any natural or synthetic substance that may have a beneficial cosmetic effect and / or a beneficial therapeutic effect for the skin disease acne and its associated symptoms and manifestations. "Anti-acne active agents" used in this context may, for example, reduce or control the number of acne blemishes, acne papules, blackheads and whiteheads in a patient, reduce or control sebum production, reduce, control or alleviate inflammation / swelling associated with acne (e.g., reduce the reddened appearance of the skin), and / or control the development of acne. As will be appreciated by a skilled artisan, some anti-acne active agents may only provide a beneficial cosmetic effect, while other anti-acne active agents may provide a beneficial therapeutic effect or both.

[0037] As used herein, the term "cargo" refers to any compound / agent of interest that is intended to be delivered to a specific cellular destination via a delivery system to elicit a response. The cargo used in this context can be any compound or agent (such as an antimicrobial agent or an anti-acne active agent) that is intended to be transported to where Propionibacterium acnes resides and released therein by the delivery system of the present disclosure so that the cargo can interact with the bacteria to elicit a local effect.

[0038] As used herein, the term "comprising" or "including" should be interpreted as specifying the presence of the stated features, integers, steps or components mentioned, but not excluding the presence or addition of one or more features, integers, steps or components or groups thereof. However, in the context of the present disclosure, the term "comprising" or "including" also includes "consisting of". Variations of the word "comprising" (such as "comprise" and "comprises") and variations of "including" (such as "include" and "includes") have correspondingly varied meanings.

[0039] As used herein, the terms "effective amount" and "effective amount" are used interchangeably and refer to an amount of a cargo and / or a composition comprising a cargo that is sufficient to achieve a beneficial or desired result against Propionibacterium acnes and / or acne. An effective amount of a composition of the present disclosure used in this context can result in, for example, killing and / or inhibiting Propionibacterium acnes, reducing inflammation in or around acne, producing a beneficial cosmetic effect on the acne, such as improving redness or appearance of skin affected by acne.

[0040] The term "functional fragment" refers to a portion of a protein that retains some or all of the activity or function (e.g., biological activity or function, such as enzymatic activity) of the full-length protein, such as the ability to bind to and / or interact with or modulate another protein or nucleic acid. A functional fragment can be of any size, provided that the fragment retains the ability to, for example, bind to and interact with another protein or nucleic acid.

[0041] As used herein, the term "variant" refers to an amino acid sequence that is altered by one or more amino acids of a non-variant reference sequence, but retains the ability to recognize its target and affect its function. For example, a targeting peptide variant is altered by one or more amino acids of a non-variant targeting peptide reference sequence, but retains the ability to recognize and bind to the cell wall of Propionibacterium acnes. Variants can have "conservative" changes, in which the substituted amino acids have similar structural or chemical properties (e.g., replacing leucine with isoleucine). More rarely, variants can have "non-conservative" changes (e.g., replacing glycine with tryptophan). Similar minor variations can also include amino acid deletions or insertions or both. Computer programs well known in the art, such as The software (DNASTAR, Inc., Madison, WI, USA) provides guidance in determining which amino acid residues can be substituted, inserted, or deleted without destroying biological activity.

[0042] The term "nucleotide" refers to naturally occurring ribonucleotides or deoxyribonucleotide monomers, as well as non-naturally occurring derivatives and analogs thereof. Nucleotides may include, for example, nucleotides comprising naturally occurring bases (e.g., adenosine, thymidine, uridine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine or deoxycytidine) and nucleotides comprising modified bases known in the art. Therefore, the term "polynucleotide" herein generally refers to polyribonucleotides and polydeoxyribonucleotides, which may be non-modified RNA or DNA or modified RNA or DNA.

[0043] As used herein, "peptide", "polypeptide" and "protein" are used interchangeably to refer to a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). The term "protein" encompasses naturally occurring as well as artificial (e.g., engineered or variant) full-length proteins as well as functional fragments of proteins.

[0044] As used herein, the term "recombinant" means that a molecule (e.g., a nucleic acid or polypeptide) has been artificially or synthetically (i.e., non-naturally) altered by human intervention. The alteration can be performed on a molecule within its natural environment or state, or on a molecule removed from its natural environment or state.

[0045] A description of exemplary, non-limiting embodiments of the invention follows.

[0046] The present disclosure is based in part on the development of recombinant peptides that have been engineered to favorably bind to Propionibacterium acnes, a bacterial species closely associated with acne. In this regard, the inventors have successfully modified a natural lysin derived from a bacteriophage named phage Supernova and improved its solubility and its binding affinity to the cell wall of Propionibacterium acnes. Thus, the engineered targeting peptides disclosed herein are highly soluble and have enhanced specificity for Propionibacterium acnes compared to their natural counterparts. The amino acid and polynucleotide sequences of the natural lysin and the peptides isolated and developed therefrom are shown in Table 1.

[0047] Table 1. Amino acid and nucleotide sequences of natural and engineered lysins of the invention

[0048]

[0049]

[0050]

[0051] As described herein, the modified targeting peptides of the present disclosure act as homing mechanisms to deliver the cargo of interest to its designated location, and can therefore be coupled with a cellular delivery system to obtain a more precisely targeted drug delivery system. Thus, the nanoparticles, compositions, and methods of the present disclosure have been designed to advantageously target only P. acnes without targeting any other bacteria, thereby protecting the overall skin microbiome without significant off-target effects.

[0052] In one aspect, a composition is provided, comprising: i) nanoparticles; ii) a targeting peptide that binds to Propionibacterium acnes; and iii) a cargo comprising one or more antimicrobial agents and / or anti-acne active agents, wherein the nanoparticles encapsulate the cargo and the targeting peptide is a component of the surface of the nanoparticles.

[0053] The compositions described herein enable efficient, target-directed delivery of cargo of interest (e.g., antimicrobial agents) directly to Propionibacterium acnes. In particular, the nanoparticles and compositions disclosed herein can be coated with targeting peptides to provide enhanced selectivity for Propionibacterium acnes. Both covalent and non-covalent methods of coating the peptides can be employed.

[0054] In some embodiments, the targeting peptide comprises the amino acid sequence shown in SEQ ID NO: 3, or a functional fragment or variant thereof having P. acnes cell wall binding activity.

[0055] As will be appreciated by those skilled in the art, the function of a protein is directly related to its structure and sequence, and there is a positive correlation between sequence identity and functional similarity. In this regard, methods for determining protein sequence identity are known in the art. Therefore, the sequence of the targeting peptide disclosed herein can vary substantially, as long as the targeting peptide maintains its functionality and can exhibit the desired activity (e.g., the targeting peptide is able to bind to the cell wall of Propionibacterium acnes).

[0056] Therefore, in some embodiments, the targeting peptide can comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 3. In particular, the targeting peptide can consist of the amino acid sequence shown in SEQ ID NO: 3.

[0057] In some embodiments, the targeting peptide can comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, the targeting peptide can consist of the amino acid sequence shown in SEQ ID NO: 5.

[0058] In another aspect, an isolated recombinant Propionibacterium acnes targeting peptide comprising the following amino acid sequence and having Propionibacterium acnes cell wall binding activity is provided, wherein the amino acid sequence is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:5.

[0059] As will be appreciated by those skilled in the art, peptides can be encoded by nucleotide sequences that are read in groups of three nucleotides (referred to as codons). Therefore, in some embodiments, targeting peptides can be encoded by a polynucleotide comprising a nucleic acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the nucleic acid sequence shown in SEQ ID NO:4 due to the redundancy of the genetic code.

[0060] In some embodiments, the targeting peptide can be encoded by a polynucleotide comprising a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleic acid sequence shown in SEQ ID NO:6 due to the redundancy of the genetic code.

[0061] In another aspect, an isolated recombinant DNA molecule is provided, comprising a DNA sequence encoding a P. acnes targeting peptide as disclosed herein. In some embodiments, the DNA sequence encoding the targeting peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% nucleic acid sequence identity with the nucleic acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 6 due to the redundancy of the genetic code.

[0062] In another aspect, an expression vector comprising a recombinant DNA molecule of the present disclosure is provided. It should be understood that an expression vector is a construct designed for gene expression in a cell and is generally used to produce a protein. Methods for constructing an expression vector are well known in the art.

[0063] Therefore, in another aspect, there is provided use of the expression vector disclosed herein for recombinant production of a P. acnes targeting peptide.

[0064] In another aspect, a method for producing a recombinant Propionibacterium acnes targeting peptide of the present disclosure is provided, the method comprising the steps of: (i) culturing in a culture medium eukaryotic or prokaryotic cells transfected with a recombinant DNA molecule or expression vector as disclosed herein, and (ii) recovering the expressed recombinant Propionibacterium acnes targeting peptide from the cells or the culture medium.

[0065] As described herein, the compositions of the present disclosure can be loaded with any cargo that is expected to be delivered to and interact with Propionibacterium acnes. For example, the cargo can exhibit antimicrobial properties, anti-acne properties, anti-inflammatory properties, and / or exhibit a therapeutic effect against Propionibacterium acnes. Those skilled in the art will appreciate that any molecule or agent that can provide a beneficial effect (whether therapeutic, cosmetic, or other beneficial effect) in the control, improvement, and / or treatment of acne can be appropriately selected as a cargo.

[0066] In some embodiments, the goods may include one or more antimicrobial agents. For example, the antimicrobial agent may be a small molecule. The antimicrobial agent may include, but is not limited to, benzoyl peroxide, sulfur, azelaic acid, and antibiotics such as ozenoxacin, nadifloxacin, doxycycline, minocycline, azithromycin, erythromycin, clindamycin, and dapsone. In some embodiments, the one or more antimicrobial agents may include benzoyl peroxide, azelaic acid, antibiotics such as erythromycin, clindamycin, dapsone, and / or a combination thereof.

[0067] In some embodiments, the cargo may contain one or more anti-acne actives. Anti-acne actives may include, but are not limited to, alpha hydroxy acids, such as glycolic acid and lactic acid; beta hydroxy acids, such as salicylic acid; retinoids, such as adapalene, tretinoin, isotretinoin, tazarotene, alitretinoin, bexarotene, resorcinol, retinyl esters, retinaldehyde, retinal, and retinol; flavonoids and / or vitamin derivatives, such as niacinamide and resorcinol. In some embodiments, the cargo may contain one or more antimicrobial agents and / or anti-acne actives.

[0068] In some embodiments, anti-acne actives may include compounds and / or extracts that may have cosmetic benefits in managing and / or improving acne, such as tea tree oil, propolis extract, green tea extract, rice extract, astringents, anti-inflammatory compounds, or mixtures thereof.

[0069] Thus, in some embodiments, the compositions disclosed herein may be suitable for use as cosmetics, based on the appropriate cargo selected.

[0070] To facilitate delivery of cargo to where bacteria reside, the cargo can be encapsulated in nanoparticles. It is understood that nanoparticles have been used as drug carriers in the medical / pharmaceutical field by encapsulating or attaching therapeutic molecules and delivering them to target tissues more precisely and with controlled release.

[0071] In this regard, nanoparticles are generally submicron (<1 μm) colloidal particles that exhibit unique structural, chemical, mechanical, magnetic, electrical and biological properties. Nanoparticles can be made of biocompatible and biodegradable materials, such as lipids, natural polymers (e.g., gelatin, albumin, alginate, chitosan) or synthetic polymers (e.g., polyvinyl alcohol, poly-L-lactic acid, polyethylene glycol, poly-(lactic acid-co-glycolic acid) polylactide, polyalkyl cyanoacrylates, etc.). In some embodiments, nanoparticles include lipid-based structures, such as liposomes or micelles. In some embodiments, the nanoparticles may comprise poly(lactic acid) (PLA); polyglycolic acid (PGA); poly(D,L-lactide-co-glycolide) (PLGA); or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol sodium salt (DMPG) or dipalmitoylphosphatidylcholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

[0072] It will be appreciated by those skilled in the art that the selection and / or preparation of suitable nanoparticles are based on factors such as the biophysical and biochemical properties of the goods of interest and the target location. For example, goods can be encapsulated by nanoparticles via hydrophobic interactions, electrostatic interactions and / or covalent conjugation, depending on the physicochemical characteristics of the goods. Therefore, those skilled in the art can adjust the preparation of suitable nanoparticles accordingly.

[0073] In some embodiments, the nanoparticles may have an anionic surface charge, a cationic surface charge, or a neutral surface charge. In particular, the nanoparticles may have an anionic surface charge.

[0074] Therefore, in another aspect, a method for producing P. acnes targeting nanoparticles is provided, the method comprising mixing a cargo comprising one or more antimicrobial agents and / or anti-acne active agents with poly(D,L-lactide-co-glycolide) (PLGA), then combining the mixture with polyvinyl alcohol (PVA) and sonicating to form anionic nanoparticles; extracting the PLGA+cargo nanoparticles; mixing the P. acnes targeting peptide of the present disclosure with the PLGA+cargo nanoparticles until the nanoparticles are coated with the targeting peptide. In some embodiments, PLGA and PVA are substituted with: 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol sodium salt (DMPG) or dipalmitoylphosphatidylcholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

[0075] In some embodiments, the nanoparticles and compositions disclosed herein may further include a pharmaceutically acceptable carrier. Suitable pharmaceutical carriers will generally contain inert ingredients that do not interact with the medicament or active ingredient. Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg / ml benzyl alcohol), phosphate buffered saline, Hank's solution, Ringer's lactate, etc. The formulation may also include a small amount of substances that enhance the effectiveness of the active ingredient (e.g., emulsifiers, solubilizers, pH buffers, wetting agents). Methods for encapsulating compositions (such as in a coating of hard gelatin or cyclodextran) are known in the art. For inhalation, the medicament can be dissolved and loaded into a dispenser suitable for administration (e.g., a sprayer or atomizer or a pressurized aerosol dispenser).

[0076] For in vivo delivery, the nanoparticles and compositions disclosed herein can be delivered to subjects in need by a variety of administration routes, including, for example, oral, dietary, topical, transdermal or parenteral (e.g., intra-arterial, intravenous, intramuscular, subcutaneous, intradermal) administration routes. Administration can be local or systemic. The actual dosage and treatment regimen of the nanoparticles and / or compositions herein can be determined by a skilled physician in consideration of the nature of the disease being treated and the patient's characteristics.

[0077] In some embodiments, the compositions as disclosed herein may preferably be formulated for topical administration. Preferably, the topical formulation may be in the form of a liquid solution or mixture, dispersion, suspension, gel, lotion, emulsion, paste, cream, ointment, milk, pomade, spray, or medicated bandage, pad, or mask. It should be understood that methods for preparing topical formulations are known in the art and are based on standard principles and methods described in various pharmaceutical literature.

[0078] In another aspect, the use of the composition of the present disclosure in the manufacture of a medicament for treating or preventing acne is provided. In some embodiments, the compositions and medicaments disclosed herein are used to selectively kill and / or target Propionibacterium acnes on human skin. In some embodiments, the medicament is in the form of a cream, gel, or ointment.

[0079] In another aspect, provided is a method of treating or preventing acne, the method comprising administering to a subject in need of such treatment an effective amount of a composition of the present disclosure.

[0080] Unless otherwise indicated or otherwise clearly meant by the context and the understanding of those of ordinary skill in the art, the values ​​expressed as ranges in the various embodiments may assume any specific value or sub-range within the range, unless the context clearly indicates other meanings, or are accurate to one decimal place after the unit of the lower limit of the range. Unless otherwise stated or by other clear meanings according to the context, "about" involving numerical values ​​generally refers to a range of values ​​that falls within ±10% of the value, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%. In any embodiment in which there is an "about" modification before the numerical value, an embodiment in which the exact value is listed is provided. If an embodiment in which there is no "about" modification before the numerical value is provided, an embodiment in which there is an "about" modification before the value is also provided. If there is an "about" modification before the range, then unless the context clearly indicates other meanings, an embodiment in which "about" applies to the lower and upper limits of the range or to the lower or upper limits is provided. If a phrase such as "at least," "up to," "no more than," or a similar phrase precedes a series of numbers, it is understood that, unless the context clearly indicates otherwise, in various embodiments the phrase applies to each number in the list (it is understood that, depending on the context, 100% of a value (e.g., a value expressed as a percentage) may be an upper limit). For example, in various embodiments, "at least 1, 2, or 3" is understood to mean "at least 1, at least 2, or at least 3." It is also understood that any and all reasonable lower and upper limits are expressly contemplated.

[0081] Having now generally described the invention, the same will be more readily understood by reference to the following examples which are provided by way of illustration and are not intended to be limiting of the invention.

[0082] Example

[0083] Standard molecular biology techniques known in the art and not specifically described are generally followed, as described in Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2012).

[0084] Example 1: Generation of Propionibacterium acnes Targeted Engineered Lysins

[0085] A natural lysin named Supernova was selected as the starting sequence template to generate the engineered lysin. The Supernova lysin was obtained from a phage named phage Supernova that targets P. acnes. Then based on the results of BLAST and JPRED websites, it was determined that the C-terminal cell wall binding domain (CBD) of Supernova, NovaC, is from amino acids 171 to 287. However, the expression of NovaC is low, and it shows very weak binding to P. acnes, as shown in confocal microscopy data ( Figure 2 A and Figure 2 B).

[0086] To improve the solubility of NovaC, the structure of NovaC was modeled using the software I-TASSER and visualized using the software VMD. Based on the computer-simulated structure, an engineered lysin called SmartNovaC was designed by truncating amino acids 2 to 18 of NovaC. This removes the alpha helix that does not interact with the rest of the protein.

[0087] The gene (Genbank accession number ATN91960.1) of the synthetic natural lysin Supernova is cloned into the pNIC28-Bsa4 plasmid. The engineered lysin gene (NovaC and SmartNovaC) containing other enhanced green fluorescent protein (EGFP) at the N-terminus is synthesized and cloned into the pET-22b (+) plasmid. All nucleotide sequences are codon optimized to improve the efficiency of soluble expression in Escherichia coli (E.coli). The amino acid and nucleotide sequences of natural lysin and engineered lysin are provided in Table 1.

[0088] To produce recombinant lysin, the plasmid with the gene of interest was transformed into E. coli competent cells and the protein was overexpressed using IPTG induction. The protein was purified by using immobilized metal affinity chromatography and size exclusion chromatography.

[0089] To examine the binding profile of the engineered lysin SmartNovaC, 2.5 mg / ml SmartNovaC was applied to four strains of P. acnes, Enterococcus faecalis strain OG1RF, Staphylococcus epidermidis strain PCI1200, and Pseudomonas aeruginosa strain PA14. Since SmartNovaC was cloned in a plasmid that co-expressed the EGFP tag, the fluorescent lysin could be visualized using confocal microscopy. Green fluorescent SmartNovaC showed specific binding to all four strains of P. acnes ( Figure 1 , A1-3, B1-3, C1-3, D1-3), while it could not bind to E. faecalis OG1RF ( Figure 1 , E1-3), Pseudomonas aeruginosa PA14 ( Figure 1 , F1-3) or Staphylococcus epidermidis PCI1200 ( Figure 1 , G1-3). This result clearly demonstrated the activity of SmartNovaC in specific binding to Propionibacterium acnes.

[0090] To further examine whether SmartNovaC is the only lysin that specifically binds P. acnes, the binding of NovaC synthesized with co-expressed EGFP and another lysin DukeC2Rap was tested. The DukeC2Rap lysin was obtained as the CBD of the Doucette lysin targeting the species Propionibacterium freudenreichii, which is from the same genus as P. acnes. The nucleotide and amino acid sequences of DukeC2Rap are provided below.

[0091] Table 2. Nucleotide and amino acid sequences of DukeC2Rap lysin

[0092]

[0093] NovaC and DukeC2Rap were tested for binding to two strains of P. acnes at concentrations of 2.5 mg / ml and 1.3 mg / ml, respectively. Although NovaC showed some binding to P. acnes ( Figure 2 , A1-3, B1-3), but the binding signal generated was higher than that of SmartNovaC ( Figure 1 , A1-3, B1-3, C1-3, D1-3) are much weaker. This strongly suggests that the engineered SmartNovaC lysin has enhanced binding specificity to P. acnes. DukeC2Rap did not show any binding to P. acnes ( Figure 2 , C1-3, D1-3). Therefore, among the different lysins tested, only the engineered lysin SmartNovaC has specific binding to P. acnes species.

[0094] Example 2: Preparation of therapeutic compositions

[0095] Since SmartNovaC itself does not kill bacteria directly, it is necessary to combine SmartNovaC with an antibacterial agent. For example, to apply this technology to develop anti-acne products, SmartNovaC can be paired with an anti-acne active ingredient such as benzoyl peroxide (BPO). As a result, a method such as Figure 3The novel SmartArrow system shown: active ingredients are loaded into lipid / polymer-based nanoparticles and the surface of the nanoparticles is coated with SmartNovaC to selectively target the antibacterial agent to Propionibacterium acnes.

[0096] Preparation of anionic liposomes

[0097] An embodiment of the structure of the composition of the present invention is shown in Figure 3 Exemplary anionic liposomes are prepared from 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol sodium salt (DMPG) at a molar ratio of 10:1. The size and stability of the liposomes can be optimized by varying the molar ratio, using different lipid types, and adding cholesterol, polyethylene glycol (PEG), and other additives that can alter the behavior of the liposomes.

[0098] The advantage of SmartArrow is that it delivers active ingredients to where the bacteria reside. As a result, a much lower dose of bioactive substances can be loaded into SmartArrow to achieve high bacterial killing compared to non-targeted liposomes. Only 0.002% BPO can achieve complete killing of Propionibacterium acnes ( Figure 4 ). Considering that the concentrations of BPO found in the retail market range from 2.5%-10%, which is approximately 1000 times higher than the concentration required to kill bacteria, it is not surprising that most users of these retail products typically suffer from several side effects such as dry and flaky skin. By using the SmartArrow approach, we can conservatively load 0.02% BPO (which is 100 times less than the retail product) into the liposomes to achieve complete bacterial kill and minimize side effects.

[0099] Preparation of PLGA+BPO Nanoparticles

[0100] To load BPO into the nanoparticles, poly(D,L-lactide-co-glycolide) (PLGA) was chosen because it is known to form nanoparticles that are excellent for loading hydrophobic compounds such as BPO.

[0101] PLGA particles containing BPO were prepared using a single emulsion technique as previously described (Jain RA. 2000. Biomaterials 21: 2475-2490). Briefly, 5 g of PLGA and 5 g of BPO were each dissolved in 500 μl of chloroform. PLGA and BPO were then added to 5 ml of 1% polyvinyl alcohol (PVA), and the mixture was immediately sonicated at 23% amplitude for 5 minutes. Figure 5As shown, uniform emulsified nanoparticles are formed after ultrasonic treatment of the oil phase (containing PLGA and BPO) and the aqueous phase (containing PVA). In order to extract and remove the organic solvent, the mixture containing the emulsified PLGA+BPO nanoparticles was stirred at 700 rpm in a fume hood at room temperature for 6 hours. In order to purify the PLGA+BPO nanoparticles, the mixture was centrifuged at 6,000 rpm for 15 minutes to obtain a precipitate containing nanoparticles. The precipitate was resuspended with 2 ml of ultrapure water and centrifuged to remove all free BPO. The final precipitate of the purified PLGA+BPO nanoparticles was resuspended with 2 ml of ultrapure water and stored at 4°C until use.

[0102] Dynamic light scattering (DLS) and mass spectrometry were used to characterize PLGA+BPO nanoparticles. The average size of the nanoparticles measured by DLS was 150 nm, and the zeta potential was -18 mV. The amount of BPO loaded in the PLGA particles was quantitatively determined by mass spectrometry, as shown in Figure 5. Figure 6 We have shown that we can effectively load up to 5 mg of BPO into 5 mg of PLGA particles. For the SmartArrow application of the present invention, it is likely that much less amounts can be loaded.

[0103] Preparation of DPPC+DSPE+BPO Nanoparticles

[0104] DPPC+DSPE nanoparticles containing BPO were prepared using 4 mg of DPPC and 1 mg of DSPE and 1 mg of BPO. In brief, 4+1 mg of DPPC+DSPE lipids and 1 mg of BPO were dissolved in 500 μl of chloroform, respectively. DPPC+DSPE lipids and BPO were then added to 4 ml of chloroform, and the mixture was placed in a fume hood overnight to evaporate the chloroform. The next day, 5 ml of 1x phosphate buffered saline (PBS) was added to rehydrate the dried lipid film. The solution was ultrasonically treated at 70 ° C for 5 minutes for three rounds. The mixture will undergo a 400 nm extruder step as a filtration step and form uniform nanoparticles. The filtered sample will be subjected to tangential flow filtration (TFF) using ultrapure water to separate free BPO from the nanoparticles loaded with BPO. The final product will be stored at 4 ° C until use.

[0105] SmartNovaC was coated on anionic liposomes and PLGA nanoparticles via a non-covalent method.

[0106] Using charge-based binding, anionic liposomes and PLGA nanoparticles were coated with positively charged SmartNovaC targeting peptides (both GFP fusion and free forms). The coating was performed by mixing SmartNovaC peptide and liposome / PLGA nanoparticles at a ratio of 2: 1 and vortexing the mixture for 2 hours. In order to purify the protein-bound nanoparticles, the well-vortexed mixture was centrifuged at 6,000xg for 15 minutes. The precipitate was resuspended with 300μl ultrapure water and settled via centrifugation to remove all unbound proteins. The final precipitate was resuspended with 100μl of 1x phosphate buffered saline (PBS) and stored at 4°C until use.

[0107] The resulting particles were characterized using dynamic light scattering (DLS). The size of SmartNovaC-coated liposomes increased and the zeta potential decreased ( Figure 7 A). This is consistent with the fact that positively charged SmartNovaC coated on anionic liposomes will reduce the overall charge of the particles. Figure 7 C shows the decrease in zeta potential of PLGA nanoparticles after SmartNovaC coating. Figure 7 B and Figure 7 D shows the increase of GFP fluorescence signal, thus GFP-fused SmartNovaC is bound to and coated on the nanoparticles.

[0108] like Figure 7 The coating method shown is based on the principle of charge-based electrostatic interactions, but more directed protein-nanoparticle bioconjugation can also be performed to ensure that SmartNovaC maintains its selective binding on P. acnes.

[0109] SmartNovaC is coated on lipid- or polymer-based nanoparticles via site-directed covalent conjugation.

[0110] Conjugation was performed using a thiol-maleimide reaction, where the thiol group was present in the SmartNovaC protein and the maleimide group was covalently linked to a lipid (e.g., DSPE and DPPC lipids) or a polymer (e.g., PLGA). In the nanoparticles, only 20%-30% of the lipid / polymer substrate contained maleimide groups to avoid overcrowding of the conjugated protein, which could affect its targeting properties. According to DLS, the size of the nanoparticles was in the range of 150-400nm when loaded with BPO and coated with conjugated SmartNovaC. Figure 8 Confocal microscopy images in grayscale of SmartNovaC-coated, Nile Red-loaded polymer nanoparticles bound to P. acnes are shown. GFP ( Figure 8 A) and Nile Red ( Figure 8 B) The two signals are combined into a single image Figure 8 Overlay in D demonstrates the binding of nanoparticles to bacterial cells. Fig. 9 It was shown that the SmartArrow loaded with BPO successfully exhibited bacterial killing, ie, 90% killing of bacteria at 0.01% BPO and 99% killing of bacteria at 0.1% BPO.

[0111] Summarize

[0112] Provided herein is SmartNovaC, a 11kDa cell wall binding protein derived from bacteriophage lysin. It has been conclusively shown that SmartNovaC can specifically bind to various strains of Propionibacterium acnes (Cutibacterium acnes / C.acnes) and does not bind to other bacteria. A delivery system comprising SmartNovaC and an active ingredient (such as benzoyl peroxide) will be able to selectively target and kill Propionibacterium acnes, thereby avoiding damage to the skin microbiome. By incorporating SmartNovaC into a product formulation, it is estimated that the effective concentration of the active ingredient can be reduced by 100 times compared to existing anti-acne products on the market.

[0113] The SmartArrow targeted delivery system involves loading anti-acne active ingredients into polymer / lipid-based nanoparticles and coating the surface of the nanoparticles with SmartNovaC peptides. Both covalent and non-covalent methods can be used for coating using SmartNovaC. Therefore, SmartArrow can be advantageously used in the skin care industry as a targeted delivery system for topical cosmetic or therapeutic acne treatments.

[0114] References

[0115] Jain RA.2000.The manufacturing techniques of various drug loaded biodegradable poly(lactide-co-glycolide)(PLGA)devices.Biomaterials 21:2475-2490.

[0116] Phage supernova genome:worldwideweb.ncbi.nlm.nih.gov / nuccore / MF919533

Claims

1. A composition comprising: i) nanoparticles; ii) a targeting peptide that binds to the cell wall of Cutibacterium acnes; and iii) goods containing one or more antimicrobial and / or anti-acne active agents, The nanoparticle encapsulates the cargo, and the targeting peptide is a component of the surface of the nanoparticle.

2. The composition according to claim 1, wherein the targeting peptide comprises the following amino acid sequence: MPGPWFPWDKFMAVVNGHGGGSSSEELTVADVKALHNQIKQLSAQLSGSVNKLHHDVGVVQVQNGDLSKRVDALSWVKNPVTGKLWRTKDALWS VWYYVLECRSRIDRLESAVNGLKK(SEQ ID NO:3), or a functional fragment or variant thereof having P. acnes cell wall binding activity.

3. compositions according to claim 2, wherein said targeting peptide is by the polynucleotide encoding comprising following nucleotide sequence, and described nucleotide sequence has at least 80%, at least 85%, at least 90%, at least 95% or 100% identity with following nucleotide sequence due to the redundancy of genetic code: 5'-ATGCCGGGTCCGGTGGTTCCCGTGGGATAAATTCATGGCGGTTGTTAACGG TCACG GTGGTGGTTCTTCTTCTGAAGAACTGACCGTTGCTGATGTTAAAGCGCTGCACAACCAGATCAAACAGCTGTCTGCGCAGCTGAGCGGTTCTGTTAACAAACTGCACCACGATGTTGGCGTTGTTCAGGTTCAGAACGGTGATCTGAGCAAACG TGTTGATGCGCTGTCTTGGGTTAAAAACCCGGTTACCGGTAAACTGTGGCGTACCAAAGATGCTCTGTGGTCTGTTTGGTACTATGTTCTGGAATGCCGTAGCCGTATTGATCGTCTGGAAAGCGCGGTTAACGGTCTGAAAAAATAA-3'(SEQ ID NO:4).

4. The composition of claim 2, wherein the targeting peptide comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the following amino acid sequence: MGGGSSSEELTVADVKALHNQIKQLSAQLSGSVNKLHHDVGVVQVQN GDLSKRVDALSWVKNPVTGKLWRTKDALWSVWYYVLECRSRIDRLES AVNGLKK (SEQ ID NO: 5).

5. compositions according to claim 4, wherein said targeting peptide is by the polynucleotide encoding comprising following nucleotide sequence, and described nucleotide sequence has at least 80%, at least 85%, at least 90%, at least 95% or 100% identity with following nucleotide sequence due to the redundancy of genetic code: 5'-ATGGGTGGTTGGTTCAAGCTCTGAAGAACTGACTGTGGCTGATGTTAAAG CACTGC ACAATCAGTTAAACAGTTAAGCGCACAACTGAGCGGTTCTGTTAATAAACTGCATCACGATGTTGGTGTTGTTCAGGTTCAGAACGGTGATCTGAGCAAACGTGTTGATGCTTCTGTCCTGGGTTAAAA ATCCGGTTACCGGTAAACTGTGGCGTACTAAAGACGCGCTGTGGAGTGTTTGGTATTACGTTCTGGAATGTCGTTCTCGTATTGATCGTCTGGAAAGTGCGGTTAACGGTCTGAAAAAAATAA-3'(SEQ ID NO:6).

6. The composition of any one of claims 1 to 5, wherein the antimicrobial agent is a small molecule.

7. The composition of any one of claims 1 to 6, wherein the one or more antimicrobial agents comprise benzoyl peroxide, azelaic acid, erythromycin, clindamycin, dapsone, and / or combinations thereof.

8. The composition according to any one of claims 1 to 7, wherein the nanoparticles are selected from liposomes, micelles, other lipid-based nanoparticles and other polymer-based nanoparticles.

9. The composition of claim 8, wherein the nanoparticles have an anionic surface charge.

10. The composition of claim 9, wherein the nanoparticles comprise: i) poly(D,L-lactide-co-glycolide) (PLGA), poly(lactic acid) (PLA) or polyglycolic acid (PGA); or ii) 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol sodium salt (DMPG); or iii) dipalmitoylphosphatidylcholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

11. The composition of any one of claims 1 to 10, wherein the cargo comprises an anti-acne active selected from: i) alpha hydroxy acids, such as glycolic acid and lactic acid, and / or beta hydroxy acids, such as salicylic acid; and / or ii) retinoids such as adapalene, isotretinoin, tazarotene, retinal and retinol; and / or iii) Flavonoids and / or vitamin derivatives.

12. The composition according to any one of claims 1 to 11, further comprising a pharmaceutically acceptable carrier.

13. Use of the composition according to any one of claims 1 to 12 in the manufacture of a medicament for treating or preventing acne.

14. The use according to claim 13, wherein the drug is used to selectively kill and / or target Propionibacterium acnes on human skin.

15. The use according to claim 13 or 14, wherein the medicament is in the form of a cream, a gel or an ointment.

16. A method of treating or preventing acne, the method comprising administering to a subject in need of such treatment an effective amount of a composition according to any one of claims 1 to 12.

17. An isolated recombinant DNA molecule comprising a DNA sequence encoding the Propionibacterium acnes targeting peptide according to any one of claims 1 to 5.

18. according to the recombinant DNA molecule of separation described in claim 16, wherein the described DNA sequence dna encoding the described targeting peptide has at least 80%, at least 85%, at least 90%, at least 95% or 100% nucleic acid sequence identity with the nucleotide sequence shown in SEQ ID NO:4 or SEQ ID NO:6 due to the redundancy of genetic code.

19. An expression vector comprising the recombinant DNA molecule according to claim 17 or 18.

20. Use of the expression vector according to claim 19 for recombinant production of a Propionibacterium acnes targeting peptide.

21. An isolated recombinant Propionibacterium acnes targeting peptide comprising an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:5 and having Propionibacterium acnes cell wall binding activity.

22. A method for producing a recombinant Propionibacterium acnes targeting peptide as defined in any one of claims 1 to 5, the method comprising the following steps: (i) culturing in a culture medium a eukaryotic or prokaryotic cell transfected with a recombinant DNA molecule according to claim 17 or 18 or an expression vector according to claim 19, and (ii) recovering the expressed recombinant Propionibacterium acnes targeting peptide from the cells or the culture medium.

23. A method for producing Propionibacterium acnes targeting nanoparticles, the method comprising: i) mixing a cargo comprising one or more antimicrobial agents and / or anti-acne active agents with poly(D,L-lactide-co-glycolide) (PLGA), and then combining the mixture with polyvinyl alcohol (PVA) and sonicating to form anionic nanoparticles; extracting the PLGA+cargo nanoparticles; Mixing the P. acnes targeting peptide as defined in claim 2 or 4 with the PLGA+cargo nanoparticles until the nanoparticles are coated with the targeting peptide; or ii) mixing a cargo comprising one or more antimicrobial agents and / or anti-acne active agents with lipids to form anionic liposomes+cargo nanoparticles, The P. acnes targeting peptide as defined in claim 2 or 4 is mixed with the cargo nanoparticles until the nanoparticles are coated with the targeting peptide.

24. The method of claim 23, wherein the PLGA and the PVA are substituted by: 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol sodium salt (DMPG); or dipalmitoylphosphatidylcholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

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