EP4719445A1 - Phage-extracellular vesicle conjugate - Google Patents

Phage-extracellular vesicle conjugate

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Publication number
EP4719445A1
EP4719445A1 EP24813608.7A EP24813608A EP4719445A1 EP 4719445 A1 EP4719445 A1 EP 4719445A1 EP 24813608 A EP24813608 A EP 24813608A EP 4719445 A1 EP4719445 A1 EP 4719445A1
Authority
EP
European Patent Office
Prior art keywords
conjugate
phage
types
linker
polymer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24813608.7A
Other languages
German (de)
French (fr)
Inventor
Thi Thu Hien DUONG
Wojciech Chrzanowski
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Sydney
Original Assignee
University of Sydney
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2023901709A external-priority patent/AU2023901709A0/en
Application filed by University of Sydney filed Critical University of Sydney
Publication of EP4719445A1 publication Critical patent/EP4719445A1/en
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
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    • C08F293/00—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule
    • C08F293/005—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule using free radical "living" or "controlled" polymerisation, e.g. using a complexing agent
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    • C08F2438/00—Living radical polymerisation
    • C08F2438/03—Use of a di- or tri-thiocarbonylthio compound, e.g. di- or tri-thioester, di- or tri-thiocarbamate, or a xanthate as chain transfer agent, e.g . Reversible Addition Fragmentation chain Transfer [RAFT] or Macromolecular Design via Interchange of Xanthates [MADIX]
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Abstract

The present invention relates to phage-extracellular vesicle (EV) conjugates, wherein the phage is linked with an EV by a linker, wherein the phage and the EV are independently conjugated to the linker by one or more types of cleavable bond, and methods for preparing phage-EV conjugates. The present invention also relates to methods of using the phage-EV conjugates, in particular for promoting tissue repair in an individual having damaged tissue such as associated with a bacterial infection.

Description

Phage-extracellular vesicle conjugate
Field of the invention
[0001] The present invention relates to phage-extracellular vesicle (EV) conjugates. The present invention also relates to methods for preparing phage-EV conjugates. The present invention further relates to methods of using the phage-EV conjugates.
Related application
[0002]This application claims priority from Australian provisional application
AU 2023901709, the entire contents of which are hereby incorporated by reference.
Background of the invention
[0003] Current approaches to combat infections that impair wound healing are ineffective.
[0004] The misuse and overuse of antibiotics has significantly increased the emergence of antimicrobial resistance. Bacteria can develop defences against antimicrobial therapies, rendering these lifesaving drugs ineffective. The problem of antimicrobial resistance is posing a high risk for health and healthcare systems globally. Antimicrobial-resistant pathogens of particular concern include the ESKAPE pathogens - Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. - which are responsible for causing various infections in humans, especially in hospital admitted patients and immunocompromised people. These pathogens can potentially evade the host immune defence system and resist antibiotics. New antibiotics are urgently needed; however, the development of new antibiotics is typically a lengthy and costly process and is quickly undermined by antimicrobial resistance.
[0005] For example, biofilm formation and antibiotic resistance are major causes of non-healing diabetic foot ulcers. Infections may also be present in up to 73% of failures of implantable orthopaedic devices and contribute to aseptic loosening.
[0006] The success of antimicrobial approaches is hampered by the absence of integrated strategies that (1) inhibit bacteria growth, (2) prevent biofilm formation, (3) activate the immune system, and (4) promote tissue repair. Efforts to address this challenge have failed to provide a satisfactory solution for at least three reasons. Firstly, they focus on only one aspect of the problem, addressing the antimicrobial activity or biofilm formation or immunomodulation. Secondly, they offer no integrated way to target infection sites and rapidly mitigate its spread. Thirdly, they do not take into consideration the recovery of the function of infected tissues.
[0007] Current approaches to overcome infections primarily focus on one aspect of the problem: inhibition of the bacteria growth and disruption of the biofilm - and offer no integrated approaches to mitigation of infections.
[0008] The primary mechanism of action of these strategies is (1 ) the disruption of the bacteria cell membrane and cell wall, (2) inference in the bacteria metabolism, (3) denaturation and coagulation of proteins and enzymes in cytoplasm and (4) DNA damage.
[0009] There is a need for a strategy that integrates different compounds that simultaneously provide antimicrobial activity, activate the immune system, and promote tissue repair of the infected tissue.
[0010] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.
Summary of the invention
[0011] The present inventors have developed phage-extracellular vesicle (EV) conjugates which are capable of promoting tissue repair in an individual having damaged tissue, wherein the tissue is in an individual who has, or is at risk of, impaired tissue repair associated with a bacterial infection.
[0012] Accordingly, in one aspect the present invention provides a conjugate of a phage linked with an extracellular vesicle (EV) by a linker, wherein the phage and the EV are independently conjugated to the linker by one or more types of cleavable bond.
[0013] In some embodiments, at least one of the one or more types of cleavable bond is pH labile. In preferred embodiments, at least one of the one or more types of cleavable bond are labile in acidic conditions, preferably labile at a pH of from about 4.0 to about 6.0, more preferably labile at about pH 5.5.
[0014] In preferred embodiments, at least one of the one or more types of cleavable bond is an imine.
[0015] In preferred embodiments, the one or more types of cleavable bond are the same. The cleavable bond is preferably an imine.
[0016] In some embodiments, the linker comprises a polymer which comprises the one or more types of cleavable bond. The polymer is preferably prepared by reversible addition-fragmentation chain-transfer (RAFT) polymerisation.
[0017] In some embodiments, the polymer comprises a diblock copolymer comprising a first block and a second block. In these embodiments, one of the first block and the second block may comprise the one or more types of cleavable bond, preferably the second block.
[0018] In preferred embodiments, the first block comprises poly(oligo(ethylene glycol) methyl ether acrylate) (POEGA).
[0019] In preferred embodiments, the second block comprises a polymer composed of repeating monomer units comprising one type of cleavable bond. The monomer unit preferably has the following structure: wherein
R is independently -CHO or -C=NZ; and
Z is independently a phage or an EV.
[0020] In preferred embodiments, the conjugate has the following structure: wherein
R is independently -CHO or -C=NZ;
Z is independently a phage or an EV; n is 9; x is 10 to 30, preferably 20 to 28, more preferably 20; and y is 5 to 15, preferably 10.
[0021] In some embodiments, the phage is capable infecting one or more pathogens selected from Escherichia coli, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. In preferred embodiments, the phage is capable of infecting Escherichia coli or Pseudomonas aeruginosa. In one preferred embodiment, the phage is capable of infecting Escherichia coli. In another preferred embodiment, the phage is capable of infecting Pseudomonas aeruginosa.
[0022] In preferred embodiments, the phage is selected from a filamentous phage, an MS2 phage and a caudovirus. In preferred embodiments, the phage is a caudovirus.
[0023] In some embodiments, the EV is derived from the group comprising: eukaryotic cells, bacteria, probiotics and combinations thereof. In a preferred embodiment, the EV is derived from: a stem cell, a probiotic and combinations thereof. Preferably, the EV derived from a stem cell is derived from a mesenchymal stromal cell.
[0024] In some embodiments, the conjugate comprises a phage linked with one type of EV. For example, the phage may be linked with an EV derived from a stem cell. In another example, the phage may be linked with an EV derived from a probiotic. [0025] In other embodiments, the phage may be linked with more than one type of EV. For example, the phage may be linked with an EV derived from a stem cell and an EV derived from a probiotic.
[0026] In some embodiments, the conjugate comprises, or further comprises, one or more types of active agent conjugated to the linker. Preferably, the one or more types of active agent may comprise one or both of (i) one or more antibacterial agents and (ii) one or more antibiofilm agents. Preferably, the one or more antibiofilm agents comprise a nitroxide group.
[0027] In some embodiments, the conjugate comprises, or further comprises, one or more types of imaging agent conjugated to the linker. In embodiments where an imaging agent is also suitable for use in therapy, the imaging agent and the active agent may be the same.
[0028] In another aspect, the present invention provides a method for preparing a conjugate of a phage linked with an EV by a linker, the method comprising: providing a linker comprising one or more types of reactive functionality capable of forming a cleavable bond; conjugating an EV to the linker via at least one of the one or more types of reactive functionality; and conjugating a phage to the linker via at least one of the one or more types of reactive functionality; thereby providing the conjugate, wherein the phage and the EV are each independently conjugated to the linker by one or more types of cleavable bond.
[0029] In some embodiments, at least one of the one or more types of reactive functionality is capable of forming a cleavable bond that is pH labile. In preferred embodiments, at least one of the one or more types of reactive functionality is capable of forming a cleavable bond that is labile in acidic conditions, preferable labile at a pH of from about 4.0 to about 6.0, more preferably labile at about pH 5.5.
[0030] In some embodiments, at least one of the one or more types of reactive functionality is capable of forming an imine. [0031] In preferred embodiments, at least one of the one or more types of reactive functionality is an aldehyde.
[0032] In preferred embodiments, the one or more types of reactive functionality are the same. The reactive functionality is preferably an aldehyde.
[0033] The method described herein may comprise a step of preparing a linker comprising one or more types of reactive functionality capable of forming a cleavable bond.
[0034] In some embodiments, the linker comprises a polymer which comprises the one or more types of reactive functionality. The polymer is preferably prepared by reversible addition-fragmentation chain-transfer (RAFT) polymerisation.
[0035] In some embodiments, the polymer comprises a diblock copolymer comprising a first block and a second block. In these embodiments, one of the first block or the second block may comprise the one or more types of reactive functionality capable of forming a cleavable bond, preferably the second block.
[0036] In preferred embodiments, the first block comprises poly(oligo(ethylene glycol) methyl ether acrylate) (POEGA).
[0037] In preferred embodiments, the second block comprises poly(3- vinylbenzaldehyde) (PVBA).
[0038] In preferred embodiments, the diblock copolymer comprises POEGA-b/oc - PVBA (POEGA-b-PVBA).
[0039] In preferred embodiments, the linker has the following structure: wherein n is 9; x is 10 to 30, preferably 20 to 28, more preferably 20; and y is 5 to 15, preferably 10.
[0040] In another aspect, the present invention provides a conjugate prepared by the method described herein.
[0041] In another aspect, the present invention provides a pharmaceutical composition comprising the conjugate described herein or prepared by the method of described herein, and a pharmaceutically acceptable diluent, excipient or carrier.
[0042] In one embodiment, the pharmaceutical composition comprises one type of conjugate. Preferably, the pharmaceutical composition comprising one type of conjugate comprises one type of phage and one type of EV.
[0043] In another embodiment, the pharmaceutical composition comprises a combination of at least two types of conjugates. The type of phage and/or the type of EV may be different between the at least two types of conjugates.
[0044] In another aspect, the present invention provides a method of promoting tissue repair in an individual having damaged tissue, the method comprising administering a therapeutically effective amount of the conjugate described herein, the conjugate prepared by the method described herein, or the pharmaceutical composition described herein to the individual in need thereof, thereby promoting tissue repair in the individual.
[0045] In another aspect, the present invention provides a method of reducing inflammation in an individual in need thereof, the method comprising administering a therapeutically effective amount of the conjugate described herein, the conjugate prepared by the method described herein, or the pharmaceutical composition described herein to the individual in need thereof, thereby reducing inflammation in the individual. Preferably, the inflammation is associated with damaged tissue, bacterial infection, or a combination thereof.
[0046] Preferably, the individual has, or is at risk of, impaired tissue repair associated with a bacterial infection. [0047] Accordingly, in another aspect, the present invention provides a method of preventing and/or treating a bacterial infection in an individual in need thereof, the method comprising administering a therapeutically effective amount of the conjugate described herein, the conjugate prepared by the method described herein, or the pharmaceutical composition described herein to the individual in need thereof, thereby preventing and/or treating a bacterial infection in the individual.
[0048] In one embodiment, the present invention provides a method of decreasing the damaged tissue area or volume of damaged tissue in an individual, the method comprising administering a therapeutically effective amount of the conjugate described herein, the conjugate prepared by the method described herein, or the pharmaceutical composition described herein to the individual in need thereof, thereby decreasing the damaged tissue area or volume of damaged tissue in the individual.
[0049] In one embodiment, the present invention provides a method of accelerating the rate of tissue repair, or decreasing the time to completion of tissue repair in an individual, the method comprising administering a therapeutically effective amount of the conjugate described herein, the conjugate prepared by the method described herein, or the pharmaceutical composition described herein to the individual in need thereof, thereby accelerating the rate of tissue repair, or decreasing the time to completion of tissue repair in the individual.
[0050] In one embodiment, the present invention provides a method of inducing or promoting or initiating a tissue repair mechanism in damaged tissue in an individual, the method comprising administering a therapeutically effective amount of the conjugate described herein, the conjugate prepared by the method described herein, or the pharmaceutical composition described herein to the individual in need thereof, thereby inducing or promoting or initiating a tissue repair mechanism in damaged tissue in the individual.
[0051] The damaged tissue may be in an individual who has, or is at risk of impaired tissue repair associated with a bacterial infection.
[0052] In one embodiment, the tissue may be selected from the group comprising: lung, bone, dermal, cutaneous, skin tissue and combinations thereof. [0053] In one embodiment, the present invention provides a method of promoting wound healing in an individual, the method comprising administering a therapeutically effective amount of the conjugate described herein, the conjugate prepared by the method described herein, or the pharmaceutical composition described herein to the individual in need thereof, thereby promoting wound healing in the individual.
[0054] In one embodiment, the present invention provides a method for the treatment of a dermal or cutaneous wound, the method comprising administering a therapeutically effective amount of the conjugate described herein, the conjugate prepared by the method described herein, or the pharmaceutical composition described herein to the individual in need thereof, thereby treating the dermal or cutaneous wound in the individual.
[0055] The wound is a chronic or an acute wound. In some embodiments, the wound arises from pressure, laceration, burn, incision, maceration, crushing, puncture abrasion or like injury. In some embodiments, the wound is associated with diabetes mellitus. In some embodiments, the individual has a prosthetic medical device.
[0056] In another aspect, the present invention provides the use of the conjugate described herein or prepared by the method described herein for:
- promoting tissue repair;
- reducing inflammation;
- treating a bacterial infection;
- decreasing the damaged tissue area or volume of damaged tissue;
- accelerating the rate of tissue repair, or decreasing the time to completion of tissue repair;
- inducing or promoting or initiating a tissue repair mechanism in damaged tissue;
- promoting wound healing; and/or
- the treatment of a dermal or cutaneous wound. [0057] The present invention also provides the use of the conjugate described herein or prepared by the method described herein for preventing and/or treating a bacterial infection.
[0058] In another aspect, the present invention provides the use of the conjugate described herein or prepared by the method described herein in the manufacture of a medicament for:
- promoting tissue repair;
- reducing inflammation;
- treating a bacterial infection;
- decreasing the damaged tissue area or volume of damaged tissue;
- accelerating the rate of tissue repair, or decreasing the time to completion of tissue repair;
- inducing or promoting or initiating a tissue repair mechanism in damaged tissue;
- promoting wound healing; and/or
- the treatment of a dermal or cutaneous wound.
[0059] The present invention also provides the use of the conjugate described herein or prepared by the method described herein in the manufacture of a medicament for preventing and/or treating a bacterial infection.
[0060] In another aspect, the present invention provides the conjugate described herein or prepared by the method described herein for use in:
[0061] - promoting tissue repair;
- reducing inflammation;
- treating a bacterial infection;
- decreasing the damaged tissue area or volume of damaged tissue; - accelerating the rate of tissue repair, or decreasing the time to completion of tissue repair;
- inducing or promoting or initiating a tissue repair mechanism in damaged tissue;
- promoting wound healing; and/or
- the treatment of a dermal or cutaneous wound.
[0062] The present invention also provides the conjugate described herein or prepared by the method described herein for use in preventing a bacterial infection.
[0063] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.
Brief description of the drawings
[0064] Figure 1. Particle size distribution of phage and EVs coated with the polymer and measured using nanoflow cytometry: (A) phage only, (B) PEV only, (C) DEV only, (D) polymer-coated phage, (E) polymer-coated PEV, and (F) polymer-coated DEV. An increase in particle size after polymer conjugation indicates successful attachment of the polymer to the EV and phage.
[0065] Figure 2. Overlaid 1H NMR spectra of (a) POEGA-b-PVBA and (b) POEGA-b- PVBA conjugated with EV. The conjugation of EV with copolymer POEGA-b-PVBA was determined by 1H NMR in deuterated dimethyl sulfoxide (DMSO-de) solvent.
[0066] Figure 3. TEM images of PEVs (A), phage Pae7 (B) and BiobotP (C) (scale bar = 50 nm). The samples were loaded onto carbon film copper grids and then negatively stained with 2% uranyl acetate.
[0067] Figure 4. Results of cytotoxicity for phage, EVs, polymers, BiobotP and BiobotD using epithelial cells and two assays: (A) Metabolic activity assay - PrestoBlue, and (B) DNA-based proliferation assay CyQUANT. An increase in metabolic activity and proliferation indicated non-cytotoxicity of biobots and any of the biobot components. [0068] Figure 5. (A) Results of cell-growth assays for phage, EVs, polymer, BiobotP and BiobotD at two concentrations of 105 and 107 particles/mL show no cytotoxicity of Biobots and their individual components up to the concentration 107 particles/mL. (B) The assessment of the morphology of epithelial cells treated with BiobotP, BiobotD, phage, DEV, PEV, and polymer shows no significant changes to morphology, which indicates no cytotoxicity.
[0069] Figure 6. Single-cell morphology and structure assessment using holotomography. Endothelial cells treated phage, DEV, PEV, polymer, BiobotP, and BiobotD. The reduction of vacuole and physiological morphology and structure of cells after the treatment with Biobots indicates improvements of cellular function and no cytotoxicity of biobots.
[0070] Figure 7. Results of cell growth assays for keratinocytes treated with BiobotP, BiobotD, phage, PEVs, DEVs, at three concentrations (1 k, 100k, 10000k particles per cell). An increase in cell growth for cells treated with BiobotD and BiobotP; the improvement of the cell growth was concentration dependent.
[0071] Figure 8. Results of quantifying of lactate dehydrogenase (LDH) expression, cell cytotoxicity assays, for keratinocytes treated with BiobotP, BiobotD, phage, PEVs, DEVs, at three concentrations (1 k, 100k, 10000k particles per cell). A significant reduction of LDH for cells treated with BiobotD and BiobotP indicates substantial improvement in cell function/growth.
[0072] Figure 9. Results of the wound closure assay using keratinocytes (HEK) treated with A) DEV, B) BiobotD, C) DEV + phage, and D) no treatment. An increase in wound closure for wounds treated with BiobotD and DEV+phage.
[0073] Figure 10. Results of the wound closure assay using keratinocytes (HEK) treated with A) PEV, B) BiobotP, C) PEV + phage, and D) phage only. An increase in wound closure for wounds treated with BiobotP and PEV+phage.
[0074] Figure 11. Results of the wound density assessment for keratinocytes (HEK) treated with DEV, PEV, phage, BiobotD, BiobotP, PEV+phage, DEV+phage. A significant increase in wound density (re-cellurisation of wound) for wounds treated with BiobotP, PEV+phage and BiobotD. [0075] Figure 12. Results of the wound closure rate assessment for keratinocytes (HEK) treated with DEV, PEV, phage, BiobotD, BiobotP, PEV+phage, DEV+phage. A significant increase in wound closure rate (a speed of re-cellularization of the wound) for wounds treated with BiobotP, PEV+phage and BiobotD.
[0076] Figure 13. Results of the wound closure assay using fibroblasts (HDF) treated with A) DEV, B) BiobotD, C) DEV + phage, and D) no treatment. An increase in wound closure for wounds treated with BiobotD and DEV+phage.
[0077] Figure 14. Results of the wound closure assay using fibroblasts (HDF) treated with A) PEV, B) BiobotP, C) PEV + phage, and D) phage only. An increase in wound closure for wounds treated with BiobotP and PEV+phage.
[0078] Figure 15. Results of the wound density assessment for fibroblasts (HDF) treated with DEV, PEV, phage, BiobotD, BiobotP, PEV+phage, DEV+phage. A significant increase in wound density (re-cellurisation of wound) for wounds treated with BiobotP, PEV+phage and BiobotD.
[0079] Figure 16. Results of the wound closure rate assessment for fibroblasts (HDF) treated with DEV, PEV, phage, BiobotD, BiobotP, PEV+phage, DEV+phage. A significant increase in wound closure rate (a speed of re-cellularization of the wound) for wounds treated with BiobotP, PEV+phage and DEV+phage, BiobotD.
[0080] Figure 17. Graph illustrating the antibacterial effects of EVs (103-107 EVs/mL), phages (103-107 PFU/mL), and biobots (ratios of EVs and phages of 1 :1 , 103-107 EVs/mL, 103-107 PFU/mL) against P. aeruginosa PAO1. A bacterial culture medium with 105-106 CFU/mL was prepared freshly in broth and added to a 2-fold serial dilution of different formulations in 96-well microplates. The microplates were incubated at 37°C for 24 h.
[0081]Figure 18. Graph illustrating the antibacterial effects of EVs (107 EVs/mL), phages (107 PFU/mL), biobots (107 EVs/mL, 107 PFU/mL) against E.coli JIE 3454. A bacterial culture medium with 105-106 CFU/mL was prepared freshly in broth and added to a 2-fold serial dilution of different formulations in microplates. The microplates were incubated at 37 °C for 24 hours. [0082] Figure 19. Graph illustrating the effects of different formulations on P. aeruginosa biofilm inhibition. Inoculated cultures were added with DEV (107 EVs/mL), PEV (107 EVs/mL), Pae7 phages (102 and 106 PFU/mL), BiobotD (107 EVs/mL, 102 PFU/mL or 106 PFU/mL) and BiobotP (107 EVs/mL, 102 PFU/mL or 106 PFU/mL) for 6 hours.
[0083] Figure 20. Demonstration of the difference in uptake of BiobotD and BiobotP in keratinocytes (cellular uptake in HEK, BiobotD vs. BiobotP). Biobots were labelled with AcoDyes-600 fluorescent probe in the cell uptake experiment. (A) HEK + BiobotD (cellular uptake of fluorescent-labeled BiobotD in HEK). (B) HEK + BiobotP (cellular uptake of fluorescent-labeled BiobotP in HEK).
[0084] Figure 21. Demonstration of the difference in uptake of BiobotD in different cells (cellular uptake of BiobotD in HEK vs. in HDF). Biobots were labelled with AcoDyes-600 fluorescent probe in the cell uptake experiment. (A) HEK + BiobotD (cellular uptake of fluorescent-labeled BiobotD in HEK). (B) HDF + BiobotD (cellular uptake of fluorescent- labeled BiobotD in HDF).
[0085] Figure 22. Demonstration of the distribution of biobots in different cells (distribution of biobots - intensity maps). (A) HEK + BiobotD (cellular uptake of fluorescent-labelled BiobotD in HEK). (B) HEK + BiobotP (cellular uptake of fluorescent- labeled BiobotP in HEK). (C) HDF + BiobotD (cellular uptake of fluorescent-labelled BiobotD in HDF). Stacks of images were taken from top to the bottom level of the cells.
[0086] Figure 23. Demonstration of the 3D distribution of Biobots in different cells (3D distribution of biobots - Z stack images). Images taken from HEK treated with BiobotD. Stacks of images taken from the bottom to the top level of the cells (increment of depth between images: 1 .2 pm), at 2.8 pm (A); 4.0 pm (B); 5.2 pm (C); 6.4 pm (D); 7.6 pm (E); and 8.8 pm (F).
[0087] Figure 24. Demonstration of the 3D distribution of Biobots in different cells (3D distribution of biobots - Z stack images). Images taken from HEK treated with BiobotP. Stacks of images taken from the bottom to the top level of the cells (increment of depth between images: 1 .2 pm), at 2.8 pm (A); 4.0 pm (B); 5.2 pm (C); 6.4 pm (D); 7.6 pm (E); and 8.8 pm (F).
[0088] Figure 25. Demonstration of the 3D distribution of Biobots in different cells (3D distribution of biobots - Z stack images). Images taken from HDF treated with BiobotD. Stacks of images taken from the bottom to the top level of the cells (increment of depth between images: 1 .2 pm), at 2.8 pm (A); 4.0 pm (B); 5.2 pm (C); 6.4 pm (D); 7.6 pm (E); and 8.8 pm (F).
[0089] Figure 26. Demonstration of Biobots’ healing effect on cells’ barrier function via impedance measurement. HEK damaged with acetone. Day 0 was measured just before acetone was added. Acetone was added at day 0 (dashed line at day 0) for 24 hours. Biobots were added as treatment at day 1 (dashed line at day 1 ).
[0090] Figure 27. Demonstration of Biobots’ healing effect on cells’ barrier function via impedance measurement. HEK damaged with hydrogen peroxide. Day 0 was measured just before hydrogen peroxide was added. Hydrogen peroxide was added at day 0 (dashed line at day 0) for 24 hours. Biobots were added as treatment at day 1 (dashed line at day 1 ).
[0091]Figure 28. Demonstration of Biobots’ healing effect on cells’ barrier function via impedance measurement. HDF damaged with acetone. Day 0 was measured just before acetone was added. Acetone was added at day 0 (dashed line at day 0) for 24 hours. Biobots were added as treatment at day 1 (dashed line at day 1 ).
[0092] Figure 29. Demonstration of Biobots’ healing effect on cells’ barrier function via impedance measurement. HDF damaged with hydrogen peroxide. Day 0 was measured just before hydrogen peroxide was added. Hydrogen peroxide was added at day 0 (dashed line at day 0) for 24 hours. Biobots were added as treatment at day 1 (dashed line at day 1 ).
[0093] Figure 30. Demonstration of the effect of Biobots on the stiffness of damaged cells. HDF were injured with bleomycin before treatment. HDF stiffness was measured after 24 hours of Biobot treatment.
[0094] Figure 31. Demonstration of Biobots’ effect on the morphology of damaged cells (HEK).
[0095] Figure 32. Demonstration of Biobots’ effect on the morphology of damaged cells (HDF).
Detailed description of the embodiments [0096] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.
[0097] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.
[0098] All of the patents and publications referred to herein are incorporated by reference in their entirety.
[0099] For the purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.
[0100] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are described. For the purposes of the present disclosure, the following terms are defined below.
[0101] The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “a reactive group” means one reactive group or more than one reactive group.
[0102] As used herein, the term “and/or”, e.g., “X and/or Y” will be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.
[0103] As used herein, the term “about” refers to a quantity, value, dimension, size, or amount that varies by as much as 10%, 5%, 1% or 0.1 % to a reference quantity, value, dimension, size, or amount. [0104] Throughout the present disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
[0105] As used herein, unless the context requires otherwise, the term “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
[0106] As used herein, the term “tissue repair” refers to healing of damaged tissue and includes the restoration of tissue architecture and function after an injury. Tissue repair encompasses tissue regeneration and tissue replacement.
[0107] Typically the injury is one arising from insult to lung, bone, dermal, cutaneous or skin tissue.
[0108] As used herein, the term "impaired tissue repair" refers to the repair of damaged tissue that does not heal at expected rates including slow-healing damaged tissue, delayed-healing damaged tissue, incompletely repaired damaged tissue, and chronic damaged tissue.
[0109] As used herein, the phrase " damaged tissue that does not heal at expected rates" refers to damaged tissue that is delayed or difficult to repair. Examples of damaged tissue that does not heal at expected rates include infected tissue, tissue adjacent to a prosthesis, and ulcers.
[0110] A “subject” herein is preferably a human subject. It will be understood that the terms “subject” and “individual” are interchangeable in relation to an individual requiring administration of the aqueous formulation of the present disclosure. [0111]The phrase “therapeutically effective amount” generally refers to an amount of one or more conjugates of the present invention that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.
[0112] “Extracellular vesicles” (EVs) generally refer to membrane-surrounded nanostructures secreted ubiquitously by cells (cellular nanoparticles). EVs typically contain cargo, which may be a therapeutic or drug cargo, for example one or more membrane proteins, cytosolic and nuclear proteins, extracellular matrix proteins, lipids, metabolites, and nucleic acids including DNA and RNA such as mRNA and non-coding RNA species. The EV may be a naive EV or an engineered EV. The term “naive EV” as used herein will be understood to mean an unmodified EV that is naturally produced by cells. EVs may be derived from any type of cell that secretes EVs including, but not limited to, eukarotyic cells, bacteria, probiotics and combinations thereof. Examples of suitable cells from which naive EVs can be derived include stem cells such as mesenchymal cells (MSCs); platelets; human induced pluripotent stem cells (hiPSCs), e.g., hiPSC derived neural stem cells; cells from body fluids including milk, such as human or bovine milk, and urine. The term “engineered EV” as used herein will be understood to mean vesicles that have been modified to express a targeting molecule on their surface and/or to carry a specific drug cargo. EVs mediate communication between cells and influence/control cellular function. EVs typical have a diameter from about 20 to 1000 nm. Preferably less than about 200 nm. A “single type” of EV generally refers to EVs derived from a single source, for example from a particular cell line.
[0113] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text. All of these different combinations constitute various alternative aspects of the invention.
[0114] The present inventors have developed conjugates comprising a phage and an EV independently conjugated to a cleavable linker. EVs reduce oxidative stress, activate the immune system and promote tissue regeneration. Phages have high and specific antimicrobial activity and are employed as a carrier system for selectively delivering the EV(s) to the damaged tissue. Advantageously, the phage-EV conjugate provides targeted dual activity. The phage component of the phage-EV conjugate is capable of preventing and/or treating bacterial infection at a damaged tissue site, thereby reducing impaired tissue repair associated with infection. The EV component of the phage-EV conjugate promotes tissue repair. Administering an EV using a phagebased carrier can allow for controlled, site specific delivery of the EV, thereby achieving therapeutic local concentration at the site of the damaged tissue and/or infection. The conjugate described herein has the potential for wide application by conjugating different EVs and different phages to target different diseases, including a wide variety of bacterial infections. The conjugate has particular application in preventing and/or treating bacterial infections that impair wound healing and thereby promote wound repair. The efficacy of the phage-EV conjugate may be further enhanced by conjugating one or more types of active agents to the linker. The active agent may be an antibacterial agent and/or an antibiofilm agent. This has particular application in biofilm infections where common antibiotics lack efficacy. Biofilm infections are associated with prosthetic devices infections as well as infections of bone, heart valves, urinary tract, and particularly in chronic respiratory infections (e.g., cystic fibrosis).
[0115] The present invention uses a cleavable linker for conjugating the phage and EV. Irreversible conjugation of the EV to the phage may be undesirable, for example because the bound phage and/or EV may be less effective compared to their usual free (unconjugated) form. The use of cleavable (reversible) linker in the conjugate described herein can advantageously allow for controlled release of the EV from the conjugate at the site of concentration of the phage.
Phage-EV conjugate
[0116] The present invention provides a conjugate of a phage linked with an EV by a linker, wherein the phage and the EV are independently conjugated to the linker by one or more types of cleavable bond. Each of the one or more types of cleavable bond is capable of degrading such that the phage or EV to which it is conjugated is released upon degradation. Advantageously, this allows the conjugate to function as a targeted delivery system.
Cleavable bond [0117] The conjugate comprises one or more types of cleavable (or degradable) bond. In this context, ‘cleavable bond’ refers to a chemical bond or a chemical group/structure that is able to be cleaved or degraded. The term ‘cleavable bond’ may alternatively be referred to as a ‘cleavable linkage’ or ‘cleavable group’. Each cleavable bond functions to conjugate either a phage or an EV to the linker, while being capable of degrading such that the phage or EV to which it is conjugated is released upon degradation.
[0118] Each of the one of more types of cleavable bond may be any suitable cleavable bond. The cleavable bond (or cleavable group) may comprise a covalent bond or a non-covalent interaction. In one embodiment, the cleavable bond is a covalent bond. In another embodiment, the cleavable bond is a non-covalent interaction, preferably the non-covalent interaction comprises a strong non-covalent interaction (e.g. an electrostatic interaction such as an ionic interaction). Suitable cleavable bonds include bonds that are pH labile, i.e., susceptible to degradation in certain pH conditions. In some embodiments, at least one of the one or more types of cleavable bond is labile (or susceptible to degradation) in acidic conditions. Suitable acid-labile cleavable bonds include hydrazones, imines, acetals, ketals, and esters such as boronate esters. Each cleavable bond may be independently labile at a pH of from about 0.5 to about 6.5, for example at a pH of about 0.5, 1 .0, 1 .5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or 6.5. Any minimum and maximum can be combined to form a range provided that the range is between 0.5 to 6.5, such as a pH of from about 4.0 to about 6.0. In preferred embodiments, at least one of the one or more types of cleavable bond is labile at about pH 5.5.
[0119] In some embodiments, at least of the one or more types of cleavable bond is selected from a hydrazone, an imine, an acetal, a ketal, and an ester (e.g., a boronate ester). In preferred embodiments, at least one of the one or more types of cleavable bond is an imine.
[0120] The one or more types of cleavable bond present in the conjugate may all be the same. That is, the conjugate may comprise one type of cleavable bond, to which the phage and the EV are each independently conjugated to the linker. In preferred embodiments, the cleavable bond is an imine. [0121] Accordingly, in some aspects, the present invention provides a conjugate of a phage linked with an EV by a linker, wherein the phage and the EV are each independently conjugated to the linker by one type of cleavable bond, preferably an imine.
Linker
[0122] The conjugate comprises a linker, to which the phage is conjugated via one or more types of cleavable bond, and to which the EV is conjugated via one or more types of cleavable bond. The skilled person will understand that such a linker comprising one or more types of cleavable bond to be a reversible linker. The linker reversibly conjugates either a phage or an EV to the linker.
[0123]The linker may comprise, or consist of, a polymer which comprises the one or more types of cleavable bond. In this context, the term “consist of” means that the linker is entirely composed of the polymer comprising the one or more types of cleavable bond. The polymer may be a homopolymer or a multiblock co-polymer (e.g., a diblock, triblock or tetrablock copolymer). The polymer may comprise any natural polymer, modified natural polymer or synthetic polymer. Suitable natural polymers include polysaccharides such as dextran. Suitable modified natural polymers include modified polysaccharides (e.g., dextran), for example polysaccharides in which hydroxyl groups have been modified to groups capable of forming a cleavable bond (e.g., aldehyde groups). Suitable synthetic polymers include poly(oligo(ethylene glycol) methyl ether acrylate), polyacrylamide, poly(oligo(ethylene glycol) methyl ether methacrylate), poly(3- vinylbenzaldehyde), and modified derivatives thereof.
[0124] The polymer may be prepared by any suitable method known in the art. Suitable methods include, but are not limited to, living radical polymerisation techniques, also referred to as controlled radical polymerisation. Controlled radical polymerisation includes but is not limited to: reversible addition/fragmentation chain transfer (RAFT) polymerisation, atom transfer radical polymerisation (ATRP), and nitroxide-mediated polymerisation (NMP). In some embodiments, the polymer is prepared by RAFT polymerisation, for example by the methods described herein. The RAFT agent may be 2-(butylthiocarbonothioylthio) propionic acid (PABTC) or 2-(propylthiocarbonothioylthio)- 2-methylpropionoic acid (BPTA), preferably PABTC. In embodiments where the polymer is prepared by RAFT polymerisation, it will be understood that the linker may comprise, or consist of, a polymer having end caps corresponding to the RAFT agent used (e.g., where the RAFT agent is PABTC, a -S-C(=S)-S-C4Hg cap and a -C(CH3)-CO2H cap).
[0125] In some embodiments, the polymer comprises, or consists of, a triblock copolymer comprising a first block, a second block and a third block. In some embodiments, the polymer comprises, or consists of, a diblock copolymer comprising a first block and a second block. In this context, the term “consist of” means that the polymer is entirely composed of the diblock polymer (including end caps, if the polymer is prepared by RAFT polymerisation). In embodiments where the polymer is prepared by RAFT polymerisation, it will be understood that the first block and the second block will each independently comprise an end cap corresponding to the RAFT agent used (e.g., where the RAFT agent is PABTC, either a -S-C(=S)-S-C4Hg cap or a -C(CH3)- CO2H cap).
[0126] The first block and the second block may each be independently composed of a repeating monomer unit (and an end cap, if the polymer is prepared by RAFT polymerisation). The monomer may be any suitable monomer for forming the desired polymer known in the art, including those described herein. In some embodiments, the first block and the second block each independently comprise, or consist of, from 5 to 50 monomer units, for example 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 monomer units. Any minimum and maximum can be combined to form a range provided that the range is between 5 to 50, such as from 5 to 30 monomer units.
[0127] One or both of the first block and the second block may comprise the one or more types of cleavable bond, to which the phage and the EV are each independently conjugated. In some embodiments, one of the first block and the second block comprise the one or more types of cleavable bond. In preferred embodiments, the second block comprises the one or more types of cleavable bond.
[0128] One or both of the first block and the second block may be useful for tuning one or more properties of the conjugate. For example, one or both of the first and second block may be selected to tune one or more properties of the conjugate relating to its therapeutic use, such as biocompatibility and water solubility. In some embodiments, the first block is selected to tune one or more properties of the conjugate, such as water solubility and biocompatibility. In some embodiments, the first block is hydrophilic. In some embodiments, the first block is biocompatible.
[0129] In some embodiments, the first block comprises, or consists of, a polymer composed of 10 to 30 repeating monomer units, for example 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24,25, 26, 27, 28, 29 or 30 monomer units. Any minimum and maximum can be combined to form a range provided that the range is between 10 to 30, such as from 20 to 28 monomer units. In this context, the term “consists of” means the first block is entirely composed of the polymer composed of the 10 to 30 repeating monomer units (including end cap, if the polymer is prepared by RAFT polymerisation). In embodiments where the polymer is prepared by RAFT polymerisation, it will be understood that the first block comprises an end cap corresponding to the RAFT agent used (e.g., where the RAFT agent is PABTC, either a -S-C(=S)-S-C4Hg cap or a - C(CH3)-CO2H cap, preferably a -C(CH3)-CO2H cap). In some embodiments, the first block comprises, or consists of, a polymer composed of 20 monomer units.
[0130] In some embodiments, the first block comprises, or consists of, a polymer selected from poly(oligo(ethylene glycol) methyl ether acrylate) (POEGA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) and polyacrylamide. In some embodiments, the first block comprises, or consists of, POEGA, which is composed of oligo (ethylene glycol) methyl ether acrylate (OEGA) as a repeating monomer unit. The OEGA may have an average Mn of 480 g/mol or 2000 g/mol, preferably 480 g/mol. Advantageously, the POEGA may be useful for conferring the conjugate with improved hydrophilicity and/or biocompatibility properties. In some embodiments, the first block comprises, or consists of, POEGMA, which is composed of oligo (ethylene glycol) methyl ether methacrylate (OEGMA) as a repeating monomer unit. The OEGMA may have an average Mn of 300 g/mol. In some embodiments, the first block comprises, or consists of, polyacrylamide, which is composed of acrylamide as a repeating monomer unit.
[0131] In some embodiments, the first block comprises, or consists of, POEGA which is composed of 10 to 30 OEGA units, for example 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24,25, 26, 27, 28, 29 or 30 OEGA units. Any minimum and maximum can be combined to form a range provided that the range is between 10 to 30, such as from 20 to 28 repeating OEGA units. In preferred embodiments, the first block comprises, or consists of, 20 repeating OEGA units.
[0132] In some embodiments, the second block comprises, or consists of, a polymer composed of 5 to 15 repeating monomer units, for example 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15 monomer units. Any minimum and maximum can be combined to form a range provided that the range is between 5 and 15, such as a range of 5 to 13 monomer units. In this context, “consist of” means the second block is entirely composed of the repeating monomer unit comprising the one or more types of cleavable bond (including end cap, if the polymer is prepared by RAFT polymerisation). In embodiments where the polymer is prepared by RAFT polymerisation, it will be understood that the second block comprises an end cap corresponding to the RAFT agent used (e.g., where the RAFT agent is PABTC, either a -S-C(=S)-S-C4Hg cap or a -C(CH3)-CO2H cap, preferably a -S-C(=S)-S-C4Hg cap). In some embodiments, the second block comprises, or consists of, 10 repeating monomer units.
[0133] The second block may comprise, or consist of, a repeating monomer unit comprising the one or more types of cleavable bond, to which the phage and the EV are each independently conjugated. It will be understood that each monomer unit may independently i) be conjugated to either the phage or the EV of the conjugate, or ii) contain an unreacted reactive functionality (e.g., where conjugation efficiency is below 100%). It will be appreciated that, in these embodiments, the second block will comprise at least one conjugated phage and one conjugated EV.
[0134] In some embodiments, the second block comprises, or consists of, a repeating monomer unit comprising one type of cleavable bond. Preferably the cleavable bond is an imine.
[0135] In some embodiments, the second block comprises, or consists of, a polymer composed of a repeating monomer unit having the following structure: wherein R is independently -CHO or -C=NZ; and
Z is independently a phage or an EV.
[0136] In some embodiments, the second block comprises, or consist of, a polymer composed of 5 to 15 repeating monomer units having the following structure: wherein
R is independently -CHO or -C=NZ; and
Z is independently a phage or an EV, for example 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 of the monomer units. Any minimum and maximum can be combined to form a range provided that the range is between 5 to 15, such as a range of 5 to 12 monomer units. In preferred embodiments, the second block comprises, or consists of, 10, of the monomer units.
[0137] In preferred embodiments, the conjugate has the following structure: wherein
R is independently -CHO or -C=NZ;
Z is independently a phage or an EV; n is 9; x is 10 to 30, preferably 20 to 28, more preferably 20; and y is 5 to 15, preferably 10.
It will be appreciated that the conjugate will comprise at least one conjugated phage and at least one conjugated EV.
Phage
[0138] The conjugate comprises a phage, which is conjugated to the linker via a cleavable bond. Phages (also called bacteriophages) are viruses that infect and replicate within bacteria and archaea. Phages have been used directly as antibacterial therapies. Phages have also been used as drug carriers or nanobots for targeted drug delivery, due to their drug loading capability as well as their ability to selectively target bacteria and/or to be engineered to selectively target specific cell types.
Advantageously, the phage of the conjugate described herein may allow for targeted delivery of the EV to the wound site comprising specific bacteria or cell type targeted by the phage.
[0139] The phage of the conjugate described herein may be any phage suitable for use in delivery of an EV, for example targeted EV delivery. The phage may be suitably selected depending on the intended target for delivery of the EV. For example, in cases where the intended target is a bacteria (e.g., a pathogenic bacteria), the phage may be capable of targeting that bacteria. Methods for genetically modifying phages are known in the art.
[0140] In some embodiments, the phage is capable of infecting a pathogenic bacteria. Examples of suitable pathogenic bacteria include Escherichia coli, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. In preferred embodiments, the phage is capable of infecting Pseudomonas aeruginosa. In preferred embodiments, the phage is capable of infecting Escherichia coli.
[0141] The phage may be selected from a filamentous phage, an MS2 phage and a caudovirus (e.g. a myovirus such as T4 phage or a siphovirus such as lambda phage). In some embodiments, the phage is a caudovirus, for example a myovirus.
[0142] Caudoviruses, such as myoviruses, comprise a head portion and a tail portion (tail fibres). Typically the head portion exhibits an overall net negative charge, and the tail portion exhibits an overall net positive charge. In embodiments of the conjugate described herein where the phage is a caudovirus (e.g. myovirus), preferably the head of the phage is conjugated to the linker. This may advantageously allow the tail portion of the caudovirus to be oriented outwards, which may facilitate target (host) recognition.
EV
[0143] The conjugate comprises an EV conjugated to the linker by one or more types of cleavable bond.
[0144] The EV may comprise one or more types of complementary reactive functionality capable of forming a cleavable bond with the one or more types of reactive functionality of the linker.
[0145] Preferably, at least one of the one or more types of reactive functionality is capable of forming an imine.
[0146] In preferred embodiments, at least one of the one or more types of reactive functionality is an aldehyde.
[0147] In preferred embodiments, at least one of the one or more types of complementary reactive functionality is an amine.
[0148] The EV may be released from the conjugate by degradation of the cleavable bond.
[0149] The EV may be any suitable EV. EVs useful in the invention include EVs that reduce oxidative stress, activate the immune system and/or promote tissue regeneration. Examples of EVs that are useful in the invention include, but are not limited to, EVs derived from: eukaryotic cells, bacteria, probiotics, and combinations thereof. Preferably, the EVs are derived from: stem cells, probiotics, and combinations thereof. Preferably, EVs derived from stem cells include EVs derived from mesenchymal stromal cells.
[0150] EVs may be derived from a single source, for example from a single cell line.
EVs derived from a single source are herein referred to as a “single type of EV”. [0151] EVs may be derived from more than one source, for example from multiple cell lines. EVs derived from more than one source may be harvested from a heterogeneous cell line mixture. Alternatively, EVs derived harvested from a first cell line may be combined with EVs harvested from a second cell line to provide an EV mixture from more than one source.
[0152] In some embodiments, the conjugate comprises a phage linked with one type of EV. For example, the phage may be linked with an EV derived from a stem cell. In another example, the phage may be linked with an EV derived from a probiotic.
[0153] In other embodiments, the phage may be linked with more than one type of EV. For example, the phage may be linked with an EV derived from a stem cell and an EV derived from a probiotic.
Active agent
[0154] The conjugate may comprises, or further comprises, one or more types of active agents (or drugs) conjugated to the linker.
[0155] In one embodiment, the one or more types of active agent are each independently conjugated to the linker by one or more types of cleavable bond. In such embodiments, the one or more types of active agent comprise one or more types of complementary reactive functionality capable of forming a cleavable bond with the one or more types of reactive functionality of the linker.
[0156] Preferably, at least one of the one or more types of reactive functionality is capable of forming an imine.
[0157] In preferred embodiments, at least one of the one or more types of reactive functionality is an aldehyde.
[0158] In preferred embodiments, at least one of the one or more types of complementary reactive functionality is an amine.
[0159] The one or more types of active agent may be released from the conjugate by degradation of the cleavable bond. [0160] The one or more types of active agent may be hydrophilic, hydrophobic, or a combination thereof. The one or more types of active agent may comprise none, one or more types of complementary reactive functionality, preferably an amine, capable of forming a cleavable covalent bond with the one or more types of reactive functionality of the linker.
[0161] In one embodiment, the one or more types of active agent may be hydrophilic. The one or more types of hydrophilic active agent may comprise one or more types of complementary reactive functionality, preferably an amine, capable of forming a cleavable covalent bond with the one or more types of reactive functionality of the linker. In another embodiment, the one or more types of hydrophilic active agent do not comprise one or more types of complementary reactive functionality. The one or more types of hydrophilic active agent that do not comprise one or more types of complementary reactive functionality may form a cleavable charge interaction bond with the one or more types of reactive functionality of the linker.
[0162] In another embodiment, the one or more types of active agent may be hydrophobic. The one or more types of hydrophobic active agent may comprise one or more types of complementary reactive functionality, preferably an amine, capable of forming a cleavable covalent bond with the one or more types of reactive functionality of the linker. In another embodiment, the one or more types of hydrophobic active agent do not comprise one or more types of complementary reactive functionality. The one or more types of hydrophobic active agent that do not comprise one or more types of complementary reactive functionality may be indirectly bound to the linker.
[0163] In one embodiment, the one or more types of active agent are each indirectly bound to the linker. In such embodiments, the one or more types of active agent do not comprise one or more types of complementary reactive functionality capable of forming a cleavable bond with the one or more types of reactive functionality of the linker. In such embodiments, preferably the one or more types of active agent do not comprise an amine.
[0164] In preferred embodiments, the one or more types of active agent that do not comprise one or more types of complementary reactive functionality, may be each encapsulated within a nanostructure comprising the linker. Preferably, the one or more types of active agent that do not comprise one or more types of complementary reactive functionality form a self-assembled nanostructure with the linker wherein the one or more types of active agent are each indirectly bound to the linker.
[0165] The one or more types of active agents may be any suitable active agent. Each active agent may independently be a small molecule drug or a macromolecular drug. In some embodiments, the one or more types of active agent comprise one or more small molecule drugs. In some embodiments, the one or more types of active agent are small molecule drugs. The small molecule drug(s) may be any suitable organic compound having a low molecular weight (less than about 900 daltons) and that can regulate a biological process to treat a particular disease or condition. In some embodiments, the one or more types of active agent comprise one or more macromolecular drugs. Macromolecular drugs useful in the invention include large molecules (molecular weight more than about 900 daltons) such as proteins, polysaccharides and nucleic acids and that can regulate a biological process to treat a particular disease or condition.
[0166]The active agent(s) may be suitably selected, for example, depending on the intended target for delivery of the active agent(s) and/or the intended application of the conjugate. For example, in cases where the intended target may be a bacteria (e.g., a pathogenic bacteria), the one or more types of active agent may comprise one or more antibacterial agents and/or an antibiofilm agents, including those known in the art. Advantageously, the capability of the conjugate for targeted delivery may allow for safe administration of otherwise toxic drugs, for example second-line antibiotic drugs. Accordingly, in some embodiments, the one or more types of active agent comprise one or more second-line antibiotic drugs, including those described herein.
[0167] In some embodiments, the one or more types of active agent comprise one or more antibacterial agents. Any suitable antibacterial agent may be used, including those known in the art. The one or more antibacterial agents may comprise an antibacterial agent useful for treating a disease and/or an infection caused by an antibiotic-resistant bacteria. The one or more antibacterial agents may comprise a second-line antibacterial agent. Examples of suitable antibacterial agents include aminoglycoside antibiotics such as amikacin, gentamicin, neomycin, kanamycin, neomycin, tobramycin and streptomycin; antimicrobial peptides including octapeptides, migainins, and polymyxins such as polymixin B and polymyxin E (colistin); and fluoroquinolone antibiotics such as ciprofloxacin and gemifloxacin. In some embodiments, the one or more antibacterial agents comprise one or more of amikacin, colistin, gentamicin and ciprofloxacin.
[0168] The one or more antibacterial agents may comprise an antibiotic useful for treating an infection caused by a pathogenic bacteria selected from Escherichia coli, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. In preferred embodiments, the one or more antibacterial agents comprise an antibiotic useful for treating an infection of caused by Pseudomonas aeruginosa. In preferred embodiments, the one or more antibacterial agents comprise an antibiotic useful for treating an infection of caused by Escherichia coli.
[0169] In some embodiments, the one or more types of active agent comprise one or more antibiofilm agents. Any suitable antibiofilm agent may be used, including those known in the art. The one or more antibiofilm agents may each be independently capable of one or more of the following: inhibiting (or preventing) biofilm formation, dispersing biofilm, and treating (or eradicating) biofilm. The one or more antibiofilm agents may comprise one or more of the following: one or more biofilm-inhibiting agents, one or more biofilm-dispersing agents and one or more biofilm eradication agents. Examples of suitable antibiofilm agents include nitric oxide (NO) donors and nitroxide-containing antibiofilm agents such as 4-amino-2,2,6,6-tetramethyl- piperidinyloxyl (4-amino TEMPO). In some embodiments, the one or more antibiofilm agents comprise a nitroxide-containing antibiofilm agent, preferably 4-amino TEMPO.
[0170] The one or more antibiofilm agents may comprise an antibiofilm agent useful for inhibiting, dispersing and/or treating biofilm formed by a pathogenic bacteria selected from Escherichia coli, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. In preferred embodiments, the one or more antibiofilm agents comprise an antibiofilm agent useful for inhibiting, dispersing and/or treating biofilm formed by Pseudomonas aeruginosa. In preferred embodiments, the one or more antibiofilm agents comprise an antibiofilm agent useful for inhibiting, dispersing and/or treating biofilm formed by Escherichia coli.
[0171] In some embodiments, the one or more types of active agent comprise one or more disinfectant agents. Imaging agent
[0172] The conjugate of the present invention may comprise, or may further comprise, one or more types of imaging agent conjugated to the linker.
[0173] The one or more types of imaging agent may be any suitable agent that can allow for visualisation of the conjugate via appropriate imaging techniques known in the art. Suitable imaging agents include chromophores, fluorophores and radionuclides. The imaging agent may additionally be suitable for therapeutic purposes. It will therefore be appreciated that in some embodiments the imaging agent and the active agent may be the same.
[0174] The one or more types of imaging agent may be linked to the conjugate by one or more types of cleavable (reversible) bond, including the one or more types of cleavable bond described herein. In these embodiments, the one or more types of imaging agent may be released from the conjugate by degradation of the cleavable bond. Additionally, or alternatively, the one or more types of imaging agent may be linked to the conjugate by one or more types of non-cleavable (irreversible) bond, for example a bond that is not pH labile. In some embodiments, each of the one or more types of imaging agent are independently linked to the conjugate by a non-cleavable bond, preferably a bond that is not pH labile. The one or more types of imaging agent may be directly linked to the cleavable bond or indirectly linked (e.g., via a spacer or chelator moiety that is linked to the cleavable bond).
[0175] In embodiments where the linker of the conjugate is a polymer, the one or more types of imaging agent may be linked to the conjugate via a monomer unit of the polymer. In embodiments where the polymer is prepared by RAFT polymerisation, the one or more types of imaging agent may additionally or alternatively be conjugated to an end cap of the polymer corresponding to the RAFT agent used to prepare the polymer. It will be appreciated that in embodiments where the one or more types of imaging agent are linked to the conjugate by a non-cleavable bond, the one or more types of imaging agent may preferably be linked to the polymer via a different portion of the polymer than the phage and the one or more EVs. For example, each of the one or more types of imaging agent may be independently conjugated to a different polymer block (e.g., in embodiments where the polymer is a multiblock polymer, such as a diblock or triblock copolymer) or an end cap of the polymer (e.g., in embodiments where the polymer is prepared by RAFT polymerisation).
Methods of preparation
[0176] The present invention also provides a method for preparing a conjugate of a phage linked with an EV by a linker.
[0177] The method comprises a step of providing a linker comprising one or more types of reactive functionality capable of forming a cleavable bond. The method may additionally, or alternatively, comprise a step of preparing the linker.
[0178] The one or more types of reactive functionality may comprise a reactive functionality capable of forming a cleavable bond (or degradable bond) that is pH labile. In some embodiments, the one or more types of reactive functionality comprise a reactive functionality capable of forming a cleavable bond that is labile (or susceptible to degradation) in acidic conditions. Suitable acid-labile cleavable bonds include hydrazones, imines, acetals, ketals, and esters such as boronate esters. Suitable reactive functionalities for forming acid-labile cleavable bonds include aldehydes, ketones, hydrazines, amines (e.g., primary amines), alcohols, and carboxylic acids. The cleavable bond may be labile at a pH of from about 0.5 to about 6.5, for example at a pH of about 0.5, 1 .0, 1 .5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or 6.5. Any minimum and maximum can be combined to form a range provided that the range is between 0.5 to 6.5, such as a pH of from about 4.0 to about 6.0. In preferred embodiments, the cleavable bond is labile at about pH 5.5. In some embodiments, at least one of the one or more types of reactive functionality is capable of forming a cleavable bond selected from a hydrazone, an imine, an acetal, a ketal, and an ester (e.g., a boronate ester). In preferred embodiments, at least one of the one or more types of reactive functionality is capable of forming an imine.
[0179] In some embodiments, the one or more types of reactive functionality comprise one or more of an aldehyde, a ketone, a hydrazine, an amine (e.g., a primary amine), an alcohol, and a carboxylic acid. In preferred embodiments, the one or more types of reactive functionality comprise an aldehyde. In these embodiments, the aldehyde may react with an amine of the phage and/or the EV to form an imine. Accordingly, in some embodiments, the phage and/or the EV may each independently comprise an amine, preferably a primary amine.
[0180] The one or more types of reactive functionality of the linker may all be the same. That is, the linker may comprise one type of reactive functionality, which subsequently reacts with a complementary reactive functionality of the phage and the EV to form a cleavable bond. In preferred embodiments, the reactive functionality is an aldehyde. In preferred embodiments, the phage comprises an amine, preferably a primary amine. In some preferred embodiments, the EV comprise an amine, preferably a primary amine.
[0181] The linker may comprise, or consist of, a polymer which comprises the one or more types of reactive functionality. In this context, the term “consist of” means that the linker is entirely composed of the polymer comprising the one or more types of reactive functionality. Any polymerisation technique known in the art may be used to form the polymer. The polymerisation technique may, for example, be a radical polymerisation, preferably radical addition fragmentation chain transfer (RAFT) polymerisation. RAFT polymerisation typically involves the polymerisation of a monomer or combination of monomers in solution in the presence of a radical initiator, chain transfer agent (RAFT agent) and heat. Some suitable RAFT conditions are described in the reviews Moad, G.; Rizzardo, E.; Thang, S. H. Accounts of Chemical Research 2008, 41 , 1 133 and Moad, G.; Rizzardo, E.; Thang, S. H. Polymer 2008, 49, 1079. The person skilled in the art will be able to determine appropriate conditions, including solvent selection, temperature for reaction and combination of radical initiator and chain transfer agent depending on the monomer or combination of monomers selected.
[0182] In some embodiments, the polymer is prepared by RAFT polymerisation in the presence of a RAFT agent. Any suitable RAFT agent may be used. Examples of suitable RAFT agents include thiocarbonylthio compounds such as dithioesters, dithiocarbamates, trithiocarbonates, and xanthates. In some embodiments, the RAFT agent is selected from a dithioester (e.g., dithiobenzoate) and a trithiocarbonate (e.g., 2- (propylthiocarbonothioylthio)-2-methylpropionoic acid (BPTA) or 2- (butylthiocarbonothioylthio) propionic acid (PABTC), preferably PABTC). In embodiments where the polymer is prepared by RAFT polymerisation, it will be understood that the linker may comprise, or consist of, a polymer having end caps corresponding to the RAFT agent used (e.g., where the RAFT agent is PABTC, a -S- C(=S)-S-C4Hg cap and a -C(CH3)-CO2H cap).
[0183] The polymer may comprise, or consist of, a diblock copolymer comprising a first block and a second block. In this context, the term “consist of” means that the polymer is entirely composed of the diblock polymer (including end caps, if the polymer is prepared by RAFT polymerisation). In embodiments where the polymer is prepared by RAFT polymerisation, it will be understood that the first block and the second block will each independently comprise an end cap corresponding to the RAFT agent used (e.g., where the RAFT agent is PABTC, either a -S-C(=S)-S-C4Hg cap or a -C(CH3)- CO2H cap). The first block and the second block may each be independently composed of a repeating monomer unit (and an end cap, if the polymer is prepared by RAFT polymerisation).
[0184] One or both of the first block and the second block may comprise the one or more types of reactive functionality capable of forming a cleavable bond. In some embodiments, one of the first block and the second block comprise the one or more types of reactive functionality. In preferred embodiments, the second block comprises the one or more types of reactive functionality.
[0185] In preferred embodiments, the first block comprises, or consists of, poly(oligo(ethylene glycol) methyl ether acrylate) (POEGA). In this context, “consists of” means the first block is entirely composed of the POEGA polymer (including end cap, if the polymer is prepared by RAFT polymerisation). In embodiments where the polymer is prepared by RAFT polymerisation, it will be understood that the first block comprises an end cap corresponding to the RAFT agent used (e.g., where the RAFT agent is PABTC, either a -S-C(=S)-S-C4Hg cap or a -C(CH3)-CO2H cap).
[0186] In preferred embodiments, the first block comprises, or consists of, oligo (ethylene glycol) methyl ether acrylate (OEGA) as a repeating monomer unit. In this context, “consist of” means the first block is entirely composed of repeating OEGA monomer units (including end cap, if the polymer is prepared by RAFT polymerisation). In some embodiments, the first block comprises, or consists of, 10 to 30 repeating OEGA units, for example 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24,25, 26, 27, 28, 29 or 30 repeating OEGA units. Any minimum and maximum can be combined to form a range provided that the range is between 10 to 30, such 20 to 28 repeating OEGA units. In preferred embodiments, the first block comprises, or consists of, 20 repeating OEGA units.
[0187] The second block may comprise, or consist of, a repeating monomer unit comprising the one or more types of reactive functionality. In this context, “consist of” means the second block is entirely composed of the repeating monomer unit comprising the one or more types of reactive functionality (including end cap, if the polymer is prepared by RAFT polymerisation). In some embodiments, the second block comprises, or consists of 5 to 15 repeating monomer units, for example 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 repeating monomer units. Any minimum and maximum can be combined to form a range provided that the range is between 20 to 35, such as a range of 25 to 30 monomer units. In preferred embodiments, the second block comprises, or consists of, 28 to 30, more preferably 28, repeating monomer units.
[0188] In some embodiments, the second block comprises, or consists of, a repeating monomer unit comprising one type of reactive functionality. Preferably the reactive functionality is an aldehyde.
[0189] In preferred embodiments, the second block comprises, or consists of, poly(3- vinylbenzaldehyde) (PVBA). In this context, the term “consists of” means the second block is entirely composed of the PVBA polymer (including end cap, if the polymer is prepared by RAFT polymerisation). In embodiments where the polymer is prepared by RAFT polymerisation, it will be understood that the second block comprises an end cap corresponding to the RAFT agent used (e.g., where the RAFT agent is PABTC, either a -S-C(=S)-S-C4Hg cap or a -C(CH3)-CO2H cap).
[0190] In preferred embodiments, the second block comprises, or consists of, 3- vinylbenzaldehyde (VBA) as a repeating monomer unit. In this context, “consist of” means the second block is entirely composed of repeating VBA monomer units (including end cap, if the polymer is prepared by RAFT polymerisation). In preferred embodiments, the second block comprises, or consists of, 5 to 15, preferably 10, repeating VBA units.
[0191] Accordingly, in some embodiments, the linker is a diblock copolymer which is prepared by: reacting a RAFT agent with a first monomer to provide a first block polymer; and reacting the first block polymer with a second monomer to provide the diblock copolymer.
[0192] Further, in some embodiments, the method further comprises a step of preparing a linker which is a diblock copolymer. The step of preparing the diblock copolymer may comprise: reacting a RAFT agent with a first monomer to provide a first block polymer; and reacting the first block polymer with a second monomer to provide the diblock copolymer.
[0193] In these embodiments, the RAFT agent may be any suitable RAFT agent known in the art, including those described herein. The first monomer and the second monomer may be any monomer known in the art suitable for use with the conjugate, including the monomers described herein. The percentage conversion of the first and second monomers can be determined by methods known in the art, including those described herein.
[0194] In some embodiments, the first monomer is OEGA.
[0195] In some embodiments, the first block polymer is a POEGA polymer (including end caps corresponding to the RAFT agent used).
[0196] In some embodiments, the POEGA polymer has an average molecular weight of from about 4000 g mol-1 to about 15000 g mol-1 , for example an average molecular weight of about 4500 g mol-1 , 5000 g mol-1 , 5500 g mol-1, 6000 g mol-1, 6500 g mol-1 , 7000 g mol-1, 7500 g mol-1 , 8000 g mol-1 , 8500 g mol-1, 9000 g mol-1, 9500 g mol-1 , 10000 g mol-1 , 10500 g mol-1 , 1 1000 g mol-1 , 1 1500 g mol-1 , 12000 g mol-1 , 12500 g mol-1, 13000 g mol-1 , 13500 g mol-1 , 14000 g mol-1 , 14500 g mol-1 , or 15000 g mol-1. Any minimum and maximum can be combined to form a range provided that the range is between 4500 g mol-1 to about 15000 g mol-1, such as an average molecular weight of from about 9000 g mol-1 to about 12000 g mol-1. In preferred embodiments, the POEGA polymer has an average molecular weight of about 9000 g mol-1. The molecular weight of the POEGA polymer can be determined by methods known in the art, for example by 1 H NMR spectroscopy analysis and size exclusion chromatography (SEC) as described herein.
[0197] The POEGA polymer preferably has a low polydispersity index (PDI). In some embodiments, the POEGA polymer has a PDI of from about 1 .05 to about 1 .30, for example about 1.05, 1.06, 1.07, 1.08, 1.09, 1.10, 1.1 1 , 1.12, 1.13, 1.14, 1.15, 1.16, 1 .17, 1 .8, 1 .19, 1 .20, 1 .21 , 1 .22, 1 .23, 1 .24, 1 .25, 1 .26, 1 .27, 1 .28, 1 .29, or 1 .30. Any minimum and maximum can be combined to form a range provided that the range is between 1 .05 to 1 .30, such as a polydispersity of from about 1 .05 to 1 .25. In preferred embodiments, the POEGA polymer has a polydispersity of about 1.19. The PDI or molecular weight distribution of the POEGA polymer can be determined by methods known in the art, including those described herein, and may be calculated from the following equation: PDI = Mw/Mn, where Mw is the weight-average molecular weight and Mn is the number-average molecular weight.
[0198] In some embodiments, the second monomer is VBA.
[0199] In some embodiments, the diblock copolymer is POEGA-b/oc -PVBA (POEGA-b-PVBA) (including end caps corresponding to the RAFT agent used).
[0200] In some embodiments, the POEGA-b-PVBA has an average molecular weight of from about 5000 g mol-1 to about 17000 g mol-1 , for example an average molecular weight of about 5000 g mol-1 , 5500 g mol-1 , 6000 g mol-1, 6500 g mol-1, 7000 g mol-1 , 7500 g mol-1, 8000 g mol-1 , 8500 g mol-1 , 9000 g mol-1, 9500 g mol-1, 10000 g mol-1 , 10500 g mol-1 , 1 1000 g mol-1 , 1 1500 g mol-1 , 12000 g mol-1 , 12500 g mol-1 , 13000 g mol-1, 13500 g mol-1 , 14000 g mol-1 , 14500 g mol-1 , 15000 g mol-1 , 15500 g mol-1, 16000 g mol-1, 16500 g mol-1 , or 17000 g mol-1. Any minimum and maximum can be combined to form a range provided that the range is between 5000 g mol-1 to 17000 g mol-1, such as an average molecular weight of from about 5500 g mol-1 to about 15000 g mol-1. In some embodiments, the POEGA-b-PVBA has an average molecular weight of about 1 1000 g mol-1. In preferred embodiments, the POEGA-b-PVBA has an average molecular weight of about 15000 g mol-1. The molecular weight of the POEGA-b-PVBA can be determined by methods known in the art, for example by 1H NMR spectroscopy analysis and size exclusion chromatography (SEC) as described herein. [0201] The POEGA-b-PVBA polymer preferably has a low PDI. In some embodiments, the POEGA-b-PVBA polymer has a PDI of from about 1 .05 to about 1 .30, for example about 1.05, 1.06, 1.07, 1.08, 1.09, 1.10, 1.1 1 , 1.12, 1.13, 1.14, 1.15, 1.16, 1 .17, 1 .8, 1 .19, 1 .20, 1 .21 , 1 .22, 1 .23, 1 .24, 1 .25, 1 .26, 1 .27, 1 .28, 1 .29, or 1 .30. Any minimum and maximum can be combined to form a range provided that the range is between 1 .05 to 1 .30, such as a polydispersity of from about 1 .05 to 1 .25. The PDI of the POEGA-b-PVBA polymer can be determined by methods known in the art including those described herein.
[0202] Size/conjugation may be measured using techniques known in the art, including dynamic light scattering (DLS), flow cytometry and transmission electron microscopy (TEM).
[0203] In preferred embodiments, the linker has the following structure: wherein n is 9; x is 10 to 30, preferably 20 to 28, more preferably 20; and y is 5 to 15, preferably 10.
[0204] The method of the invention also comprises the steps of: conjugating an EV to the linker via at least one of the one or more types of reactive functionality; and conjugating a phage to the linker via at least one of the one or more types of reactive functionality; thereby providing the conjugate, wherein the phage and the EV are each independently conjugated to the linker by one or more types of cleavable bond.
[0205] It will be understood that each of the one or more types of reactive functionality of the linker independently react with a respective complementary reactive functionality of the phage or the EV, so as to form a cleavable bond which conjugates the phage or EV respectively to the linker. The complementary reactive functionality of the phage and the EV may be the same or different. In preferred embodiments, the complementary reactive functionality of the phage and the EV is the same.
[0206] The phage may be any phage as described herein. In preferred embodiments, the phage comprises an amine, preferably a primary amine, as a complementary reactive functionality for reacting with a reactive functionality of the linker to form a cleavable bond. In some embodiments, the complementary reactive functionality of the phage is present on the head portion of the phage.
[0207] The EV may be any EV as described herein. In preferred embodiments, the EV comprises an amine, preferably a primary amine, as a complementary reactive functionality for reacting with a reactive functionality of the linker to form a cleavable bond.
[0208] The one or more types of cleavable bond may be any cleavable bond as described herein. Preferably, the cleavable bond is an imine.
[0209] The steps of conjugating the phage and the EV to the linker may be conducted in any order.
[0210] Accordingly, in some embodiments, the method comprises: conjugating an EV to the linker via at least one of the one or more types of reactive functionality, to provide a conjugate of the linker and the EV; and conjugating a phage to the conjugate of the linker and the EV via at least one of the one or more types of reactive functionality, to thereby provide the conjugate of the phage linked with the EV by the linker.
[0211] In other embodiments, the method comprises: conjugating a phage to the linker via at least one of the one or more types of reactive functionality, to provide a conjugate of the linker and the phage; and conjugating the EV to the conjugate of the linker and the phage via at least one of the one or more types of reactive functionality, to thereby provide the conjugate of the phage linked with the EV by the linker.
[0212] In some embodiments, the step of conjugating the phage is conducted before the step of conjugating the EV. In some embodiments, the step of conjugating the EV to the linker is conducted before the step of conjugating the phage. This may advantageously prevent phage-phage and/or EV-EV conjugation by removing the excess of the linker prior to conjugating the EVs.
[0213] Any concentration (or amount) of linker, phage and EV(s) for preparing the conjugate of the invention may be used to achieve a desired level of conjugation of the phage and/or EV to the linker, and the person skilled in the art will be able to determine appropriate concentrations and conditions. Typically, the concentration of phage and/or EV reacted with the linker may be selected based on the concentration of the polymer (in particular, the concentration of the reactive functionalities present in the linker).
[0214] It will be appreciated that the conjugation efficiency (i.e., the extent to which the reactive functionalities present in the linker react with the phage and the one or more EV) may vary depending on the amounts of reagents used. The conjugation efficiency may range from i) at least one reactive functionality of the linker has reacted with at least one phage and at least one reactive functionality of the linker has reacted with at least one EV, to ii) 100% conjugation efficiency (i.e., all reactive functionalities present on the linker have reacted with one or more phages and one or more EVs). For example, the conjugation efficiency may be at least one reactive functionality of the linker has reacted with at least one phage and at least one reactive functionality of the linker has reacted with at least one active agent, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% conjugation efficiency. Any minimum and maximum percentage may be combined to form a range. The conjugation efficiency may also be described herein in terms of the proportion of reactive functionalities present on the linker that have reacted with either i) one or more phages, ii) one or more EVs, or iii) if present, one or more active agents and/or imaging agents. 100% conjugation efficiency may be achieved by using an excess concentration of phage and/or EV(s) relative to the concentration of the linker (in particular, the concentration of reactive functionalities present in the linker). It will be understood that where the conjugation efficiency is below 100%, the actual percentage conjugation efficiency will depend on the number of reactive functionalities present in the linker. It will also be appreciated that where conjugation efficiency is below 100%, the conjugate produced by the method may comprise one or more unreacted reactive functionalities.
[0215] The conjugation of the phage and the EV to the linker can be determined by methods known in the art, for example by 1H NMR analysis and Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy as described herein.
[0216] As described herein, the conjugate may further comprise one or more active agents and/or imaging agents conjugate to the linker. Accordingly, in some embodiments, the method further comprises a step of conjugating one or more types of active agent and/or imaging agent to the linker. The one or more types of active agent and/or imaging agent may be any active agent and/or imaging agent as described herein. The step of conjugating the one or more active agents and/or imaging agents may comprise conjugating at least one of the one or more types of active agent and/or imaging agent to the linker via at least one of the one or more types of reactive functionality capable of forming a cleavable (reversible) bond. Additionally, or alternatively, the step may comprise conjugating at least one of the one or more types of imaging agent to the linker via a non-cleavable (irreversible) bond. The one or more types of active agent and/or imaging agent may be conjugated to the linker before, at the same time or after conjugation of one or both of the phage and the EV to the linker.
[0217] Also provided herein is a conjugate prepared by the method described herein.
[0218] In preferred embodiments, the conjugate prepared by the method has the following structure: wherein
R is independently -CHO or -C=NZ;
Z is independently a phage or an EV; n is 9; x is 10 to 30, preferably 20 to 28, more preferably 20; y is 5 to 15, preferably 10.
As described herein, it will be appreciated that the conjugate will comprise at least one conjugated phage and at least one conjugated EV.
Pharmaceutical compositions
[0219] The present invention also provides a pharmaceutical composition comprising the conjugate described herein or prepared by the method described herein, and a pharmaceutically acceptable diluent, excipient or carrier.
[0220] The conjugate or pharmaceutical composition described herein may be administered, or formulated for administration by, any route described herein. As used herein, the term “administered” means administration of a therapeutically effective dose of the compound described herein to the subject. As used herein, the term “formulated for administration” means a therapeutically effective dose of the compound described herein is formulated in such a way that is suitable for the route of administration. In preferred embodiments, the conjugate (or pharmaceutical composition) is administered orally, topically, by nasal administration or parenterally, more preferably topically. In other preferred embodiments, the conjugate (or pharmaceutical composition) is formulated for oral administration, topical administration, nasal administration or parenteral administration, more preferably for topical administration.
[0221] Pharmaceutical compositions may be formulated for any appropriate route of administration including, for example, topical (for example, transdermal or ocular), oral, inhalable, buccal, nasal, vaginal, rectal or parenteral administration. The term parenteral as used herein includes subcutaneous, intradermal, intravascular (for example, intravenous), intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial and intraperitoneal injection, as well as any similar injection or infusion technique. In certain embodiments, compositions in a form suitable for oral use or parenteral use, especially parenteral use, are preferred. Suitable oral forms include, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Within yet other embodiments, compositions provided herein may be formulated as a lyophilisate.
[0222] As yet another example, the conjugate can be topically administered by application of a transdermal device. Either "passive" or "active" transdermal devices can be employed for administration of one or more compositions of the invention, the selection of which will depend in part upon the location for application of the device (e.g., at or proximal to the site of epithelial damage for local administration of, for example, rapidly metabolized compositions, or distal to the site for systemic composition administration). Examples of passive transdermal devices include reservoir-type patches (e.g., in which the composition is provided within a walled reservoir having a permeable surface) and matrix-type patches (in which the composition is dispersed within a polymeric composition).
[0223] Active transdermal devices include, but are not limited to, devices employing iontophoresis (e.g., a low voltage electrical current), electroporation (e.g., short electrical pulses of higher voltage), sonophoresis (e.g., low frequency ultrasonic energy), or thermal energy for delivery of the composition. Typically, passive-type transdermal devices would be utilized for application at a current site of epithelial damage, since additional mechanisms for overcoming the epithelial barrier provided by active-type transdermal devices is not necessary. For a review of various transdermal technologies, see Ghosh, Pfister and Yum Eds. (1997) Transdermal and Topical Drug Delivery Systems (CRC Press, London); Potts and Guy (Eds.) (1997) Transdermal Drug Delivery (Marcel Dekker, New York); and Potts and Cleary (1995) Transdermal drug delivery: useful paradigms. J Drug Targ. 3:247-251 .
[0224] As yet another example, the conjugate can be topically administered by introduction of a foam (e.g., a biologically inert or pharmaceutically acceptable foam) or other carrier comprising the conjugate to an epithelial-lined cavity comprising the wound.
[0225] It will be evident that various means of administration can be combined, for the same or different conjugates. Thus, for example, the conjugate can be administered both topically and orally or topically and by injection, simultaneously or sequentially, as indicated by the nature and severity of the damaged tissue to be treated.
[0226] The various dosage units are each preferably provided as a discrete dosage tablet, capsules, lozenge, dragee, gum, or other type of solid formulation. Capsules may encapsulate a powder, liquid, or gel. The solid formulation may be swallowed, or may be of a suckable or chewable type (either frangible or gum-like). The present invention contemplates dosage unit retaining devices other than blister packs; for example, packages such as bottles, tubes, canisters, packets. The dosage units may further include conventional excipients well-known in pharmaceutical formulation practice, such as binding agents, gellants, fillers, tableting lubricants, disintegrants, surfactants, and colorants; and for suckable or chewable formulations.
[0227] Compositions intended for oral use may further comprise one or more components such as sweetening agents, flavouring agents, colouring agents and/or preserving agents in order to provide appealing and palatable preparations. Tablets contain the active ingredient in admixture with physiologically acceptable excipients that are suitable for the manufacture of tablets. Such excipients include, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate, granulating and disintegrating agents such as corn starch or alginic acid, binding agents such as starch, gelatine or acacia, and lubricating agents such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate may be employed. [0228] Formulations for oral use may also be presented as hard gelatine capsules wherein the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or as soft gelatine capsules wherein the active ingredient is mixed with water or an oil medium such as peanut oil, liquid paraffin or olive oil.
[0229] Aqueous suspensions contain the active ingredient(s) in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as naturally-occurring phosphatides (for example, lecithin), condensation products of an alkylene oxide with fatty acids such as polyoxyethylene stearate, condensation products of ethylene oxide with long chain aliphatic alcohols such as heptadecaethyleneoxycetanol, condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol mono-oleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides such as polyethylene sorbitan monooleate. Aqueous suspensions may also comprise one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin.
[0230] Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and/or flavouring agents may be added to provide palatable oral preparations. Such suspensions may be preserved by the addition of an antioxidant such as ascorbic acid.
[0231] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, such as sweetening, flavouring and colouring agents, may also be present.
[0232] Pharmaceutical compositions may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil such as olive oil or arachis oil, a mineral oil such as liquid paraffin, or a mixture thereof. Suitable emulsifying agents include naturally-occurring gums such as gum acacia or gum tragacanth, naturally- occurring phosphatides such as soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides such as sorbitan monoleate, and condensation products of partial esters derived from fatty acids and hexitol with ethylene oxide such as polyoxyethylene sorbitan monoleate. An emulsion may also comprise one or more sweetening and/or flavouring agents.
[0233] Syrups and elixirs may be formulated with sweetening agents, such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also comprise one or more demulcents, preservatives, flavouring agents and/or colouring agents.
[0234] Conjugates described herein may be formulated for local or topical administration, such as for topical application to the skin. Formulations for topical administration typically comprise a topical vehicle combined with active agent(s), with or without additional optional components.
[0235] Suitable topical vehicles and additional components are well known in the art, and it will be apparent that the choice of a vehicle will depend on the particular physical form and mode of delivery. Topical vehicles include organic solvents such as alcohols (for example, ethanol, iso-propyl alcohol or glycerine), glycols such as butylene, isoprene or propylene glycol, aliphatic alcohols such as lanolin, mixtures of water and organic solvents and mixtures of organic solvents such as alcohol and glycerine, lipid- based materials such as fatty acids, acylglycerols including oils such as mineral oil, and fats of natural or synthetic origin, phosphoglycerides, sphingolipids and waxes, proteinbased materials such as collagen and gelatine, silicone-based materials (both nonvolatile and volatile), and hydrocarbon-based materials such as microsponges and polymer matrices.
[0236] A composition may further include one or more components adapted to improve the stability or effectiveness of the applied formulation, such as stabilizing agents, suspending agents, emulsifying agents, viscosity adjusters, gelling agents, preservatives, antioxidants, skin penetration enhancers, moisturizers and sustained release materials. Examples of such components are described in Martindale - The Extra Pharmacopoeia (Pharmaceutical Press, London 1993) and Martin (ed.), Remington's Pharmaceutical Sciences. Formulations may comprise microcapsules, such as hydroxymethylcellulose or gelatine-microcapsules, liposomes, albumin microspheres, microemulsions, nanoparticles or nanocapsules.
[0237] A topical formulation may be prepared in a variety of physical forms including, for example, solids, pastes, creams, foams, lotions, gels, powders, aqueous liquids, emulsions, sprays and skin patches. The physical appearance and viscosity of such forms can be governed by the presence and amount of emulsifier(s) and viscosity adjuster(s) present in the formulation. Solids are generally firm and non-pourable and commonly are formulated as bars or sticks, or in particulate form. Solids can be opaque or transparent, and optionally can contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product. Creams and lotions are often similar to one another, differing mainly in their viscosity. Both lotions and creams may be opaque, translucent or clear and often contain emulsifiers, solvents, and viscosity adjusting agents, as well as moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product. Gels can be prepared with a range of viscosities, from thick or high viscosity to thin or low viscosity. These formulations, like those of lotions and creams, may also contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product. Liquids are thinner than creams, lotions, or gels, and often do not contain emulsifiers. Liquid topical products often contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product.
[0238] Emulsifiers for use in topical formulations include, but are not limited to, ionic emulsifiers, cetearyl alcohol, non-ionic emulsifiers like polyoxyethylene oleyl ether, PEG-40 stearate, ceteareth-12, ceteareth-20, ceteareth-30, ceteareth alcohol, PEG-100 stearate and glyceryl stearate. Suitable viscosity adjusting agents include, but are not limited to, protective colloids or nonionic gums such as hydroxyethylcellulose, xanthan gum, magnesium aluminum silicate, silica, microcrystalline wax, beeswax, paraffin, and cetyl palmitate. A gel composition may be formed by the addition of a gelling agent such as chitosan, methyl cellulose, ethyl cellulose, polyvinyl alcohol, polyquaterniums, hydroxyethylceilulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carbomer or ammoniated glycyrrhizinate. Suitable surfactants include, but are not limited to, nonionic, amphoteric, ionic and anionic surfactants. For example, one or more of dimethicone copolyol, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, lauramide DEA, cocamide DEA, and cocamide MEA, oleyl betaine, cocamidopropyl phosphatidyl PG-dimonium chloride, and ammonium laureth sulfate may be used within topical formulations.
[0239] Preservatives include, but are not limited to, antimicrobials such as methylparaben, propylparaben, sorbic acid, benzoic acid, and formaldehyde, as well as physical stabilizers and antioxidants such as vitamin E, sodium ascorbate/ascorbic acid and propyl gallate. Suitable moisturizers include, but are not limited to, lactic acid and other hydroxy acids and their salts, glycerine, propylene glycol, and butylene glycol. Suitable emollients include lanolin alcohol, lanolin, lanolin derivatives, cholesterol, petrolatum, isostearyl neopentanoate and mineral oils. Suitable fragrances and colours include, but are not limited to, FD&C Red No. 40 and FD&C Yellow No. 5. Other suitable additional ingredients that may be included in a topical formulation include, but are not limited to, abrasives, absorbents, anticaking agents, antifoaming agents, antistatic agents, astringents (such as witch hazel), alcohol and herbal extracts such as chamomile extract, binders/excipients, buffering agents, chelating agents, film forming agents, conditioning agents, propellants, opacifying agents, pH adjusters and protectants.
[0240] Typical modes of delivery for topical compositions include application using the fingers, application using a physical applicator such as a cloth, tissue, swab, stick or brush, spraying including mist, aerosol or foam spraying, dropper application, sprinkling, soaking, and rinsing. Controlled release vehicles can also be used, and compositions may be formulated for transdermal administration (for example, as a transdermal patch).
[0241] A pharmaceutical composition may be formulated as inhaled formulations, including sprays, mists, or aerosols. This may be particularly preferred for treatment of pulmonary fibrosis. For inhalation formulations, the composition or combination provided herein may be delivered via any inhalation methods known to a person skilled in the art. Such inhalation methods and devices include, but are not limited to, metered dose inhalers with propellants such as CFC or HFA or propellants that are physiologically and environmentally acceptable. Other suitable devices are breath operated inhalers, multidose dry powder inhalers and aerosol nebulizers. Aerosol formulations for use in the subject method typically include propellants, surfactants and co-solvents and may be filled into conventional aerosol containers that are closed by a suitable metering valve.
[0242] Inhalant compositions may comprise liquid or powdered compositions containing the active ingredient that are suitable for nebulization and intrabronchial use, or aerosol compositions administered via an aerosol unit dispensing metered doses. Suitable liquid compositions comprise the active ingredient in an aqueous, pharmaceutically acceptable inhalant solvent such as isotonic saline or bacteriostatic water. The solutions are administered by means of a pump or squeeze-actuated nebulized spray dispenser, or by any other conventional means for causing or enabling the requisite dosage amount of the liquid composition to be inhaled into the patient's lungs. Suitable formulations, wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient.
[0243] Pharmaceutical compositions may also be prepared in the form of suppositories such as for rectal administration. Such compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Suitable excipients include, for example, cocoa butter and polyethylene glycols.
[0244] Pharmaceutical compositions may be formulated as sustained release formulations such as a capsule that creates a slow release of modulator following administration. Such formulations may generally be prepared using well-known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Carriers for use within such formulations are biocompatible, and may also be biodegradable. Preferably, the formulation provides a relatively constant level of modulator release. The amount of modulator contained within a sustained release formulation depends upon, for example, the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.
[0245] It will be understood that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination (i.e. other drugs being used to treat the patient), and the severity of the particular disorder undergoing therapy.
Applications
[0246] The conjugate described herein may be useful as an EV delivery platform or biobot for targeted EV delivery and provide dual antimicrobial and tissue repair activity. It will be appreciated that the conjugate may be suitable for use in preventing and/or treating any disease or condition for which phage therapy is used as a treatment (eg bacterial infection). The application or intended use of the conjugate may be tailored based on the phage and/or the EV selected for use in the conjugate. For example, in cases where the phage may be selected to target a bacteria (e.g., a pathogenic bacteria), the conjugate may be useful for preventing or treating an infection by that bacteria, particularly bacterial infection associated with impaired tissue repair.
[0247] As used herein, the terms “treatment” or “treating” of a subject include the application or administration of a conjugate (or pharmaceutical composition) described herein to a subject with the purpose of delaying, slowing, stabilizing, curing, healing, alleviating, relieving, altering, remedying, less worsening, ameliorating, improving, or affecting the disease or condition, the symptom of the disease or condition, or the risk of (or susceptibility to) the disease or condition. The term “treating” refers to any indication of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; lessening of the rate of worsening; lessening severity of the disease; stabilization, diminishing of symptoms or making the injury, pathology or condition more tolerable to the subject; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject's physical or mental well-being. [0248] As used herein, the term “prevention” or “preventing” are intended to refer to at least the reduction of likelihood of the risk of (or susceptibility to) acquiring a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a patient that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease). Biological and physiological parameters for identifying such patients are provided herein and are known in the art.
[0249] Accordingly, the present invention provides a method for promoting tissue repair in an individual having damaged tissue, the method comprising administering a therapeutically effective amount of the conjugate described herein, the conjugate prepared by the method described herein, or the pharmaceutical composition described herein to the individual in need thereof, thereby promoting tissue repair in the individual.
[0250] Damaged tissue may include: injury to cells and cellular barrier, oxidative stress, or a combination thereof. Oxidative stress may be associated with damage to proteins, lipids, nucleic acids, or a combination thereof. A symptom of oxidative stress may include skin ageing.
[0251] In one embodiment, the present invention provides a method of repairing a damaged tissue, the method comprising the step of administering a therapeutically effective amount of a conjugate of a phage linked with an extracellular vesicle by a linker, wherein preferably, the damaged tissue includes: injury to cells and cellular barrier, oxidative stress, or a combination thereof. In a preferred embodiment, the damaged tissue is injury to cells and cellular barrier, and the conjugate of a phage linked with an extracellular vesicle by a linker is BiobotP. In another preferred embodiment, the damaged tissue is oxidative stress, and the conjugate of a phage linked with an extracellular vesicle by a linker is BiobotD.
[0252] In another aspect, the present invention provides a method of reducing inflammation in an individual in need thereof, the method comprising administering a therapeutically effective amount of the conjugate described herein, the conjugate prepared by the method described herein, or the pharmaceutical composition described herein to the individual in need thereof, thereby reducing inflammation in the individual. Preferably, the inflammation is associated with damaged tissue, bacterial infection, or a combination thereof. [0253] Preferably, the individual has, or is at risk of, impaired tissue repair associated with a bacterial infection.
[0254] Accordingly, in another aspect, the present invention provides a method of preventing and/or treating a bacterial infection in an individual in need thereof, the method comprising administering a therapeutically effective amount of the conjugate described herein, the conjugate prepared by the method described herein, or the pharmaceutical composition described herein to the individual in need thereof, thereby preventing and/or treating a bacterial infection in the individual.
[0255] In one embodiment, the present invention provides a method of decreasing the damaged tissue area or volume of damaged tissue in an individual, the method comprising administering a therapeutically effective amount of the conjugate described herein, the conjugate prepared by the method described herein, or the pharmaceutical composition described herein to the individual in need thereof, thereby decreasing the damaged tissue area or volume damaged tissue in the individual.
[0256] In one embodiment, the present invention provides a method of accelerating the rate of tissue repair, or decreasing the time to completion of tissue repair in an individual, the method comprising administering a therapeutically effective amount of the conjugate described herein, the conjugate prepared by the method described herein, or the pharmaceutical composition described herein to the individual in need thereof, thereby accelerating the rate of tissue repair, or decreasing the time to completion of tissue repair in the individual.
[0257] In one embodiment, the present invention provides a method of inducing or promoting or initiating a tissue repair mechanism in damaged tissue in an individual, the method comprising administering a therapeutically effective amount of the conjugate described herein, the conjugate prepared by the method described herein, or the pharmaceutical composition described herein to the individual in need thereof, thereby inducing or promoting or initiating a tissue repair mechanism in damaged tissue in the individual.
[0258] The damaged tissue may be in an individual who has, or is at risk of impaired tissue repair associated with a bacterial infection. [0259] Typically the injury is one arising from insult to lung, bone, dermal, cutaneous or skin tissue. Accordingly, in one embodiment, the tissue may be selected from the group comprising: lung, bone, dermal, cutaneous, skin tissue and combinations thereof.
[0260] In one embodiment of the present invention, the compositions and methods of the present invention are used for the treatment or pre-emptive therapy of lesions showing early signs of developing into non-healing lesions. Thus, the methods of the invention can be considered methods for preventing the onset of chronic tissue damage.
[0261] The invention may include the step of assessing an individual to determine whether the individual or injury site has one or more systemic or local risk factors described above for an impaired tissue repair process. Typically, the individual is assessed for one or more systemic or local risk factors applicable to formation of chronic tissue damage such as those described herein.
[0262] Where the individual is assessed as having one or more local or systemic risk factors for an impaired tissue repair process, the method may include the further step of selecting the individual for treatment with a conjugate as described herein, to minimise the likelihood of onset of an impaired tissue repair process.
[0263] Beneficial response to treatment with a conjugate according to a method described herein, can be assessed according to whether an individual patient experiences a desirable change in disease status. Examples of desirable change in disease status in impaired tissue repair include an increase in blood perfusion at the site of tissue injury, or of the tissue adjacent to the damaged tissue; an increase in wound closure; a decrease in inflammatory response; lessening of pain at the damaged tissue site.
[0264] In one embodiment of the present invention, the methods for prevention, alleviation and/or treatment of a disease, disorder or infection include a step of treating the tissue in need of treatment with a local anaesthetic agent, such as for example lidocaine.
[0265] In methods of the present invention, the treatments with a composition as defined herein may be combined with other types of treatment or procedures normally used in the treatment of wounds, ulcers, scars or other lesions, such as for example debridement, surgical wound revision, topical negative pressure treatment (TNPT), frequent change of wound dressing, control of diabetes and/or off-loading in order to reduce oedema.
[0266] In one preferred embodiment, the present invention provides a method of promoting wound healing in an individual, the method comprising administering a therapeutically effective amount of the conjugate described herein, the conjugate prepared by the method described herein, or the pharmaceutical composition described herein to the individual in need thereof, thereby promoting wound healing in the individual.
[0267] In one embodiment, the present invention provides a method for the treatment of a dermal or cutaneous wound, the method comprising administering a therapeutically effective amount of the conjugate described herein, the conjugate prepared by the method described herein, or the pharmaceutical composition described herein to the individual in need thereof, thereby treating the dermal or cutaneous wound in the individual.
[0268] The wound may be in an individual who has, or is at risk of impaired wound healing associated with a bacterial infection. For example, the individual may have an existing disease or condition that makes then more susceptible to, or at risk of, developing a bacterial infection. In some embodiments, the individual has a prosthetic medical device.
[0269] In the described embodiments, the dermal wound may be chronic or acute wound and may arise from laceration, burn, incision, maceration, crushing, pressure, puncture abrasion or like injury. The wound may be a chronic skin wound such as a venous stasis ulcer, a diabetic foot ulcer, a neuropathic ulcer, or a decubitus ulcer. In another class of embodiments, the wound results from surgical wound dehiscence. The methods can also be applied to other types of wounds. For example, the wound can comprise a burn, cut, incision, laceration, ulceration, abrasion, or essentially any other wound in an epithelial tissue.
[0270] The injury may arise from insult to dermal, cutaneous or skin tissue. The insult may impact on all layers of dermal tissue, for example on stratum basale (stratum germinativum), stratum spinosum, stratum granulosum, stratum lucidum. Examples of particular injury include laceration, abrasion, rupture, burn, contusion, compression. The injury may be a burn, including a 1 st, 2nd or 3rd degree burn. The injury may be a bedsore or pressure ulcer.
[0271] Chronic or “non-healing” wounds, lesions or ulcers arise when a wound generally fails to follow an appropriate timely healing process to achieve the normal sustained and stable anatomic and functional integrity of healed tissue. Generally speaking, a skin lesion which has failed to make at least substantial progress towards healing within a period of at least about three months, or which has become stable in a partially healed state for more than about three months, or a skin lesion which is unhealed after at least about six months is categorized as a chronic or non-healing wound.
[0272] In another aspect, the present invention provides the use of the conjugate described herein or prepared by the method described herein for:
- promoting tissue repair;
- reducing inflammation;
- treating a bacterial infection;
- decreasing the damaged tissue area or volume of damaged tissue;
- accelerating the rate of tissue repair, or decreasing the time to completion of tissue repair;
- inducing or promoting or initiating a tissue repair mechanism in damaged tissue;
- promoting wound healing; and/or
- the treatment of a dermal or cutaneous wound.
[0273] The present invention also provides the use of the conjugate described herein or prepared by the method described herein for preventing and/or treating a bacterial infection.
[0274] In another aspect, the present invention provides the use of the conjugate described herein or prepared by the method described herein in the manufacture of a medicament for: - promoting tissue repair;
- reducing inflammation;
- treating a bacterial infection;
- decreasing the damaged tissue area or volume of damaged tissue;
- accelerating the rate of tissue repair, or decreasing the time to completion of tissue repair;
- inducing or promoting or initiating a tissue repair mechanism in damaged tissue;
- promoting wound healing; and/or
- the treatment of a dermal or cutaneous wound.
[0275] The present invention also provides the use of the conjugate described herein or prepared by the method described herein in the manufacture of a medicament for preventing and/or treating a bacterial infection.
[0276] In another aspect, the present invention provides the conjugate described herein or prepared by the method described herein for use in:
- promoting tissue repair;
- reducing inflammation;
- treating a bacterial infection;
- decreasing the damaged tissue area or volume of damaged tissue;
- accelerating the rate of tissue repair, or decreasing the time to completion of tissue repair;
- inducing or promoting or initiating a tissue repair mechanism in damaged tissue;
- promoting wound healing; and/or - the treatment of a dermal or cutaneous wound.
[0277] The present invention also provides the conjugate described herein or prepared by the method described herein for use in preventing a bacterial infection.
[0278] In these embodiments, the phage of the conjugate is preferably capable of targeting a bacteria (e.g., a pathogenic bacteria) as described herein, and the EV is preferably capable of promoting tissue repair.
[0279] The bacterial infection may be caused by a pathogenic bacteria, including an antibiotic resistant pathogenic bacteria. In some embodiments, the bacterial infection is caused by a pathogenic bacteria selected from Escherichia coli, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp, preferably Escherichia coli and Pseudomonas aeruginosa.
[0280] The conjugate may be provided in any form suitable for its intended application, including forms described herein. The conjugate may be suitable for use in oral, inhalable, parenteral and wound dressing products.
[0281] The conjugate described herein may advantageously exhibit a comparable or greater therapeutic effect than administration of the individual conjugate components (i.e., the one or more types of EV and optionally the phage, depending on the intended application) alone. Accordingly, in some embodiments, the therapeutic effect following administration of the conjugate is comparable to or greater than that achieved by administration of the individual components of the conjugate alone. It will be understood that this is based on the conjugate and the individual conjugate components being administered in the same relative amounts and by the same mode of administration. It will be appreciated that even if the conjugate exhibits a similar effect to the individual conjugate components alone, an advantage of using conjugates is the use of the phage of the conjugate as a means to deliver the EV(s) to targeted sites while avoiding off-site effects.
[0282] As described herein, the conjugate may comprise (or further comprise) one or more types of active agent conjugated to the linker. Preferably the one or more types of active agent preferably comprise one or both of an antibacterial agent and an antibiofilm agent as described herein. [0283] As described herein, the conjugate may comprise (or further comprise) one or more types of imaging agent conjugated to the linker. Advantageously, due to the phage site-specific delivery, an imaging agent may allow for the site of concentration of the phage to be identified. In addition, the detection of the imaging agent may advantageously allow for the identification of whether an individual has a certain disease, disorder or infection, depending on the intended target of the phage. Further, the site-specific delivery of both one or more EVs and one or more imaging agents may advantageously make them suitable for application in the field of theranostics (i.e., suitable for diagnosis as well as therapy).
[0284] Accordingly, the present invention also provides a method for detecting a cell or biological target associated with a disease, disorder or infection in a subject, comprising: administering to a subject the conjugate described herein or prepared by the method described herein, the conjugate comprising one or more types of imaging agent; and detecting the conjugate in the subject, wherein presence of the conjugate indicates the presence of the cell or biological target.
[0285] The present invention also provides an in vivo method of diagnosing, monitoring or prognosing a disease, disorder or infection in a subject, comprising: administering to a subject the conjugate described herein or prepared by the method described herein, the conjugate comprising one or more types of imaging agent, and the conjugate specifically localising at a cell or biological target associated with a disease, disorder or infection; allowing the conjugate to concentrate at sites in the subject where the cell or biological target is found; and detecting the conjugate; whereby detection of the conjugate above a background or standard level indicates that the subject has the disease, disorder or infection. [0286] The present invention also provides a theranostic method for in vitro and/or in vivo visualisation, identification and/or detection of a cell or biological target associated with a disease, disorder or infection as well as a method for treating that disease, disorder or infection. In one embodiment, the present invention includes a theranostic method that comprises administering the conjugate described herein or prepared by the method described herein, the conjugate comprising one or more types of imaging agent, to an individual in need thereof.
Examples
[0287] The invention will be further described by way of non-limiting example(s). It will be understood to persons skilled in the art of the invention that many modifications may be made without departing from the spirit and scope of the invention.
Materials and methods
Materials
[0288] P. aeruginosa phages (Pae7), E.coli phages (EC84A, EC84B and EC84C) and bacteria strain, E.coli J IE 3454 were provided by the Westmead Institute for Medical Research. P. aeruginosa wild type PAO1 (WT PAO1 ) was a laboratory bacteria strain. The titers of phages Pae7, EC84A, EC84B and EC84C were 1 .0x109, 1 .3x109, 8.0x108, and 2.9x109 PFU mL-1, respectively. Cation Adjusted Mueller-Hinton (MH) broth was purchased from BD. Luria Bertani (LB) Broth and agar were purchased from Oxoid.
EV biomanufacturing and quality control
Cell and probiotic culture of EV manufacturing
[0289] Maternal mesenchymal stromal cells, DMSC23 cell line, since cell lines were preferred over primary cells for reproducibility, reducing the occurrence of MSC senescence and ease of maintenance. DMSC23 cells were cultured in MesenCult™ MSC Basal medium (Human), 10% Mesenchymal stem cell stimulatory supplement (STEMCELL Technologies, Canada), GlutaMAX™ (Life Technologies, Australia) and 1% Pen/Strep (100 units penicillin and 0.1 mg/mL streptomycin, Sigma-Aldrich, Australia). Hanks' Balanced Salt Solution (HBSS(-), Sigma-Aldrich) was used for washing DMSC23. TrypLE™ Select Enzyme (Thermo Fisher Scientific) was used as dissociation reagent for all adherent cells. [0290] The original Lactobacillus rhamnosus EVAB01 was obtained from BiomeCentric and was activated in de Man Rogosa Sharpe (MRS) broth (MRS, CM0359, Oxoid Limited, Basingstoke). The pH of MRS broth was controlled at 5.5 using 37% HCL To maintain culture in its active form, Lactobacillus rhamnosus EVAB01 were inoculated with a 12-hour active subculture. 10% of fermentation broth from the subculture was transferred into fresh MRS broth and inoculated at 37 °C. Growth was monitored by measurement of the optical density (OD) at 600 nm.
EV isolation
[0291] DMSC23 cells were cultured to 80% confluency. Cells were washed twice with HBSS before incubating cells with EV isolation media (MesenCult™ MSC Basal medium containing 10% of CDM-HD Serum replacement for 48 hours. After 48 hours, EV-containing media were collected and centrifuged at 500 x g for 5 minutes and 2,000 x g for 10 minutes to remove cells and debris. The supernatant was then transferred to TFF-Easy 20 nm pores (HansaBioMed/Lonza, Tallinn, Estonia) for EV concentration. The EVs concentration process was described in the manufacture’s protocol (HansaBioMed/Lonza) and EVs from DMSC23 (DEV) were finally diafiltrated in RNase- free PBS.
[0292] Lactobacillus rhamnosus was cultured for 72 hours and OD600 reached 0.8. EV-containing fermentation broth was centrifuged at 8000 x g (10 minutes) twice, then the EV containing supernatant was filtered (0.45 pm) and transferred to TFF-Easy 20 nm pores (HansaBioMed/Lonza, Tallinn, Estonia) for Probiotic EV (PEV) concentration.
Phage propagation and purification
[0293] Propagation of phages was carried out in liquid bacterial host cultures. Phages were added to an exponential phase of host culture at 106 CFU mL-1 and incubated at 37 °C until the culture visibly cleared. The cultures were then centrifuged at 4000 rpm for 20 minutes. The supernatant was collected and passed through a 0.22-pm pore size filter. The titer of phage was quantified using a plaque assay presented as a plaqueforming unit per millilitre (PFU mL-1).
Biobot formulation [0294] A diblock copolymer linker poly(oligo(ethylene glycol) methyl ether acrylate)- block-poly(3-vinylbenzaldehyde) (POEGA-b-PVBA) were prepared using reversible addition-fragmentation chain transfer (RAFT) polymerisation. The first block is comprised of POEGA, a PEG-like polymer that assists with colloidal stability. The second block, PVBA is responsible for the reversible conjugations of phages and EV.
Scheme 1. Synthesis of POEGA-b-PVBA diblock copolymer
[0295] Stock solutions of POEGA-b-PVBA (10 mg mL"1) were prepared by dissolving in PBS and stored at 4 °C for further use. The attachment of phages and EVs on the POEGA-b-PVBA was prepared in PBS with shaking at 120 rpm overnight. The conditions included: PEV only, DEV only, Phages only, Polymer-PEV (PPEV), Polymer- DEV (PDEV), Polymer-Pae7 Phage (PP7), BiobotD and BiobotP (Table 1).
[0296] The conjugation of EV with copolymer POEGA-b-PVBA was determined by 1H NMR in deuterated dimethyl sulfoxide (DMSO-de) solvent. The morphology of phages, EVs and BiobotP were negatively stained with 2% uranyl acetate and characterised using TEM.
Table 1. Preparation of biobot conjugates for characterisation and biological assays.
The conditions included: DEV only, PEV only, Phage Pae7 only, Polymer-DEV (PDEV), Polymer-PEV (PPEV), Polymer-Pae7 (PP7), BiobotD and BiobotP. The stock concentrations of DEV, PEV, Pae7 and linkers were 8.7x1010 EV mL~1, 3.7x1010 EV mL 1, 2.9x109 PFU mL1, and 10 mg mL~1, respectively.
Confirmation of EV-Phage conjugation
Analysis of the EV-phage conjugation using transmission electron microscopy (TEM)
[0297] The morphology of PEV, phages and BiobotP were characterised using TEM, FEI Tecnai T12, with a beam voltage of 120 kV (FEI, USA). The samples were loaded onto carbon film copper grids and then negatively stained with 2% uranyl acetate (pH 4.5). The excess stain was removed within 1 minute, and the stained specimens were air-dried for 30 minutes.
Analysis of the EV-phage conjugation using nuclear magnetic resonance (NMR) spectroscopy
[0298] The successful conjugation of the linker and PEV to form BiobotP were confirmed by 1H NMR (Varian 400-MR) at 25 °C.
Assessment of the effects of biobots on cell functioning and wound healing
Cell culture and maintenance
[0299] Human dermal keratinocyte (HEK, cell line), Human dermal fibroblast (HDF, primary cell) and A549 (another class of fibroblast, cell line) cells were all cultured in medium containing Dulbecco’s Modified Eagle’s Medium (DMEM medium-high glucose, Sigma-Aldrich, Australia) supplemented with 10% Foetal Bovine Serum (FBS, Bovogen, Australia) and 1% antibiotics (Pen/Strep). Phosphate buffer saline without Ca2+and Mg2+ (PBS, Lonza, Bella Vista, NSW, Australia) was used for A549. TrypLE™ Select Enzyme (Thermo Fisher Scientific) was used as dissociation reagent for all adherent cells. DMSC23 and probiotics were used for EV isolation. All cells were maintained at 37 °C and 5% CO2.
Analysis of cell viability using DNA-based cell proliferation assay (CyQUANT)
[0300] DNA CyQUANT® NF Cell Proliferation Assay Kit (Thermo Fisher Scientific, Scoresby, VIC, Australia) is also used for cell viability tests as per manufacturers’ protocols. A549 is seeded and cultured at a density of 5 x 103/well in 100 pL of medium into 96-well microplates (Corning, USA). Then, after the cell attach to the plate overnight, the cells were treated with different treatment including DEV, DEV+phage (Pae7), BiobotD, PEV, PEV+phage (Pae7), BiobotP and phage only (Pae7) at 3 different concentrations (107, 105 and 103 molecules per mL). Then, 100 pL of CyQUANT Direct 2X detection reagent was added to each microplate wells. After incubating the plate for 60 minutes fluorescence using excitation/emission — 508/527 nm. All experiments were performed in triplicate. The proliferation of cells was expressed by the absorbance.
Analysis of cell viability using metabolic activity assay (PrestoBlue)
[0301] PrestoBlue™ Cell Viability Reagent (Thermo Fisher Scientific, Scoresby, VIC, Australia) was also used or cell viability tests as per manufacturers’ protocols. It is done in a microplate seeded with A549, the initial cell concentration, treatment starting time and treatment condition was identical with the plate used in CyQUANT Cell Proliferation Assay. After 24 hours of treatment, 10 pL of PrestoBlue reagent was added to each well. After incubating the plate for 10 min fluorescence using excitation/emission — 560/590 nm. All experiments were performed in triplicate. The proliferation of cells was expressed by the absorbance.
Analysis of cell growth using real-time cell imaging system (IncuCyte)
[0302] Cells (fibroblast, class I) were seeded and cultured at a density of 5 x 103/well in 100 pL of medium into 96-well microplates (Corning, USA). Then, after the cell attach to the plate overnight, the cells were treated with different treatment including DEV, DEV+phage (Pae7), BiobotD, PEV, PEV+phage (Pae7), BiobotP and phage only (Pae7) at 3 different concentrations (107, 105 and 103 molecules per mL). IncuCyte ZOOM software program was used for quantitative analysis, to measure and calculate the confluency of the cells at each time point. Assessment of cell morphology using single cell holotomography (Nanolive)
[0303] A549 were seeded and cultured at a density of 3 x 104/dish in 2 ml of medium into 35 mm Glass bottom dish with 20 mm micro-well #1 .5 high performance cover glass (Cellvis, China). After the cell attach to the dish overnight, the cells were treated with different treatment including DEV, DEV+phage (Pae7), BiobotD, PEV, PEV+phage (Pae7), BiobotP and phage only (Pae7) at a concentration of 107 molecules per mL. Then cells were fixed using 4% paraformaldehyde (PFA) after 24 h-treatment. 3D Cell Explorer (Nanolive, Switzerland) was used for cell Holotomographic imaging.
Analysis of the cell viability and vitality using cell counting kit-8 (CCK-8)
[0304] Cell Counting Kit (CCK-8) (Dojindo Molecular Technologies, Inc., Santa Clara, CA, USA) were used for long term cell viability tests as per manufacturers’ protocols. Cells (HEK and HDF in separate plates) were seeded and cultured at a density of 5 x 103/well in 100 pL of medium into 96-well microplates (Corning, USA). Then, after the cell attach to the plate overnight, the cells were treated with different treatment including DEV, DEV+phage (Pae7), BiobotD, PEV, PEV+phage (Pae7), BiobotP and phage only (Pae7) at 3 different concentrations (107, 105 and 103 particles per mL). After treatment for 24 hours, 100 pL of medium was transferred to a new plate for LDH assay, pre -mixed CCK-8 solution (10% CCK-8 reagent +90% of basal medium (DMEM high glucose) was added to each well and then incubated for 2.5 hours, the plate is protected from light. All experiments were performed in triplicate. The absorbance was analysed at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA) using wells without cells as blanks. The proliferation of cells was expressed by the absorbance.
Analysis of biobot cytotoxicity using lactate dehydrogenase (LDH) Assay
[0305] Cytotoxicity LDH Assay Kit (Dojindo Molecular Technologies, Inc., Santa Clara, CA, USA) were used to assess cell death as per manufacturers’ protocols. This experiment shares the same plate with the CCK-8 plate. After treatment for 24 hours, 100 pL of medium in each well was transferred to a new 96-well microplate, including high control (cells treated with 20 pL lysis buffer, incubated for 30 minutes before transferring the medium) and low control (no treatment). Then 100 pL of working solution was added to each well, the plate was protected from light and incubated for 30 min. Then 50 pL of stopping solution was added to each well and the absorbance is measured at 490 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). All experiments were performed in triplicate. Cell death was expressed by the absorbance.
Analysis of the effect of biobots on wound healing (Wound Scratch Assay; WoundMaker and IncuCyte)
[0306] To investigate the impact of EV, phage and biobot on skin cell migration, the rates of repopulation of a two-dimensional scratch wound on a monolayer of cells were compared. HEK cells and HDF cells were seeded at 1 x 104 cells/well on 96-well plates and were cultured in normal media until 90% confluent. Then cells were scratched using a 96-pin woundmaking tool (IncuCyte® WoundMakerTM). After washing with PBS twice to remove cell debris, then fresh media were added. The cells were treated with different treatment including DEV, DEV+phage (Pae7), BiobotD, PEV, PEV+phage (Pae7), BiobotP and phage only (Pae7) at 107, 105 and 103 particles per mL for each treatment. Wound images were taken using the IncuCyte® live cell imaging system (Sartorius, Inc.) every 2 hours for 48 hours with 10 x magnification. IncuCyte ZOOM software program was used for quantitative analysis, to measure and calculate the relative density and confluence of the scratch wound at each time point.
Analysis of the effect of biobots on cell migration (single cell migration assay)
[0307] To investigate the impact of EV, phage and biobot on skin cell migration, single cell migration was used to track the movement of the skin cells at a single cell level, in response to EV, phage and biobot treatment. HEK cells and HDF cells were seeded at 500 cells/well on 96-well plates and were cultured in normal media. After the cell attach to the plate, images were taken using the IncuCyte® live cell imaging system (Sartorius, Inc.) every 2 hours for 48 hours with 10 x magnification. Cell tracking software was used for analysis of the cell migration.
Analysis of biobot uptake and 3D distribution
Preparation of cells
[0308] Human epidermal keratinocytes (HEK) and Human dermal fibroblast (HDF) were seeded in 96-well plate at the seeding densities cells/well. Cells were maintained at 37SC and 5% CO2 overnight to ensure cell adhesion. Preparation of fluorescent-labelled Biobots
[0309]To fluorescently label biobots, Acoerela AcoDyesTM (Aco-600) was used. This dye is a membrane dye that is only emissive after imbedding into the lipid bilayer of the biobots. First, the dye was diluted with sterile 1x PBS, and vortex for 1 minute to ensure it was fully dissolved. Next, the dye solution was sonicated for 15 minutes at 40°C, and the dye stock solution was then diluted to 10 pM.
[0310] Both biobots (BiobotD and BiobotP) were diluted to 109 particles/mL in separate Eppendorf tubes. Next, the dye was added to the biobots preparations at 1 :9 volume ratio (109 particles/mL biobots preparation : 10 pM dye solution). To ensure uniform mixing, biobot-dye preparation was mixed using a pipette by taking the solution up and down 5 times followed by vortexing for 15 seconds. After the mixing, the preparations were incubated in dark conditions at 37°C for 1 hour.
[0311] After the incubation, the excess of the dye was removed using exosome spin columns (Exosome Spin Column, MW3000, invitrogenTM), as per manufacturer protocol. In brief, the columns were hydrated with 650 pL of 1 x PBS made with RNase- free water. Next, the columns were caped, vortexed, and taped to remove air bubbles, and hydrated at room temperature for 5-15 min. The columns were then placed in a 2 mL Collection Tube and spun at 750 x g for 2 min at room temperature to remove excess interstitial fluid.
[0312]The Collection Tubes used to collect the excess PBS were discarded after the centrifugation and the gel bed was formed in the column. Next, 100 pL of samples were loaded directly to the centre of the gel bed at the top of the column. The Spin Columns were then placed in the 1 .5 mL Elution Tube and placed in the rotor, maintaining the previous orientation. The Spin Columns were spined at 750 x g for 2 min at room temperature. The stained biobots were then eluted into the Elution Tube. The excess dye was removed.
[0313]After staining and dye removal, biobot-dye preparations were diluted to the desired treatment concentration (5 x 107 particles/mL) using cell culture media, and then used to treat cells.
Treating cells with stained biobots [0314] After overnight maintenance of cells, cell culture media were aspirated from each well and cells were treated with the stained biobots. Cells were then maintained at 37SC and 5% CO2 for 36 hours. After treatment, the cells were washed with 1 x PBS 3 times to remove not internalized biobots, and then the cells were fixed with 4% PFA for fluorescent imaging.
Impedance measurement
[0315]To examine the electrical resistance and capacitance of cell layers, impedance measurement was performed every two days using a high-throughput automated monitoring system - Locsense (Locsense, Netherland). The impedance of cell layers was measured in a broad range of frequencies (from 1 to 100,000 Hz). The data were then exported in Microsoft Excel and analysed.
Preparation of cell barriers
[0316JHEK and HDF were seeded in celIQART 24-Well Plate 0.4 pm PET translucent (FP) transwell insert, pore size 0.4 pm, translucent optics (100 x 106 pores per cm2) at the seeding densities of 30,000 cells/well. Both the apical and basolateral compartments were filled with fresh cell culture media (300 pL of medium for the apical compartment and 1000 pL of medium for the basolateral compartment). Cells were maintained at 37SC and 5% CO2 overnight to ensure cell adhesion. Next, impedance measurements were conducted for each transwell every 2 days.
[0317] Before each measurement, the media of transwells (cells and blank) was changed to the fresh media (epical side for 1000 pL and basolateral side for 1 ml). Next, the plates were placed in biosafety cabinet to cool down for 10 minutes at room temperature. Kimwipes with ethanol were used to clean the electrodes twice before measurements. The positions of the transwells were adjusted to make sure the electrode could fit into the gaps of the transwells and conduct a measurement using the smart lid. These positions of the transwells were marked from the first day of measurement to make sure that the measurements were done in the same orientation consistently.
[0318] After each measurement, the electrodes were cleaned with Kimwipes plus ethanol twice; if it was the last experiment, the electrodes were wiped once and rinsed with a small volume of ethanol. Assessment of the change of barrier function
[0319] After each measurement, the impedance data were analyzed to check if the cell barrier had been established. Once the cell barriers were established, they were divided into 2 groups and damaged with hydrogen peroxide or acetone.
[0320] Hydrogen peroxide induces oxidative stress and results in damage to proteins, lipids and nucleic acids, and is a suitable model for skin ageing (at the used concentration and treatment time 24 h). Acetone (treatment conditions 50% acetone, 2 min) selectively removes lipids from the intercellular lipid domains, resulting in a general disruption to the organization of the lipid lamellae. These lipid structures are considered crucial to the barrier properties of the skin cell. Acetone can also induce increased transepidermal water loss; hence it may be used as a model of injury to cells and cellular barrier.
[0321] First, the cell culture media were aspirated. The media of the hydrogen peroxide group was changed with 400 pM hydrogen peroxide (diluted in cell culture media) for 24 hours (for both the apical and basolateral compartments). The media of the acetone group was changed with 50% acetone (diluted in cell culture media) for 2 minutes, then the treatment containing media were aspirated immediately, washed with cell culture media twice, and then changed with fresh cell culture media (for both the apical and basolateral compartments).
[0322] Following the protocol above, impedance measurements were conducted 24 hours after hydrogen peroxide and acetone treatment were added to check the changes in barrier function. Next, the damaged cell barriers were treated with BiobotD and BiobotP separately; the media of damaged cell barriers were changed with cell culture media that contained BiobotD or BiobotP (concentration: 109 particles/mL) immediately after the measurement, and the treatment duration was 48 hours. Impedance measurements were conducted every 2 days after biobot treatment was added to assess the recovery of the barrier function.
Nanomechanical analysis of cell stiffness via nanoindentation
[0323]To probe the nanomechanical properties of cells, a pre-calibrated force sensor probe was used in the Pavone nanoindentor (Opticsl 1 Life, Amsterdam, the Netherlands). The spring constant of the probe was 0.017 N/m and the tip radius was 3 pm.
Sample preparation
[0324] Human epidermal keratinocytes (HEK) and Human dermal fibroblast (HDF) were seeded in 96-wallplate at the seeding densities of 10,000 cells/well. Cells were maintained at 37SC and 5% CO2 overnight to ensure cell adhesion.
[0325]The wells had four different groups: healthy cells that received no treatment, damaged cells that received no treatment, damaged cells treated with BiobotD for 24 hours, and damaged cells treated with BiobotP for 24 hours. Each group had three replicate wells. The concentration of BiobotD and BiobotP treatment was 1000 particles/ cell.
[0326]After overnight cell maintenance, cell culture media were aspirated from each well. The damaged cell groups were treated with cell culture media containing 50 pg/mL of bleomycin to inflict damage, and the healthy cell group was changed with fresh cell culture media. Cells were then maintained at 37SC and 5% CO2 for 24 hours. After the cell damaging process, the cells were washed with 1 x PBS twice and then changed with the corresponding treatment for 24 hours. The groups that received no treatment were changed with fresh cell culture media.
[0327] After the treatment, the cells were washed with 1 x PBS twice and fixed with 4% PFA for nanoindentation.
Assessment of cells’ stiffness
[0328] Cells’ mechanical properties were assessed using a probe with a stiffness of 0.017 N/m and the tip radius was 3 pm. The maximum load of the indentations was 0.1 pN. All indentations were performed in 1x PBS. To determine the stiffness, effective young’s modulus was selected as the parameter, the contact point was identified using Opticsl 1 Life DataViewer software. A Hertzian contact model with a Poisson’s Ratio of 0.5 was fitted to the load vs. indentation curve corresponding to the first 600 nm of indentation. Any indentation with a fit for the Hertzian model below an R2 value of 0.95 was excluded from further analysis. Assessment of effects of biobots on bacterial growth and biofilm eradication; - antimicrobial activity of biobots
Antimicrobial assays
[0329] The antimicrobial effects of EV on bacteria P. aeruginosa PAO1 and E. coli J IE 3454 was measured using the broth microdilution technique. A bacteria-only control and a MH broth-only control were also included in the same microplate. The 96-well microplates were incubated at 37°C for 24 hours. The minimum inhibitory concentration (MIC) was determined to be the concentration of antimicrobial agents at which the optical density (ODeoo) was equal to that of a medium-only control using a multimode microplate reader (VICTOR™ X4, Perkin-Elmer). The MIC can also be recorded as the lowest concentration preventing visible bacterial growth after 24 hours.
Bacterial biofilm formation inhibition and eradication assays
[0330] P. aeruginosa PAO1 overnight bacterium culture medium was prepared by inoculating one colony in 10 mL of LB broth and incubating it on an orbital shaker at 220 rpm at 37 °C for 16-17 hours. The bacterium culture medium was diluted 200-fold to an optical density at 600 nm (ODeoo) between 0.003-0.005 in M9 minimal medium (containing 48 mM Na2HPO4, 22 mM KH2PO4, 9 mM NaCI, 19 mM NH4CI, 2 mM MgSO4, 0.1 mM CaCh, and 20 mM glucose, pH 7.0). This medium was freshly prepared for each experiment.
[0331] Various treatment formulations were added to the 24-well microtiter plates simultaneously as cell culture media placement for the biofilm inhibition assay. The microplates were incubated on an orbital shaker at 180 rpm for 6 hours at 37 °C.
[0332] The biofilm biomass was analysed by crystal violet (CV) staining. The biofilm on each well surface of the plates was washed once with 1 mL PBS before CV staining.
1 mL of 0.05 % CV solution made by 2% CV aqueous solution in PBS. The CV-stained biofilm was washed once with 1 mL PBS after 20 min incubation. The remaining CV- stained biofilm was dissolved in 33% glacial acetic acid and quantified by measuring the absorbance at 550 nm (OD550) using a multimode microplate reader.
Example 1 - Results of the conjugation of EV-phage assessment [0333] Figure 1 shows the shift of the main peaks of the size scattered signal towards larger sizes for samples functionalised with polymer, demonstrating successful attachment of the polymer to the surface (coating). Signal for the size distribution of biobots was outside the calibration range of the nano flow cytometry system, which could suggested a successful conjugation due to their larger size.
[0334] Successful conjugation of PEV on POEGA-b-PVBA was confirmed via 1H NMR analysis by monitoring the decrease in the signal intensity at 9.8 ppm, which corresponds to the aldehyde group. 1H NMR spectra of POEGA-b-PVBA and POEGA- b-PVBA conjugated with PEV are shown in Figure 2 traces (a) and (b) respectively. The conjugation efficiency of the PEV and the copolymer was 53%, as determined by comparing the intensity of the aldehyde signal at 9.8 ppm with the unchanged -CH2O ester of OEGA at 4.2 ppm before and after the reaction shown in Figure 2. The morphology of the PEV, phages and phage-PEV BiobotP were observed by TEM analysis shown in Figure 3 (A), 3 (B) and 3 (C), respectively.
Example 2 - Biobot cytotoxicity assessment
[0335] Biobots improved metabolic activity and had a positive influence on DNA production which demonstrated that both classes of biobots are not cytotoxic (safe) up to the concentration of 107 particles/mL (Figure 4).
[0336] Both types of biobots did not have a negative effect on epithelial cell growth. BiobotsD and P reduced the number of vacuole in cells and showed an improvement in some morphological features and reduction of lipid droplets (due to likely improved metabolism). These results showed collectively that biobots had positive effects on cell viability and structure (Figure 5 and Figure 6).
[0337] Figure 7 shows the biobot treatment had no negative effects on keratinocytes growth, and they were not cytotoxic. Small improvements in the cells growth with increased concentration of biobots were observed.
[0338] Figure 8 shows that both types of biobots reduced the expression of LDH, with BiobotP being more effective and concentration dependent (with higher concentration reducing LDH more effectively). These results showed that both biobots reduce cell death, thus improved cell viability/vitality. Results of the effect of biobots on wound healing
[0339] Treatment with both biobots and a combination of phage and EVs (PEVs and DEVs) showed substantial improvement of keratinocytes wound closure/healing, which was demonstrated by the reduction of time to close the wound and maintain high cell density (Figure 9 and Figure 10). BiobotD and BiobotP showed an improvement in the rate of wound closure when compared to EV only, phage only and no treatment. Cells that receive no treatment show the lowest rate of wound closure (Figure 11 and Figure 12).
[0340] Due to elongated structure of skin fibroblast and the heterogeneity in their migration (not following migration front/line) the analysis of the wound closer considers two aspects: wound repopulation (movements of cells towards the would space/gap) and cell density (the number of cells including their clustering). The analysis showed that BiobotP and the combination of PEV and phage were the most effective in enhancing wound closure (repopulation of the wound with cells), demonstrating its positive effects on cell migration. While the analysis showed that BiobotD improved wound healing (less than BiobotP), it also showed that BiobotD improved the density of cells, suggesting its positive effect on cell proliferation (Figure 13, Figure 14, Figure 15 and Figure 16).
[0341] Both results induced different biological functionalities of BiobotD and BiobotP; BiobotD promoted effective proliferation of skin fibroblasts, but may have less impact on migration of the cells. While BiobotP enhanced the migration of the fibroblast and closed the gap of the wound faster, however, the density of cells was lower, suggesting that BiobotP enhanced the migratory capability of fibroblasts.
Example 3 - Assessment of the antibacterial effects of biobots on planktonic bacteria
[0342]The antibacterial activity of EV on the growth of planktonic P. aeruginosa PAO1 and E.coli J IE 3454 were evaluated by determining the MIC assay. This assay is defined as the minimum concentration of an agent required to inhibit the visible growth of a microorganism after overnight incubation. The results shown in Figure 17 and Figure 18 are the MIC results of EV alone, phages alone and the biobots. Both PEV and DEV did not show any significant growth inhibition on the test bacteria. Combining phages and EVs may result in an additive or synergistic effect. The antimicrobial activities of the phage-EV conjugates were thus determined with different formulations. The results show that the biobots did not affect the activities of phages on inhibiting bacterial growth. In the case of the phage-EV conjugates described herein, it is essential to note that even if the combination of phages and EV agents in the conjugate form has a similar effect to the test phages alone inhibiting the growth of planktonic P. aeruginosa PAO1 and E.co/ JIE 3454.
Example 4 - Effects of biobots on the inhibition of biofilm formation assays
[0343]To assess the antibiofilm activities of the Phage-EV conjugates, the potential biofilm inhibition activity of EV alone was first determined using the CV staining assay. The results of the P. aeruginosa PAO1 biofilm inhibition study are presented in Figure 19. The results indicated that PEV, but not DEV, had inhibitory activity on biofilm formation. The PEV could cause 40-50 % biofilm inhibition. A previous study has shown Pae7 at a titer higher than 104 PFU mL-1 began to exhibit an inhibitory effect on bacterial biofilm growth. Hence, two titers of Pae7 (106 and 102 PFU mL-1) were chosen to study the potential interaction between EV and phages. Pae7_EV conjugates biobots with phages titer at 106 and 102 PFU mL-1 displayed comparable results of phage alone on biofilm inhibition assay. These results could indicate that the presence of EV in the conjugate forms did not impact phage activity on biofilm inhibition.
Example 5 - Assessment of the uptake and distribution of biobots in different cell types
[0344]AcoDyes-600 fluorescently labelled biobots were incubated with HEK or HDF for 36 hours to assess the uptake and distribution of biobots in different cell types.
[0345] Representative images of the uptake of biobots in HEK or HDF are shown in Figure 20 and Figure 21 , respectively. The area of intensity and percentage coverage of fluorescently labelled biobots are summarised in Table 2.
Table 2. Area of intensity and percentage coverage of fluorescently labelled biobots
[0346]The results demonstrated that BiobotP uptake was higher compared to BiobotD uptake in HEK, and that BiobotD uptake by HEK was higher compared to BiobotD uptake in HDF.
[0347] Furthermore, 3D distribution of AcoDyes-600 labelled biobots that were uptaken by HEK and HDF was assessed (Figure 22). Representative images of biobots in cells at 1 .2 pm increments from the bottom surface are shown in Figures 23 to 25.
[0348]The results demonstrated that BiobotP and BiobotD had different uptake kinetics in cells. BiobotP was uptaken more effectively than BiobotD in HEK. BiobotP was distributed across the entire cell volume, while BiobotD was accumulated or clustered in zones around the nuclei. Biobots also showed cell type-dependent differences in the uptake. Fluorescence intensity analysis suggested a more effective uptake of BiobotD in HEK when compared with HDF. The distribution of BiobotD in HDF was uniform across the cell volume, while in HEK BiobotD accumulated or clustered in zones around the nuclei.
Example 6 - Assessment of the biobot’s healing effect on barrier function and stiffness in different cell types
[0349] HEK or HDF were damaged by incubation with either one of acetone and hydrogen peroxide for 24 hours. The damaged cells were then treated with biobots. Barrier function of the cells were monitored via the measurement of impedance during the period between 6 days prior to acetone or hydrogen peroxide damage and 13 days after acetone or hydrogen peroxide damage, as shown in Figures 23-26.
[0350] In HEK, treatment with BiobotD or BiobotP following acetone-induced damage led to delayed deterioration of barrier function. In contrast, similar delayed deterioration was not observed in HEK following hydrogen peroxide-induced damage.
[0351] In HDF, treatment with BiobotP led to pronounced improvements in barrier function following acetone-induced damage, but not following hydrogen peroxideinduced damage. In contrast, treatment with BiobotD led to pronounced improvements in barrier function following hydrogen peroxide-induced damage, but not following acetone-induced damage. As hydrogen peroxide and acetone use different mechanisms to induce cell damage or injury, the experiments revealed different effectiveness of biobots to reduce or repair cellular damage depending on the injury type.
[0352]The effect of biobots on the stiffness of damaged cells was assessed in HDF that was injured with 24 hours of treatment with bleomycin, as shown in Figure 27. BiobotP demonstrated superior healing effect that restored the stiffness of HDF towards their healthy state.
Example 7 - Assessment of biobot’s effect on the morphology of different damaged cell types
[0353]The effect of biobots on bleomycin-damaged HEK and HDF was assessed. Representative images of the morphology of the cells are shown in Figure 28 and Figure 29.
[0354] In HEK, both BiobotD and BiobotP demonstrated healing effects by changing damaged cells’ morphology towards their normal state. In HDF, BiobotD demonstrated a slightly better healing effects by changing damaged cells’ morphology towards their normal state.

Claims

1 . A conjugate of a phage linked with an extracellular vesicle (EV) by a linker, wherein the phage and the EV are independently conjugated to the linker by one or more types of cleavable bond.
2. The conjugate of claim 1 , wherein at least one of the one or more types of cleavable bond is pH labile.
3. The conjugate of claim 1 or 2, wherein at least one of the one or more types of cleavable bond is selected from a hydrazone, an imine, an acetal, a ketal, and an ester.
4. The conjugate of any one of claims 1 to 3, wherein the linker comprises a polymer which comprises the one or more types of cleavable bond.
5. The conjugate of claim 4, wherein the polymer comprises a diblock copolymer comprising a first block and a second block.
6. The conjugate of claim 5, wherein the first block comprises poly(oligo(ethylene glycol) methyl ether acrylate) (POEGA).
7. The conjugate of claim 5 or 6, wherein the second block comprises a polymer composed of a repeating monomer unit having the following structure: wherein
R is independently -CHO or -C=NZ; and
Z is independently a phage or an EV.
8. The conjugate of any one of claims 1 to 7, wherein the conjugate has the following structure: wherein
R is independently -CHO or -C=NZ;
Z is independently a phage or an EV; n is 9; x is 10 to 30; and y is 5 to 15.
9. The conjugate of any one of claims 1 to 8, wherein the phage is capable of infecting one or more pathogens selected from Escherichia coli, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.
10. The conjugate of any one of claims 1 to 9, wherein the phage is selected from a filamentous phage, an MS2 phage and a caudovirus.
11 . The conjugate of any one of claims 1 to 10, wherein the EV is derived from the group comprising: a stem cell, a probiotic, and combinations thereof.
12. The conjugate of claim 11 , wherein the EV derived from a stem cell is derived from a mesenchymal stromal cell.
13. The conjugate of claim 11 or 12, wherein the phage is linked with one type of EV.
14. The conjugate of any one of claims 1 to 13, wherein the conjugate comprises one or more types of active agent conjugated to the linker.
15. The conjugate of claim 14, wherein the one or more types of active agent comprise one or both of (i) one or more antibacterial agents and (ii) one or more antibiofilm agents.
16. A method for preparing a conjugate of a phage linked with an EV by a linker, the method comprising: providing a linker comprising one or more types of reactive functionality capable of forming a cleavable bond; conjugating an EV to the linker via at least one of the one or more types of reactive functionality; and conjugating a phage to the linker via at least one of the one or more types of reactive functionality; thereby providing the conjugate, wherein the phage and the EV are each independently conjugated to the linker by one or more types of cleavable bond.
17. A conjugate prepared by the method of claim 16.
18. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 15 or prepared by the method of claim 16, and a pharmaceutically acceptable diluent, excipient or carrier.
19. A pharmaceutical composition according to claim 18, wherein the composition comprises one type of conjugate.
20. A pharmaceutical composition according to claim 18, wherein the composition comprises at least two types of conjugates.
21 . A method of promoting tissue repair in an individual having damaged tissue, the method comprising the step of administering a therapeutically effective amount of the conjugate of any one of claims 1 to 15, the conjugate prepared by the method of claim 16, or the pharmaceutical composition of any one of claims 18 to 20 to the individual in need thereof, thereby promoting tissue repair in the individual.
22. The method of claim 21 , wherein the individual has, or is at risk of, impaired tissue repair associated with a bacterial infection.
23. The method of claim 22, wherein the method comprises preventing or treating the bacterial infection.
24. The method of any one of claims 21 to 23, wherein the method comprises decreasing the damaged tissue area or volume of the damaged tissue.
25. The method of any one of claims 21 to 24, wherein the method comprises accelerating the rate of tissue repair, or decreasing the time to completion of tissue repair.
26. The method of any one of claims 21 to 25, wherein the tissue is selected from the group comprising: lung, bone, dermal, cutaneous, skin tissue and combinations thereof.
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