A cell wall-associated protein that specifically targets Propionibacterium acnes

Abstract

The present invention relates to recombinant targeting peptides, compositions comprising the targeting peptides, and methods for targeted delivery of therapeutic agents suitable for controlling, ameliorating, and / or treating acne. More specifically, the present invention provides engineered targeting peptides that specifically bind to the cell wall of C. acnes, which can provide a homing mechanism for transporting nanoparticles comprising therapeutic agents to Cutibaterium acnes. Thus, the present invention also provides methods and compositions comprising the recombinant targeting peptides for topical delivery of therapeutic agents to C. acnes.

Description

[Technical field]

[0001] FIELD OF THE PRESENT APPLICATION The present invention relates to recombinant targeting peptides, compositions comprising said targeting peptides, and methods for targeted delivery of therapeutic agents suitable for controlling, ameliorating and / or treating acne. More specifically, the present invention provides engineered targeting peptides that specifically bind to the cell wall of C. acnes, which can provide a homing mechanism for transporting nanoparticles comprising therapeutic agents to Cutibacterium acnes. Thus, the present invention also provides methods and compositions comprising the recombinant targeting peptides for topical delivery of therapeutic agents to C. acnes. [Background technology]

[0002] 2. Background of the Invention Acne, also known as acne vulgaris, is a chronic inflammatory disease of the pilosebaceous unit. It is one of the most common skin diseases in the world, and nearly all adolescents and adults may experience it at some point in their lives. Acne can not only cause long-term physical effects such as scarring, but it can also have a profound effect on the psychological, social, and emotional health of patients. For example, acne can reduce patients' self-esteem and self-confidence, which in turn can affect social interactions, academic and work performance. In some cases, distress caused by acne can lead to depression and suicidal thoughts.

[0003] One of the causes of acne is strongly linked to a bacterium called Cutibacterium acnes. Cutibacterium acnes is an anaerobic gram-positive rod-shaped bacterium that lives in the skin of most healthy adults. This bacterium mainly lives deep inside hair roots and pores, feeding on sebum and by-products from the surrounding skin tissue. The role of C.acnes in causing acne is complex and may involve multiple pathological processes and factors. For example, when pores are blocked by cellular debris and sebum, C.acnes may overgrow and irritate the pore lining, inducing inflammation and causing acne.

[0004] Bacterial control is a central part of acne treatment. Active ingredients such as benzoyl peroxide and antibiotics are commonly used to reduce the bacterial count on human skin. Although these active ingredients are effective, they have some undesirable side effects. Benzoyl peroxide is a strong bleach-like chemical, so a common side effect is dryness of the skin. Over-reliance on antibiotics has already created antibiotic-resistant bacteria on the skin. For example, C. acnes is becoming resistant to antibiotic therapy. Furthermore, benzoyl peroxide and antibiotics indiscriminately kill bacteria, including the beneficial bacteria that make up our skin microbiome. Long-term use of these active agents permanently disrupts the microbiome, thereby increasing acne, rashes and other skin complications.

[0005] Thus, there is a need to provide improved medicaments, compositions, and methods for making such medicaments and compositions 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] Summary of the Invention The present invention provides isolated recombinant targeting peptides that specifically target C. 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 suitable for use in the management, treatment or prevention of acne. It will be appreciated that the present invention may be used for cosmetic or therapeutic treatments, depending on the active agent loaded on the nanoparticles, and the condition or nature of the acne.

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

[0008] In a second aspect of the invention there is provided the use of a composition of the first aspect in the manufacture of a medicament for the treatment or prevention of acne.

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

[0010] A fourth aspect of the invention provides an isolated recombinant DNA molecule comprising a DNA sequence encoding a Cutibacterium acnes targeting peptide as disclosed in the first aspect.

[0011] A fifth aspect of the invention provides an expression vector comprising a recombinant DNA molecule of the fourth aspect.

[0012] A sixth aspect of the invention provides the use of an expression vector of the fifth aspect for the recombinant production of a Cutibacterium acnes targeting peptide.

[0013] A seventh aspect of the present invention provides an isolated recombinant Cutibacterium 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 set forth in SEQ ID NO:3 or SEQ ID NO:5, and having Cutibacterium acnes cell wall binding activity.

[0014] An eighth aspect of the present invention provides a method for producing a recombinant Cutibacterium acnes targeting peptide as disclosed in the first aspect, the method comprising the steps of (i) culturing in a culture medium a eukaryotic or prokaryotic cell transfected with a recombinant DNA molecule of the fourth aspect or an expression vector of the fifth aspect, and (ii) recovering the expressed recombinant Cutibacterium acnes targeting peptide from the cell or culture medium.

[0015] A ninth aspect of the invention provides a method for the preparation of Cutibacterium acnes targeted nanoparticles comprising the steps of mixing a cargo comprising one or more antibacterial and / or anti-acne actives with poly(D,L-lactide-co-glycolide), (PLGA), then combining the mixture with polyvinyl alcohol (PVA) and sonicating to form anionic nanoparticles. Extraction of PLGA and cargo nanoparticles; Mixing a Cutibacterium acnes targeting peptide as defined in the first aspect with the PLGA and cargo nanoparticles until the nanoparticles are coated with the targeting peptide.

[0016] Alternatively, Cutibacterium acnes targeted lipid nanoparticles may be produced by a method comprising the steps of mixing a cargo comprising one or more antibacterial and / or anti-acne actives with a lipid to form anionic Liposome+cargo nanoparticles, mixing a Cutibacterium acnes targeting peptide as defined in the first aspect with the liposome and cargo nanoparticles until the nanoparticles are coated with the targeting peptide. Preferably, the lipid is dipalmitoylphosphatidylcholine (DPPC) and 1,2-distearoyl-sn-glycero-3 phosphorylethanolamine (DSPE).

[0017] Advantageously, the nanoparticles and compositions of the present disclosure aid in the preservation of the human skin microbiome by selectively targeting and killing only C.acnes. More advantageously, the specificity of the engineered nanoparticles and compositions of the present disclosure also reduces the effective concentration and amount of antibacterial and / or anti-acne active agents required, thereby minimizing the undesirable side effects that may be associated with said agents. These and other advantages of the present disclosure will become readily apparent to those skilled in the art from the detailed description that follows.

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

[0019] [Figure 1] Figure 1 shows the binding spectra of SmartNovaC. Confocal microscopy images of SmartNovaC with Cutibacterium acnes 6919 (A1-3), C. acnes 11828 (B1-3), C. acnes 11827 (C1-3), C. acnes HC038-PAI (D1-3), Enterococcus faecalis OG1RF (E1-3), Pseudomonas aeruginosa (F1-3), and Staphylococcus epidermidis (G1-3) are shown. [Diagram 2] Figure 2 shows confocal microscopy images of NovaC with C. acnes 6919 (A1-3) and C. acnes 11828 (B1-3); DukeC2Rap with C. acnes 6919 (C1-3) and C. acnes 11828 (D1-3). White bars indicate 10 μm. [Diagram 3]Figure 3 shows a schematic diagram of SmartArrow, which is composed of lipid- or polymer-based nanoparticles that encapsulate / load anti-acne active ingredients, and the surface of the nanoparticles is coated with SmartNovaC. [Figure 4] Figure 4 shows the minimum inhibitory concentration (MIC) value of benzoyl peroxide (BPO) against Cutibacterium acnes. In this experiment, an over-the-counter product containing 10% BPO was used. [Diagram 5] FIG. 5 shows a schematic diagram of the single emulsion technique. [Figure 6] Figure 6 shows a direct correlation between the BPO dosage used experimentally and the amount of BPO loaded into the PLGA nanoparticles as measured by mass spectrometry. The PLGA dosage was 5 mg. [Figure 7] Figure 7 shows SmartNovaC coated on liposomes and polymer nanoparticles. (Figure 7A, Figure 7C) When positively charged SmartNovaC is coated on the surface of nanoparticles, it effectively reduces the net charge of the nanoparticles as reflected by the zeta potential value. (Figure 7B, Figure 7D) Coating of nanoparticles with GFP-SmartNovaC can be quantified by measuring the fluorescence signal. [Figure 8] Figure 8 shows confocal microscopy images of SmartArrow nanoparticles coated with SmartNovaC loaded with Nile Red fluorescent dye binding onto C. acnes bacterial cells. Treated cells were imaged in (Figure 8A) green channel, (Figure 8B) red channel, and (Figure 8C) bright field. The image in (Figure 8D) was generated by combining the images from (Figure 8A)-(Figure 8C). [Figure 9]Figure 9 shows the efficacy of BPO loading and SmartNovaC coated SmartArrow nanoparticles. The number of colony forming units (CFLI) in each sample was determined by plating 10-fold serial dilutions and compared to the number in buffer-treated controls by two-tailed Student's t-test with Welch's correction. *p<0.05; *p<0.01. [Figure 10] Figure 10 shows the results of predicting the secondary structure positions of supernova using JPRED. JPRED, a secondary structure prediction web server, suggests that residue 171 is a suitable starting residue to define the CBD of supernova. [Figure 11] Figure 11 shows a picture of NovaC visualized using the software VMD. The dark helix (white arrow) consisting of 17 residues was removed to create SmartNovaC. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The references mentioned in this specification are listed for convenience in the form of a reference list and added at the end of the examples. The entire contents of such references are incorporated herein by reference, but their mention in this specification does not mean that they form part of the common general knowledge.

[0021] definition For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Generally, technical, scientific and medical terms used herein have the same meaning as 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 are not intended to limit the scope of the present invention to these terms alone, but are presented to better understand the following description. As used herein, "a" or "an" may mean one or more, unless indicated to the contrary or clear from the context.

[0022] 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. As used in this context, an "antimicrobial agent" preferably kills or inhibits the growth of the bacteria C. acnes.

[0023] As used herein, "anti-acne actives" refers to any natural or synthetic substance that may have a beneficial cosmetic effect and / or beneficial therapeutic effect on the skin disease acne and its associated symptoms and conditions. As used in this context, an "anti-acne active" may, for example, reduce or control the number of acne spots, blackheads, and whiteheads, reduce or control sebum production, reduce, control or soothe acne-related inflammation / swelling (e.g., reducing the appearance of redness on the skin), and / or control the occurrence of acne in a patient. As will be appreciated by those skilled in the art, some anti-acne actives may only provide a beneficial cosmetic effect, while other anti-acne actives may provide a beneficial therapeutic effect or both a beneficial therapeutic effect and a beneficial cosmetic effect.

[0024] As used herein, the term "cargo" refers to any compound / drug that is intended to be delivered to a specific cell via a delivery system and induce a response.As used in this context, the cargo can be any compound or drug, such as an antibacterial agent or an anti-acne active agent, that is intended to be transported by the delivery system of the present disclosure to a location where C.acnes is present and released within the system so that the cargo can interact with the bacteria and induce a local effect.

[0025] As used herein, the term "comprise" or "containing" should be interpreted as specifying the presence of a stated feature, integer, step or component, but does not exclude the presence or addition of one or more features, integers, steps or components, or groups thereof. However, in the context of this disclosure, the term "comprise" or "containing" also includes "consisting of." Variations of the term "comprising," such as "comprise" and "comprises," and variations of the term "including," such as "include" and "includes," have correspondingly modified meanings.

[0026] As used herein, the terms "efficacious amount" and "effective amount" are used interchangeably and refer to an amount of a cargo and / or composition containing a cargo sufficient to provide a beneficial or desired result against C. acnes and / or acne. As used in this context, an effective amount of a composition of the present disclosure can provide a beneficial cosmetic effect against acne, such as, for example, killing and / or inhibiting C. acnes, reducing inflammation in or around acne, and improving the redness or appearance of acne-affected skin.

[0027] The term "functional fragment" refers to a portion of a protein that retains some or all of the activity or function (e.g., a biological activity or function, such as an enzymatic activity) of the full-length protein, e.g., the ability to bind and / or interact with or modulate another protein or nucleic acid. A functional fragment can be of any size, so long as it retains the ability to bind and interact with, e.g., another protein or nucleic acid.

[0028] The term "variant" as used herein refers to an amino acid sequence that is modified by one or more amino acids of the non-mutant reference sequence, but retains the ability to recognize and affect its target function. For example, a targeting peptide variant is modified by one or more amino acids of the non-mutant reference sequence, but retains the ability to recognize and bind to the cell wall of C. acnes. A variant may have a "conservative" change, where the substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have a "non-conservative" change (e.g., replacement of glycine with tryptophan). Similar minor variations can also include amino acid deletions or insertions, or both. Guidance on determining which amino acid residues can be substituted, inserted, or deleted without impairing biological activity can be found using computer programs well known in the art, such as DNASTAR® software (DNASTAR, Inc., Madison, Wis., USA).

[0029] The term "nucleotide" refers to naturally occurring ribonucleotide or deoxyribonucleotide monomers, as well as non-naturally occurring derivatives and analogs thereof. Nucleotides can include, for example, nucleotides that contain naturally occurring bases (e.g., adenosine, thymidine, guanosine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine, or deoxycytidine), as well as nucleotides that contain modified bases known in the art. Thus, the term "polynucleotide" herein generally refers to polyribonucleotides and polydeoxyribonucleotides, which may be unmodified RNA or DNA, or modified RNA or DNA.

[0030] 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" includes naturally occurring full-length proteins and artificial (e.g., synthetic or mutated) full-length proteins, as well as functional fragments of proteins.

[0031] As used herein, the term "recombinant" means that a molecule (e.g., a nucleic acid or polypeptide) has been artificially or synthetically altered by human intervention (i.e., non-naturally). Alterations can be made to the molecule in its natural environment or condition or removed from its natural environment or condition.

[0032] Exemplary, non-limiting embodiments of the present invention are described below.

[0033] The present disclosure is based in part on the development of recombinant peptides engineered to favorably bind to Cutibacterium acnes, a bacterial species closely related to P. acnes. In this regard, the inventors have successfully modified a natural lysin from a bacteriophage named phage Supernova to improve its solubility and binding affinity to the cell wall of C. acnes. Thus, the engineered targeting peptides disclosed herein have high solubility and high specificity for C. 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.

[0034] [Table 1] TIFF2025507835000003.tif133165

[0035] As described herein, the modified targeting peptides of the present disclosure act as a homing mechanism to deliver the cargo of interest to its designated location, and thus can be linked to a cell delivery system for a more precise and targeted drug delivery system. Thus, the nanoparticles, compositions and methods of the present disclosure are designed to advantageously target only C. acnes and not any other bacteria, thereby protecting the entire skin microbiome without significant off-target effects.

[0036] In one aspect, a composition is provided that comprises: i) a nanoparticle; ii) a targeting peptide that binds to the cell wall of Cutibacterium acnes; and iii) a cargo comprising one or more antibacterial and / or anti-acne active agents, wherein the nanoparticle encapsulates the cargo, and the targeting peptide is one of the surface components of the nanoparticle.

[0037] The compositions described herein allow for efficient targeted delivery of cargo of interest (e.g., antibacterial agents) directly to C. acnes. In particular, the nanoparticles and compositions disclosed herein can be coated with targeting peptides to enhance selectivity against C. acnes. Both covalent and non-covalent methods can be used to coat the peptides.

[0038] In some embodiments, the targeting peptide comprises the amino acid sequence set forth in SEQ ID NO:3, a functional fragment or variant thereof having Cutibacterium acnes cell wall binding activity.

[0039] As those skilled in the art understand, 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 the sequence identity of a protein are known in the art.Therefore, the sequence of the targeting peptide disclosed herein can be varied sufficiently as long as the targeting peptide maintains its functionality and can exhibit the required activity (e.g., the targeting peptide can bind to the cell wall of C. acnes).

[0040] Thus, in certain embodiments, the targeting peptide can comprise amino acids that are 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 amino acid sequence set forth in SEQ ID NO: 3. In particular, the targeting peptide can consist of the amino acid sequence set forth in SEQ ID NO:3.

[0041] In some embodiments, the targeting peptide can comprise amino acids 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 set forth in SEQ ID NO: 5. In some embodiments, the targeting peptide can consist of the amino acid sequence set forth in SEQ ID NO:5.

[0042] In another embodiment, the isolated recombinant Cutibacterium acnes-targeting peptide can comprise amino acids that have at least 85%, at least 90%, at least 95% or 100% identity to the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:5 and have Cutibacterium acnes cell wall binding activity.

[0043] As those skilled in the art will understand, peptides may be coded by a sequence of nucleotides, which are read in groups of three nucleotides known as codons.Thus, in some embodiments, targeting peptides may be coded by polynucleotides that contain 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 set forth in SEQ ID NO:4 due to redundancy in the genetic code.

[0044] In certain embodiments, the targeting peptide may be encoded by a polynucleotide comprising a nucleic acid sequence having 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 to the nucleic acid sequence set forth in SEQ ID NO:6 due to redundancy in the genetic code.

[0045] In another embodiment, the present invention provides an isolated recombinant DNA molecule comprising a DNA sequence encoding a Cutibacterium acnes-targeting peptide disclosed herein. In some embodiments, the DNA sequence encoding the targeting peptide has 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 to the nucleic acid sequence set forth in SEQ ID NO: 4 or 6 due to redundancy in the genetic code.

[0046] In a further embodiment, an expression vector is provided that comprises the recombinant DNA molecule of the present disclosure.It should be understood that expression vector is a construct designed for gene expression in cells, and is typically used for protein production.The method of constructing expression vector is well known in the art.

[0047] Thus, in another aspect, there is provided the use of an expression vector disclosed herein for the recombinant production of a Cutibacterium acnes-targeting peptide.

[0048] In a further embodiment, a method is provided for the production of a recombinant Cutibacterium acnes-targeting peptide of the present disclosure, comprising the steps of: (i) culturing eukaryotic or prokaryotic cells transfected with a recombinant DNA or expression vector in a culture medium disclosed herein, and (ii) recovering the expressed recombinant Cutibacterium acnes-targeting peptide from the cells or culture medium.

[0049] As described herein, the compositions of the present disclosure may be loaded with any cargo that is desired to be delivered to and interact with C. acnes. For example, the cargo may exhibit antimicrobial properties, anti-acne properties, anti-inflammatory properties and / or therapeutic effects against C. acnes. It will be understood by those skilled in the art that any molecule or agent that may provide beneficial effects may be appropriately selected as a therapeutic, cosmetic or other cargo in controlling, improving and / or treating acne.

[0050] In some embodiments, the cargo may include one or more antibacterial agents. For example, the antibacterial agent may be a small molecule. The antibacterial agent may include, but is not limited to, benzoyl peroxide, sulfur, azelaic acid, and may include antibiotics such as ozenoxacin, nadifloxacin, doxycycline, minocycline, azithromycin, erythromycin, clindamycin, and dapsone. In some embodiments, the one or more antibacterial agents may include antibiotics such as benzoyl peroxide, azelaic acid, erythromycin, clindamycin, dapsone, and / or combinations thereof.

[0051] In some embodiments, the cargo can include one or more anti-acne actives. Anti-acne actives can include, but are not limited to, alpha hydroxy acids such as glycolic acid, lactic acid; beta hydroxy acids such as salicylic acid; retinoids such as adapalene, tretinoin, isotretinoin, tazarotene, alitretinoin, bexarotene, resorcinol, retinyl esters, retinal, retinol; flavonoids such as niacinamide, resorcinol, and / or niacinamide. In some embodiments, the cargo can include one or more antibacterial and / or anti-acne actives.

[0052] In certain embodiments, the anti-acne actives can include compounds and / or extracts that may have cosmetic benefits in managing and / or ameliorating acne, such as tea tree oil, propolis extract, green tea extract, rice extract, astringents, anti-inflammatory compounds, or mixtures thereof.

[0053] Thus, in some embodiments, the compositions disclosed herein may be suitable for cosmetic use based on the appropriate cargo selected.

[0054] To facilitate delivery of the cargo to the location of the bacteria, the cargo can be encapsulated in nanoparticles. It will be appreciated that nanoparticles have been utilized in the medical / pharmaceutical field as drug carriers by encapsulating or attaching therapeutic molecules and delivering them more precisely to the target tissue with controlled release.

[0055] In this regard, nanoparticles are typically submicron (<1 μm) colloidal particles that exhibit unique structural, chemical, mechanical, magnetic, electrical, and biological properties. Nanoparticles can be made from biocompatible and biodegradable materials such as lipids, natural polymers (e.g., gelatin, albumin, alginate, chitosan) or synthetic polymers (polylactic acid-co-glycolic acid, polyalkylcyanoacrylates, etc.). In some embodiments, nanoparticles comprise lipid-based structures such as liposomes or micelles. In some embodiments, the nanoparticles can include polylactic 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-phosphorylglycerol sodium salt (DMPG), or dipalmitoylphosphatidylcholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE).

[0056] Those skilled in the art will appreciate that the selection and / or preparation of suitable nanoparticles is based on factors such as the biophysical and biochemical properties of the cargo of interest and the target location. For example, the cargo may be encapsulated by the nanoparticles via hydrophobic effects, electrostatic interactions and / or covalent bonds, depending on the physicochemical properties of the cargo. Thus, the preparation of suitable nanoparticles may be adapted by those skilled in the art accordingly.

[0057] In some embodiments, the nanoparticles can have an anionic surface charge, a cationic surface charge, or a neutral surface charge. In particular, the nanoparticles can have an anionic surface charge.

[0058] Thus, in a further aspect, there is provided a method for producing Cutibacterium acnes targeted nanoparticles comprising the steps of mixing a cargo comprising one or more antibacterial and / or anti-acne actives 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; and mixing a Cutibacterium acnes targeting peptide of the present disclosure with the PLGA+cargo nanoparticles until the nanoparticles are coated with said targeting peptide. In some embodiments, the PLGA and PVA are replaced by 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol sodium salt (DMPG), or dipalmitoylphosphatidylcholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE).

[0059] In some embodiments, the nanoparticles and compositions disclosed herein can further comprise a pharma- ceutically acceptable carrier. A suitable pharmaceutical carrier typically contains inactive ingredients that do not interact with the drug or active ingredient. Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, saline, bacteriostatic saline (saline containing about 0.9% mg / ml benzyl alcohol), phosphate buffered saline, Hank's solution, lactated Ringer's solution, and the like. The formulation can also contain small amounts of substances that enhance the effectiveness of the active ingredient (e.g., emulsifiers, solubilizers, pH buffers, wetting agents). Methods of encapsulating compositions (e.g., coatings of hard gelatin or cyclodextran) are known in the art. For inhalation, the drug can be solubilized and loaded into a suitable dispenser for administration (e.g., a nebulizer, inhaler, or pressurized aerosol dispenser).

[0060] For in vivo delivery, the nanoparticles and compositions disclosed herein can be delivered to a subject in need thereof by a variety of administration routes, including, for example, oral, dietary, topical, transdermal, or parenteral (intra-arterial, intravenous, intramuscular, subcutaneous, intradermal, etc.) routes of administration. Administration can be local or systemic. The actual dosage and treatment regimen of the nanoparticles and / or compositions disclosed herein can be determined by a skilled physician, taking into account the nature of the condition being treated and the characteristics of the patient.

[0061] In some embodiments, the compositions disclosed herein can be preferably formulated for topical application.Preferably, topical formulation can be in the form of liquid solution or mixture, dispersion, suspension, gel, lotion, emulsion, paste, cream, ointment, milk, pomade, spray or medicated dressing, pad or mask.It will be understood that the method of preparing topical formulation is based on standard principles and methods known in the art and described in various pharmaceutical literatures.

[0062] In another aspect, there is provided a use of the disclosed compositions in the manufacture of a medicament for the treatment or prevention of acne. In some aspects, the compositions and medicaments disclosed herein are for selectively killing and / or targeting Cutibacterium acnes on human skin. In some aspects, the medicament is in the form of a cream, gel, or ointment.

[0063] In a further aspect, there is provided a method of treating or preventing acne comprising administering to a subject in need of such treatment an effective amount of a composition of the present disclosure.

[0064] Unless otherwise indicated or the context and understanding of one of ordinary skill in the art dictates otherwise, values ​​expressed as ranges can assume any particular value or subrange within the stated range in various embodiments, to the tenth of the unit of the lower limit of the range, unless the context dictates otherwise. "About" with respect to a numerical value generally refers to a range of values ​​that falls within ±10%, in some embodiments ±5%, in some embodiments ±1%, and in some embodiments ±0.5% of the value, unless otherwise indicated or the context dictates otherwise. In any embodiment in which a numerical value begins with "about", an embodiment is provided in which the exact value is recited. When an embodiment is provided in which a numerical value does not begin with "about", an embodiment in which the value begins with "about" is also provided. When a range begins with "about", an embodiment is provided in which "about" applies to the lower and upper limits of the range, or to either the lower or upper limit, unless the context clearly dictates otherwise. When "at least," "up to," "less than," or similar words precede a series of numbers, it is to be understood that the words apply to each number in the list in various embodiments (understanding that, depending on the context, 100% of a percentage value may be an upper limit, for example). For example, "at least 1, 2, or 3" is to be understood to mean "1 or more, 2 or more, or 3 or more" in various embodiments. It is also to be understood that all reasonable lower and upper limits are expressly contemplated.

[0065] Having generally described the invention above, 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. EXAMPLES

[0066] Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (2012).

[0067] Example 1: Production of lysins targeting C. acnes A natural lysin, named Supernova, was selected as the starting sequence template to generate the artificial lysin. Supernova lysin was obtained from a bacteriophage, named Supernova, that targets Cutibacterium acnes. The C-terminal cell wall binding domain (CBD) of Supernova, i.e., NovaC, was determined to be amino acids 171 to 287 based on the results of BLAST and JPRED websites. However, the expression of NovaC was low, and its binding to C. acnes was very weak, as shown by the confocal microscopy data (Figures 2A and 2B).

[0068] To improve the solubility of NovaC, the structure of NovaC was modeled using the software I-TASSER and visualized using the software VMD. From the in silico structure, we designed an engineered lysin called SmartNovaC by truncating amino acids 2 to 18 of NovaC, which removes the α-helix that does not interact with the rest of the protein.

[0069] The gene for the native lysin Supernova (Genbank accession number ATN91960.1) was synthesized and cloned into the pNIC28-Bsa4 plasmid. Engineered lysin genes (NovaC and SmartNovaC) tagged with N-terminus enhanced green fluorescent protein (EGFP) were synthesized and cloned into the pET-22b (+) plasmid. All nucleotide sequences were codon-optimized to improve the efficiency of soluble expression in E. coli. The amino acid and nucleotide sequences of the native and recombinant lysins are shown in Table 1.

[0070] To produce recombinant lysin, a plasmid carrying the gene of interest is transformed into E. coli competent cells and the protein is overexpressed using IPTG induction and purified using immobilized metal affinity chromatography and size exclusion chromatography.

[0071] To investigate the binding spectrum of the recombinant lysin SmartNovaC, 2.5 mg / ml of SmartNovaC was applied against four C. acnes strains, Enterococcus faecalis OG1RF, Staphylococcus epidermidis PCI1200 and Pseudomonas aeruginosa PA14. SmartNovaC was cloned into a plasmid co-expressing an EGFP tag, so that the fluorescent lysin could be visualized using confocal microscopy. Green-fluorescent SmartNovaC showed specific binding to all four strains of C. acnes (Fig. 1, A1-3, B1-3, C1-3, D1-3), but failed to bind to E. faecalis OG1RF (Fig. 1, E1-3), P. aeruginosa PA14 (Fig. 1, F1-3) or S. epidermidis PCI1200 (Fig. 1, G1-3). This result clearly indicates the specific binding activity of SmartNovaC against C. acnes.

[0072] To further investigate whether SmartNovaC is the only lysin that specifically binds to C. acnes, we tested the binding of another lysin, DukeC2Rap, which was synthesized by coexpressing NovaC with EGFP. The DukeC2Rap lysin was obtained as the CBD of Doucette lysin, which targets Propionibacterium freudenreichii, a species of the same genus as C. acnes. The nucleotide and amino acid sequences of DukeC2Rap are shown below.

[0073] [Table 2]

[0074] NovaC and DukeC2Rap were tested for binding to C. acnes strains at concentrations of 2.5 mg / ml and 1.3 mg / ml, respectively. NovaC showed some binding to C. acnes (Fig. 2, A1-3, B1-3), but a much weaker binding signal than SmartNovaC (Fig. 1, A1-3, B1-3, C1-3, D1-3). This strongly suggests that the engineered SmartNovaC lysin has enhanced binding specificity to C. acnes. DukeC2Rap did not show any binding to C. acnes (Fig. 2, C1-3, D1-3). Thus, among the different lysins tested, only the engineered lysin SmartNovaC has specific binding to C. acnes species.

[0075] Example 2: Preparation of a Therapeutic Composition Because SmartNovaC itself does not directly kill bacteria, SmartNovaC needs to be combined with an antibacterial agent. For example, to apply this technology to the development of acne treatment drugs, anti-acne active ingredients such as benzoyl peroxide (BPO) can be combined with SmartNovaC. As a result, a novel SmartArrow system was created by loading the active ingredient into lipid / polymer-based nanoparticles and coating the surface of the nanoparticles with SmartNovaC to selectively target the antibacterial agent to C. acnes, as shown in Figure 3.

[0076] Preparation of anionic liposomes One embodiment of the structure of the composition of the present invention is shown in Figure 3. Exemplary anionic liposomes were made from 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol sodium salt (DMPG) in a molar ratio of 10:1. Liposome size and stability can be optimized by altering the molar ratio, using different lipid types, and adding cholesterol, polyethylene glycol (PEG) and other additives that can change the behavior of the liposomes.

[0077] The advantage of SmartArrow is that it delivers the active ingredient where the bacteria are present. As a result, much lower doses of bioactives can be loaded into SmartArrow compared to non-targeted liposomes, and high bacterial kill can be achieved. As little as 0.002% BPO can achieve complete kill of C.acnes (Figure 4). Given that BPO concentrations found in the retail market range from 2.5% -10%, this is about 1000 times higher than what is needed to kill the bacteria. It is not surprising to find that most users of these retail products suffer from several side effects, such as dry peeling of the skin, on a daily basis. Using the SmartArrow approach, we are able to conservatively load 0.02% BPO into the liposomes, which is 100 times less than the retail product. This allows us to achieve complete bacterial kill and minimize side effects.

[0078] Preparation of PLGA+BPO nanoparticles To load BPO into nanoparticles, poly(D,L-lactide-co-glycolide) (PLGA) was chosen because it is known to form nanoparticles with excellent loading of hydrophobic compounds such as BPO.

[0079] BPO-containing PLGA particles were prepared using a single emulsion technique as previously described (Jain RA.2000. Biomaterials 21:2475-2490). Briefly, 5 g PLGA and 5 g BPO were each dissolved in 500 μl chloroform. Then, PLGA and BPO were added to 5 ml of 1% polyvinyl alcohol (PVA), and the mixture was immediately sonicated at 23% amplitude for 5 min. As shown in Figure 5, after sonication of the oil phase (containing PLGA and BPO) and the aqueous phase (containing PVA), uniform emulsified nanoparticles were formed. To extract and remove the organic solvent, the mixture containing the emulsified PLGA+BPO nanoparticles was stirred at 700 rpm at room temperature for 6 h in a fume hood. To purify the PLGA+BPO nanoparticles, the mixture was centrifuged at 6,000 rpm for 15 min to obtain a pellet containing the nanoparticles. The pellet was resuspended in 2 ml of ultrapure water and spun down to remove all free BPO. The final pellet of purified PLGA+BPO nanoparticles was resuspended in 2 ml of ultrapure water and stored at 4 °C until use.

[0080] The PLGA+BPO nanoparticles were characterized using dynamic light scattering (DLS) and mass spectrometry. DLS measured the nanoparticles to have an average size of 150 nm with a zeta potential of -18 mV. The amount of BPO loaded into the PLGA particles was quantified using mass spectrometry, as shown in Figure 6. We have shown that up to 5 mg of BPO can be efficiently loaded into 5 mg of PLGA particles. Much smaller amounts may be loaded in the SmartArrow application of the present invention.

[0081] Preparation of DPPC+DSPE+BPO nanoparticles BPO-loaded DPPC+DSPE nanoparticles were prepared using 4 mg DPPC and 1 mg DSPE and 1 mg BPO. Briefly, 4+1 mg DPPC+DSPE lipids and 1 mg BPO were each dissolved in 500 μl chloroform. Then, DPPC+DSPE lipids and BPO were added to 4 ml chloroform and the mixture was left overnight in a fume hood to allow the chloroform to evaporate. The next day, 5 ml of 1x phosphate buffer solution (PBS) was added to rehydrate the dried lipid film. The solution was sonicated at 70 °C for 5 min. The mixture went through a 400 nm extruder process as a filtration step to form uniform nanoparticles. The filtered sample went through tangential flow filtration (TFF) with ultrapure water to separate the free BPO from the BPO-loaded nanoparticles. The final product was stored at 4 °C until use.

[0082] Coating of SmartNovaC onto anionic liposomes and PLGA nanoparticles by non-covalent methods. Anionic liposomes and PLGA nanoparticles were coated with positively charged SmartNovaC targeting peptide (both GFP-fused and free) using charge-based conjugation. Coating was performed by mixing SmartNovaC peptide and liposome / PLGA nanoparticles in a 2:1 ratio and vortexing the mixture for 2 h. To purify the protein-conjugated nanoparticles, the thoroughly vortexed mixture was centrifuged at 6,000 × g for 15 min. The pellet was resuspended in 300 μl of ultrapure water and spun down by centrifugation to remove all unbound protein. The final pellet was resuspended in 100 μl of 1× phosphate-buffered saline (PBS) and stored at 4 °C until use.

[0083] The resulting particles were characterized using dynamic light scattering (DLS). SmartNovaC-coated liposomes increased in size and decreased in zeta potential (Figure 7A). This is consistent with the fact that positively charged SmartNovaC coated on anionic liposomes reduces the overall charge of the particles. Similarly, Figure 7C shows a decrease in the zeta potential of PLGA nanoparticles upon SmartNovaC coating. Figures 7B and 7D show an increase in GFP fluorescence signal, thus indicating that GFP-fused SmartNovaC was bound and coated on the nanoparticles.

[0084] Although the coating approach shown in Figure 7 was based on the principle of charge-based electrostatic interactions, more directed protein-nanoparticle bioconjugation can also be performed to ensure that SmartNovaC retains selective binding to C. acnes.

[0085] Coating of SmartNovaC onto lipid- or polymer-based nanoparticles by site-specific covalent conjugation. Thiol-maleimide reaction was used for conjugation where thiol groups were found in the SmartNovaC protein and maleimide groups were covalently attached to lipids (e.g. DSPE and DPPC lipids) or polymers (e.g. PLGA). In nanoparticles, only 20-30% of the lipid / polymer substrate contained maleimide groups to avoid overcrowding of conjugated proteins, which may affect the targeting performance. According to DLS, the size of nanoparticles when loaded with BPO and coated with conjugated SmartNovaC ranges from 150-400 nm. Figure 8 shows the confocal microscopy images in greyscale of SmartNovaC-coated, Nile Red-loaded polymer nanoparticles binding to C. acnes. Both GFP (Figure 8A) and Nile Red (Figure 8B) signals overlap in the merged image in Figure 8D, indicating nanoparticle binding to bacterial cells. FIG. 9 shows that the BPO-loaded SmartArrow successfully killed bacteria, ie, 90% of the bacteria at 0.01% BPO and 99% of the bacteria at 0.1% BPO.

[0086] summary Here we present SmartNovaC, an 11 kDa cell wall-binding protein derived from a bacteriophage lysin. SmartNovaC has been conclusively shown to be able to specifically bind various strains of Cutibacterium acnes (C. acnes) and not other bacteria. A delivery system containing SmartNovaC and an active ingredient such as benzoyl peroxide allows for the selective targeting and killing of C. acnes, thus respecting the skin microbiome. It is estimated that by incorporating SmartNovaC in a product formulation, the effective concentration of the active ingredient can be reduced by 100-fold compared to existing anti-acne products on the market.

[0087] The SmartArrow targeted delivery system involves loading anti-acne actives into polymer / lipid-based nanoparticles and coating the surface of the nanoparticles with SmartNovaC peptide. Both covalent and non-covalent methods can be used for coating with SmartNovaC. Thus, SmartArrow can be advantageously used in the skin care industry as a targeted delivery system for topical cosmetic or therapeutic acne treatments.

[0088] References Jain RA. 2000. The manufacturing techniques of various drug loaded biodegradable poly (lactide-co-glycolide) (PLGA) devices. Biomaterials 21: 2475-2490. Phage supernova genome: worldwideweb.ncbi.nlm.nih.gov / nuccore / MF919533

Claims

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

2. The targeting peptide has an amino acid sequence that has at least 90%, at least 95% or 100% identity to the following amino acid sequence: FMAVVNGHGGGSSSEELTVADVKALHNQIKQLSAQLSGSVNKLHHDVGVVQVQNGDLSKRVDALSWVKNPVTGKLWRTKDALWSVWYYVLECRSRIDRLESAVNGLKK (SEQ ID NO: 3) or a functional fragment or variant thereof having Cutibacterium acnes cell wall binding activity.

3. The targeting peptide of claim 2, wherein the targeting peptide has the amino acid sequence: FMAVVNGHGGGSSSEELTVADVKALHNQIKQLSAQLSGSVNKLHHDVGVVQVQNGDLSKRVDALSWVKNPVTGKLWRTKDALWSVWYYVLECRSRIDRLESAVNGLKK (SEQ ID NO: 3) or a functional fragment or variant thereof having Cutibacterium acnes cell wall binding activity.

4. The targeting peptide has the following amino acid sequence: MGGGSSSEELTVADVKALHNQIKQLSAQLSGSVNKLHHDVGVVQVQNGDLSKRVDALSWVKNPVTGKLWRTKDALWSVWYYVLECRSRIDRLESAVNGLKK (SEQ ID NO: 5) 3. The composition of claim 2, comprising an amino acid sequence having at least 85%, at least 90%, at least 95% or 100% identity to

5. The composition of claim 1 , wherein the antimicrobial agent is a small molecule.

6. 10. The composition of claim 1, wherein the one or more antibacterial agents comprise benzoyl peroxide, azelaic acid, erythromycin, clindamycin, dapsone, and / or combinations thereof.

7. The composition of claim 1, wherein the antibacterial agent is benzoyl peroxide administered at a content of 0.01% to 2.5%, 0.01% to 0.1%, or 0.02% to 0.05%.

8. 10. The composition of claim 1, wherein the nanoparticles are selected from the group consisting of 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 nanoparticles are 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-phosphorylglycerol sodium salt (DMPG); or iii) Dipalmitoylphosphatidylcholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE) 10. The composition of claim 9, comprising:

11. The cargo is 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 10. The composition of claim 1, comprising an anti-acne active agent selected from:

12. The composition of claim 1 further comprising a pharmaceutically acceptable carrier.

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

14. 14. The use according to claim 13, wherein the medicament is for selectively killing and / or targeting Cutibacterium acnes on human skin.

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

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

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

18. 18. The isolated recombinant DNA molecule of claim 17, wherein the DNA sequence encoding the targeting peptide has, due to redundancy in the genetic code, at least 80%, at least 85%, at least 90%, at least 95%, or 100% nucleic acid sequence identity to the nucleic acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:

6.

19. 18. An expression vector comprising the recombinant DNA molecule of claim 17.

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

21. An isolated recombinant Cutibacterium 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 set forth in SEQ ID NO: 3 or SEQ ID NO: 5, and having Cutibacterium acnes cell wall binding activity.

22. A method for producing the recombinant Cutibacterium acnes targeting peptide of any one of claims 1 to 5, comprising: (i) culturing a eukaryotic or prokaryotic cell transfected with the recombinant DNA molecule of claim 17 in a culture medium; and (ii) recovering the expressed recombinant Cutibacterium acnes targeting peptide from said cells or said culture medium.

23. 1. A method for producing Cutibacterium acnes-targeting nanoparticles, comprising: i) mixing a cargo comprising one or more antibacterial 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; 10. Mixing the Cutibacterium acnes targeting peptide of claim 2 or 4 with the PLGA+cargo nanoparticles until the nanoparticles are coated with the targeting peptide; or ii) mixing cargo comprising one or more antibacterial and / or anti-acne active agents with lipids to form anionic liposome+cargo nanoparticles; 10. A method comprising the step of mixing the Cutibacterium acnes targeting peptide of claim 2 or 4 with the cargo nanoparticles until the nanoparticles are coated with the targeting peptide.

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

Images