Charge interaction assisted lipid-encapsulation of bacteriophages

Abstract

The present invention provides use of a charged lipid for encapsulating phages in nanosize lipid particles, wherein the charged lipid is cationic or anionic; wherein the charged lipid comprises a cationic or anionic moiety and a C12 to C80 hydrocarbon chain or a polymeric chain; wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O)Rx, - C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl; wherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, - SH, C1 to C6 alkyl, and 5- or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, - NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl. The present invention further provides a method for encapsulating phages, phages encapsulated in nanosize lipid particles, compositions comprising phages encapsulated in nanosize lipid particles and their uses for example in medicine.

Description

CHARGE INTERACTION ASSISTED LIPID-ENCAPSULATION OF BACTERIOPHAGESFIELD OF THE INVENTION

[0001] The present invention relates to the field of phage therapy. However, it will be appreciated that the invention is not limited to this particular field of use.BACKGROUND OF THE INVENTION

[0002] The following discussion of the prior art is provided to place the invention in an appropriate technical context and enable the advantages of it to be more fully understood. It should be appreciated, however, that any discussion of the prior art throughout the specification should not be considered as an express or implied admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0003] Antibiotic resistance represents a global health threat, recognised as an urgent issue requiring international collaboration by the World Health Organisation (WHO) and deemed a major public health concern by the Australian Government.

[0004] Bacteriophage (phage) therapy is a promising antimicrobial to enhance or supplement the existing antibiotic arsenal to combat multi-drug resistant infections. Phage therapy is a promising bactericidal strategy to tackle antimicrobial resistance (AMR).

[0005] Phages are natural predators of bacteria that rely on the bacterial host for survival and replication. Current approaches to phage therapy are guided entirely by administering naked phages which have poor tissue penetration and are prone to degradation.

[0006] The success of phage therapy in treating lung infections, such as cystic fibrosis and pneumonia, has been limited by the short half-life of phages (4-9 hours) when administered intravenously. While inhalation aerosol delivery of phages can target infection sites in the lungs, phages may not reach bacteria within biofilms. Liposomes can penetrate biofilms and facilitate intracellular uptake, making them a suitable delivery vehicle for phages. Liposomal phages, with high encapsulation efficiency, can effectively bring these concepts together to maximize the benefits of phage-based therapy. They offer an optimal formulation and delivery strategy to target biofilm-forming P. aeruginosa in the lungs. By delivering inhalation phage aerosols, liposomal formulations enhance phage penetration into biofilms, effectively killing the bacteria.

[0007] A promising strategy is to encapsulate phages in liposomes, which not only penetrate bacterial biofilms but confer phage protection and stability, increasing its half-life. Liposomes also offer the advantage of sustained release to the infection site, extending the timefor phages to encounter bacteria, and subsequent killing by phage adsorption onto and replication inside the bacteria.

[0008] However, current strategies for encapsulating phages rely on conventional methods using standard polymer and neutral lipid formulations, which suffer from poor encapsulation efficiency and suboptimal particle sizes (microscale). Although various microfluidic devices with adjustable parameters have been explored, there is no evidence to suggest they improve phage encapsulation. Furthermore, scaling up microfluidic-based encapsulation for commercial production presents significant challenges, as these systems are limited by the low volume and slow flow rate.

[0009] It is an object of the present invention to overcome or ameliorate one or more the disadvantages of the prior art, or at least to provide a useful alternative.SUMMARY OF THE INVENTION

[0010] According to a first aspect, the present invention provides use of a charged lipid for encapsulating phages in nanosize lipid particles,wherein the charged lipid is cationic or anionic;wherein the charged lipid comprises a cationic or anionic moiety and a C12 to C80 hydrocarbon chain or a polymeric chain;wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O) Rx, -C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5-or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, -NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.

[0011] According to a second aspect, the present invention provides a method of encapsulating phages, the method comprising mixing a charged lipid formulation with a phage formulation,wherein the charged lipid formulation comprises a charged lipid;wherein the charged lipid is cationic or anionic;wherein the charged lipid comprises a cationic or anionic moiety and a C12 to C80 hydrocarbon chain or a polymeric chain;wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O) Rx, -C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5-or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, -NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.

[0012] According to a third aspect, the present invention provides a method of improving stability of a phage, the method comprising encapsulating the phage in nanosize lipid particles;wherein the lipid particles comprise a charged lipid;wherein the charged lipid is cationic or anionic;wherein the charged lipid comprises a cationic or anionic moiety and a C12 to C80 hydrocarbon chain or a polymeric chain;wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O)Rx, -C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5-or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, -NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.

[0013] According to a fourth aspect, the present invention provides a phage encapsulated in nanosize lipid particles;wherein the lipid particles comprise a charged lipid;wherein the charged lipid is cationic or anionic;wherein the charged lipid comprises a cationic or anionic moiety and a C12 to C80 hydrocarbon chain or a polymeric chain;wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O) Rx, -C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5-or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, -NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.

[0014] According to a fifth aspect, the present invention provides a composition comprising a phage encapsulated in nanosize lipid particles;wherein the lipid particles comprise a charged lipid;wherein the charged lipid is cationic or anionic;wherein the charged lipid comprises a cationic or anionic moiety and a C12 to C80 hydrocarbon chain or a polymeric chain;wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O)Rx, -C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5-or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, -NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.

[0015] According to a sixth aspect, the present invention provides a method of treating or preventing a condition or disease, comprising administering to a subject in need thereof an effective amount of a composition comprising a phage encapsulated in nanosize lipid particles;wherein the lipid particles comprise a charged lipid;wherein the charged lipid comprises a cationic or anionic moiety and a C12 to C80 hydrocarbon chain or a polymeric chain;wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O) Rx, -C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5-or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, -NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.

[0016] According to a seventh aspect, the present invention provides a method of treating a bacterial infection in the respiratory system, comprising administering to a subject in need thereof an effective amount of a composition comprising a phage encapsulated in nanosize lipid particles;wherein the lipid particles comprise a charged lipid;wherein the charged lipid comprises a cationic or anionic moiety and a C12 to C80 hydrocarbon chain or a polymeric chain;wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O)Rx, -C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5-or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted withone or more substituents selected from -OH, -NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.

[0017] The present invention uses charged lipids to encapsulate phages by employing their structural and electrostatic characteristics.

[0018] The capsid surface of certain phages carries a negative charge, while the tail is positive, depending on pH conditions. This negative charge on the capsid attracts with positively charged cationic lipids, making the liposomal encapsulation process more efficient and stable.

[0019] Unlike conventional phage encapsulation processes, in one embodiment of the present invention, the pH of the buffer for phages is modified, allowing them to carry the opposite charge to the cationic lipids, thus driving strong electrostatic attraction between the phage and the lipid. This dual approach of coupling the use of charged lipid with pH adjustment to alter the phage charge ensures higher encapsulation efficiency and stability by optimizing electrostatic interactions between the phages and lipid nanoparticles. The result is a more effective and robust liposomal encapsulation process, providing enhanced delivery and stability of the phages for therapeutic applications.

[0020] Charged interaction-assisted phage encapsulation by liposome may overcome the challenges of tackling AMR in respiratory infections more effectively than current empirical dosage regimens.

[0021] Utilizing electrostatic interactions ensures a higher proportion of active phages are effectively encapsulated into lipid nanoparticles, leading to superior product quality. This method leverages the attraction between oppositely charged lipids and phages, optimizing the incorporation process and ensuring that a greater number of viable phages are successfully encapsulated. This superior encapsulation efficiency contrasts sharply with other liposomal phage formulations that often struggle with low encapsulation efficiency. By addressing this critical issue, the electrostatic interaction method enhances the overall effectiveness of the phage product, ensuring more phages are delivered to the infection site, thereby improving therapeutic outcomes.

[0022] The charged lipid formulation is versatile and can be utilised with a range of encapsulation devices and methods. This adaptability minimises reliance on specific equipment, facilitating broader application and smoother integration into existing production lines. In contrast, other liposomal phages often necessitate specialised and inflexible production methods, which can hinder their scalability.

[0023] The skilled person will appreciate that the present invention may provide one or more significant advantages and improvements in the field and / or in view of the prior art. For example, these advantages include:(i) The ability to encapsulate or partially encapsulate a phage;(ii) The ability to stabilise a phage; and(iii) The ability to deliver phage to a location e.g. for phage therapy.DEFINITIONS

[0024] In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one having ordinary skill in the art to which the invention pertains.

[0026] Unless the context clearly requires otherwise, throughout the description and the claims, the terms “comprise”, “'comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. For example, a composition, mixture, process or method that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process or method.

[0027] Where the applicant has defined an invention or a portion thereof with an open-ended term such as "comprising", it should be readily understood that (unless otherwise stated) the description should be interpreted to also describe such an invention using the terms "consisting essentially of' or "consisting of." In other words, with respect to the terms “comprising”, “consisting of”, and “consisting essentially of’, where one of these three terms is used herein, the presently disclosed and claimed subject matter may include the use of either of the other two terms. Thus, in some embodiments not otherwise explicitly recited, any instance of “comprising” may be replaced by “consisting of’ or, alternatively, by “consisting essentially of”.

[0028] While reference may be made in this disclosure to the invention comprising a combination of a plurality of elements, it is also understood that this invention is regarded to comprise combinations which omit or exclude one or more of such elements, even if this omission or exclusion of an element or elements is not expressly stated herein, unless it isexpressly stated herein that an element is essential to the applicant' s combination and cannot be omitted. It is further understood that the related prior art may include elements from which this invention may be distinguished by negative claim limitations, even without any express statement of such negative limitations herein. It is to be understood, between the positive statements of applicant's invention expressly stated herein, and the prior art and knowledge of the prior art by those of ordinary skill which is incorporated herein even if not expressly reproduced here for reasons of economy, that any and all such negative claim limitations supported by the prior art are also considered to be within the scope of this disclosure and its associated claims, even absent any express statement herein about any particular negative claim limitations.

[0029] As used herein, with reference to numbers in a range of numerals, the terms "about," "approximately" and "substantially" are understood to refer to the range of -10% to +10% of the referenced number, preferably -5% to +5% of the referenced number, more preferably -1 % to + 1 % of the referenced number, most preferably -0.1 % to +0.1 % of the referenced number. Moreover, with reference to numerical ranges, these terms should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, from 8 to 10, and so forth.

[0030] The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0031] The complete disclosures of the patents, patent documents and publications cited herein are incorporated by reference in their entirety as if each were individually incorporated.

[0032] Unless expressly stated to the contrary, "or" refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0033] The term "and / or" used in the context of " X and / or Y" should be interpreted as " X," or " Y," or " X and Y." Similarly, "at least one of X or Y" should be interpreted as " X," or " Y," or "both X and Y."

[0034] The indefinite articles "a" and "an" preceding an element or component of the invention are intended to be non-restrictive regarding the number of instances (i.e., occurrences) of the element or component. Therefore "a" or "an" should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

[0035] As used herein, wt.% refers to the weight of a particular component relative to total weight of the referenced composition.

[0036] It will be understood that use of the term “between” herein when referring to a range of numerical values encompasses the numerical values at each endpoint of the range. For example, a temperature of between 80 °C and 150 °C is inclusive of a temperature of 80 °C and a temperature of 150 °C.

[0037] Various features of the embodiments of the invention disclosed herein are, for brevity, described in the context of a single embodiment, but may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the illustrative embodiments disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present compositions and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0038] In the foregoing paragraphs, where various ratios of components have been disclosed. It will be appreciated that these ratios of components can be combined in any disclosed combination. For example, the ratio of A: B (which may be between about 100:1 and 1:100 or any range therein), may be combined with the ratio of C: D (which may be between about 50:1 and 1:50 or any range therein), and may be combined with the ratio of E: F (which may be between about 10:1 and about 1:10 or any range therein).

[0039] The expression RT is understood to mean “Room Temperature”, which is generally accepted to be between about 20°C and about 25°C, e.g. about 22°C.

[0040] The expression “hydrocarbon chain” denotes an alkyl or alkyenyl chain.

[0041] The expression “alkyl” denotes unsubstituted alkyl unless otherwise specified. Alkyl groups may be straight chain or branched.

[0042] The expression “alkenyl” denotes unsubstituted alkenyl unless otherwise specified. Alkenyl groups may be straight chain or branched.

[0043] The expression “carbocyclyl”, unless specifically limited, denotes any ring system in which all the ring atoms are carbon. Carbocyclyl groups may be saturated or partially unsaturated, but do not include aromatic rings. A specific example of a carbocyclyl group is a cycloalkyl group. A further example of a carbocyclyl group is a cycloalkenyl group.

[0044] The expression “cycloalkyl”, unless specifically limited, denotes a C5-6 cycloalkyl group i.e. cyclopentyl or cyclohexyl.

[0045] The expression “cycloalkenyl”, unless specifically limited, denotes a C5-6 cycloalkenyl group, i.e. cyclopentenyl or cyclohexenyl.

[0046] The expression “heterocyclyl”, unless specifically limited, refers to a carbocyclyl group wherein one or more (e.g. 1, 2 or 3) ring atoms are replaced by heteroatoms selected from N, S and O. A specific example of a heterocyclyl group is a cycloalkyl group (e.g. cyclopentyl or more particularly cyclohexyl) wherein one or more (e.g. 1, 2 or 3, particularly 1 or 2, especially 1) ring atoms are replaced by heteroatoms selected from N, S or O. Exemplary heterocyclyl groups containing one hetero atom include pyrrolidine, tetra hydrofuran and piperidine, and exemplary heterocyclyl groups containing two hetero atoms include morpholine and piperazine. A further specific example of a heterocyclyl group is a cycloalkenyl group (e.g. a cyclohexenyl group) wherein one or more (e.g. 1, 2 or 3, particularly 1 or 2, especially 1) ring atoms are replaced by heteroatoms selected from N, S and O. An example of such a group is dihydropyranyl (e.g. 3,4-dihydro-2H-pyran-2-yl-).

[0047] The expression “polymeric chain” encompasses “synthetic polymeric chain” and “polysaccharide chain”.

[0048] The expression “synthetic polymeric chain” denotes a polymeric chain comprising repeating non-saccharide monomeric units (monomers), typically produced via synthetic chemical processes. Additionally, these monomers are typically derived from synthetic chemical processes and are distinct from naturally occurring polysaccharides. Synthetic polymer chains can include a wide range of non-natural building blocks. Synthetic polymer chains include homopolymers and copolymers.

[0049] The expression “polysaccharide chain” denotes a chain of monosaccharide units. The monosaccharide units are 5- and / or 6-membered rings, such as pentoses and / or hexoses. Examples of saccharide units include glucose, galactose, fructose, etc. Polysaccharide chains include homopolysaccharides and copolysaccharides.BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The aspects described above, as well as other apparent aspects, advantages, and objectives of the present invention are apparent from the detailed description below in combination with the drawing, in which:

[0051] Fig. 1 shows Transmission electron microscopy (TEM) images a and b (short-tail and long-tail bacteriophages) c and d (empty liposomes) and e and f (phages not encapsulated).

[0052] Fig. 2 shows TEM images of encapsulated short-tail phage (podovirus) and encapsulated long-tail phage (myovirus).

[0053] Fig. 3 shows log difference in titers of liposome-encapsulated phages before and after immediate release by bile salts.

[0054] Fig. 4 shows encapsulation efficiency of liposomal-encapsulated phages.

[0055] Fig. 5 plots size against flow rate ratio for liposome particles loaded with phages.

[0056] Fig. 6 plots pdi against flow rate ratio for liposome particles loaded with phages.

[0057] Fig. 7 plots zero potential against flow ratio for liposome particles without phages (control).

[0058] Fig. 8 shows average cumulative release percentage over time for liposome particles loaded with phages.

[0059] Fig. 9 compares titer loss between lipid nanoparticle-encapsulated (LNP-encap) and non-encapsulated phages (Non-encap) PEV40 and PEV2, using jet and vibrating mesh nebulizers. Comparison of liposome encapsulated and non-encapsulated phage deactivation after nebulization obtained by plaque assay. Error bars for the plaque assay represent one standard deviation of triplicate experiments. Double asterisk (**) denotes a statistically significant difference with p<0.01.

[0060] Fig. 10 shows the viable phage respirable fraction.

[0061] Fig. 11 shows the AFM-IR height image and IR spectra collected on liposomal encapsulated phage.

[0062] Fig. 12 shows Z-average measurements of phage-encapsulating liposomes using: (a) AXF-mini system, (b) CIJ mixer and (c) Microfluidic system. Double asterisk (*) denotes a statistically significant difference with p < 0.05.

[0063] Fig. 13 Zeta potential of DOTAP-based liposomes encapsulating PEV1 and PEV31 phages, (a) AXF-mini system, (b) CIJ mixer, and (c) microfluidic system. Data shown as mean ± SD (n=3).

[0064] Fig. 14 shows Encapsulation efficiency comparison between charged versus neutral lipid formulations for PEV31 and PEV1 using AXF-mini (a), CIJ mixer (b) and microfluidic system (c). Double asterisk (*) denotes a statistically significant difference with p<0.05.

[0065] Fig. 15 shows Titer difference analysis of DOTAP-based liposome-encapsulated PEV31 and PEV1 phages using AXF-mini system (a), CIJ mixer (b) and microfluidic system (c). Data presented as mean ± one standard deviation (n = 3).

[0066] Fig. 16 shows AFM-IR spectra collected from (a) pure liposome, (b) liposome encapsulating PEV1 (long-tail phage), and (c) liposome encapsulating PEV31 (short-tail phage).

[0067] Fig. 17 shows TEM images of (A) PEV40 and (B) PEV2 phages. First column arrows indicate intact phages, second column arrows empty capsids, third column arrows detached tails, and fourth column arrows contracted tails. In Liposome encapsulation column, white arrows mark lipid-encapsulated phages and black arrows empty liposomes appear. (Scale bar: 100 nm).

[0068] Fig. 18 Titre assay of liposome encapsulated PEV2 following in vitro deposition of aerosols by the MSLI (n = 3).

[0069] Fig. 19 Titre assay of liposome encapsulated PEV40 following in vitro deposition of aerosols by the MSLI (n = 3).

[0070] Fig. 20 shows live cell images of PA07 bacterial cells visualized via digital staining in green based on Refractive Index) comparing the effect of various phage treatments after 24 h: (a) untreated control; (b) PBS treated; (c) free PEV2 phage; (d) PEV2 + empty liposome physical mixture; (e) liposomal PEV2; (f) free PEV1 phage; (g) PEV1 + empty liposome physical mixture; (h) liposomal PEV1.DETAILED DESCRIPTION

[0071] The skilled addressee will understand that the invention comprises the embodiments and features disclosed herein as well as all combinations and / or permutations of the disclosed embodiments and features.

[0072] In one aspect, the present invention provides the use of a charged lipid for encapsulating phages in nanosize lipid particles,wherein the charged lipid is cationic or anionic;wherein the charged lipid comprises a cationic or anionic moiety and a C12 to C80 hydrocarbon chain or a polymeric chain;wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O) Rx, -C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5-or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, -NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.

[0073] In another aspect, the present invention provides a method of encapsulating phages, the method comprising mixing a charged lipid formulation with a phage formulation;wherein the charged lipid formulation comprises a charged lipid;wherein the charged lipid is cationic or anionic;wherein the charged lipid comprises a cationic or anionic moiety and a C12 to C80 hydrocarbon chain or a polymeric chain;wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O)Rx, -C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5-or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, -NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.Nanosize lipid particles

[0074] The lipid nanoparticles according to the invention have a diameter ranging from about 100nm to about 1000nm.

[0075] Preferably lipid nanoparticles according to the invention have a diameter ranging from about 200nm to about 900nm, e.g. from about 250nm to about 850nm, e.g. from about 300nm to about 800nm.Charged lipid

[0076] The charged lipid according to the invention comprises a cationic or anionic moiety or head group. In one embodiment, the charged lipid comprises a cationic head group. In another embodiment, the charged lipid comprises an anionic head group.

[0077] In one embodiment, the charged lipid comprises a cationic or anionic moiety and a flexible hydrocarbon chain.

[0078] In another embodiment, the charged lipid comprises a cationic or anionic moiety and a flexible polymeric chain.

[0079] The overall structure of the charged lipid must be flexible, lacking rigid or planar polycyclic elements characteristic of steroid backbones (e.g., DC-cholesterol).

[0080] Suitable charged lipids include commercially available charged lipids.

[0081] Suitable charged lipids include permanently charged cationic lipids and permanently charged anionic lipids.

[0082] Suitable charged lipids also include lipids that become charged due to protonation (e.g. of a nitrogen atom) or deprotonation (e.g. of a carboxylate group) at a certain pH. Lipids the become charged after protonation or deprotonation may be referred to as charged lipid precursors.

[0083] The pH may be suitably controlled using a buffer. pH control can afford charged lipids as a result of protonation or deprotonation of neutral lipids. For example, an amine group may be protonated to afford an ammonium group. A carboxylic acid group may be deprotonated to afford a carboxylate group.

[0084] In one embodiment, the charged lipid is stearylamine (octadecylamine) in its protonated ammonium form.

[0085] In one embodiment, the charged lipid is DOTAP (dioleoyl-3-trimethylammonium propane).

[0086] In one embodiment, the charged lipid is dicetyl phosphate.

[0087] In one embodiment, the charged lipid is DLin-MC3-DMA (also known as 4-(dimethylamino)-butanoic acid, (1 OZ, 13Z)-1-(9Z, 12Z)-9, 12-octadecadien-1 -yl-10, 13-nonadecadien-1-yl ester and dilinoleyl-methyl-4-dimethylaminobutyrate).Formula (I)

[0088] In one embodiment, the charged lipid is a lipid of Formula (I):A-L1-(L2-B)n (I)whereinA is an anionic or cationic moiety;L1 is bond or C1 to C6 alkylene;L2 is absent or -C(O)O-, -OC(O)-, -C(O)-, -NHC(O)-, -C(O)NH- -NRC(O)-, -C(O)NR- - OP(O)2O-, -O-, -OS(O)2O-, -O-C(O)NH-, -NHC(O)O-, wherein R is Ci to C6alkyl;each occurrence of B is independently selected from a Ci2to Cso hydrocarbon chain or a polymeric chain;n = 1 to 3;wherein if L1 is C1 to C6 alkylene and B is Ci2to Cso hydrocarbon chain, L2 is not absent;wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O)Rx, - C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5- or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, - NH2, -NRZ2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.

[0089] In one embodiment of formula (I), if n is more than 1, each B is an independently selected hydrocarbon chain. In another embodiment of formula (I), if n is more than 1, each B is the same hydrocarbon chain. In a further embodiment of formula (I), if n is more than 1, each B is an independently selected polymeric chain. In yet another embodiment of formula (I), if n is more than 1, each B is the same polymeric chain.Anionic moiety or cationic moiety

[0090] The charged lipid comprises a cationic or anionic moiety. In one embodiment, the charged lipid comprises a cationic moiety. In one embodiment, the charged lipid comprises an anionic moiety.

[0091] The lipid charge may be generated in situ by protonation or deprotonation at a suitable pH.

[0092] In one embodiment, the cationic moiety is an ammonium moiety.

[0093] An amine in its protonated form may be used as the cationic moiety.

[0094] Examples of ammonium moieties include quaternary ammonium, tertiary ammonium and secondary ammonium.

[0095] In one embodiment, the anionic moiety is a phosphate moiety, or a carboxylate moiety a sulfate moiety or a pyrophosphate moiety.

[0096] Examples include diacylglycerol pyrophosphate lipid, sodium dodecyl sulfate charged lipid.L1

[0097] L1 represents a bond or C1 to C6 alkylene.

[0098] In one embodiment, L1 represents a bond. In another embodiment, L1 represents C1 to C6 alkylene (e.g. Ci to C4 alkylene).L2

[0099] L2 is absent or -C(O)O-, -OC(O)-, -C(O)-, -NHC(O)-, -C(O)NH- -NRC(O)-, -C(O)NR--OP(O)2O-, -O-, -OS(O)2O-, -O-C(O)NH- or-NHC(O)O-, wherein R is Ci to C6alkyl.[000100] If L1 is C1 to C6 alkylene and B is Ci2to Cso hydrocarbon chain, L2 is not absent.[000101] If L2 is absent and L1 is a bond, A is bonded directly to B, as shown in Formula (le):A(B)n (le)wherein A, B and n are as defined above.That is, if L2 is absent and L1 is C1 to C6 alkylene, A is bonded to C1 to C6 alkylene, which is bonded to B.[000102] In one embodiment, L2 is absent or -C(O)O-, -OC(O)-, -C(O)-, -NHC(O)-, -C(O)NH--NRC(O)-, -C(O)NR-, -O-C(O)NH- or-NHC(O)O-, wherein R is Ci to C6alkyl.[000103] In one embodiment, L2 is absent or -C(O)O-, -OC(O)- or-C(O)-. In one embodiment, L2 is absent or -C(O)O- or -OC(O)-. In one embodiment, L2 is absent. In one embodiment, L2 is -C(O)O- or -OC(O)-.Hydrocarbon chain[000104] In one embodiment, with reference to Formula (I), each occurrence of B is independently selected from a C12 to C80 hydrocarbon chain.[000105] Accordingly, in one embodiment, the charged lipid of Formula (I) is represented by Formula (la):A-L1-(L2-B)n (la)whereinA is an anionic or cationic moiety;L1 is a bond or C1 to C6 alkylene;L2 is absent or -C(O)O-, -OC(O)-, -C(O)-, -NHC(O)-, -C(O)NH- -NRC(O)-, -C(O)NR- - OP(O)2O-, -O-, -OS(O)2O-, -O-C(O)NH-, -NHC(O)O-, wherein R is Ci to C6alkyl;each occurrence of B is independently selected from a C12 to C80 hydrocarbon chain;n is 1 to 3;wherein if L1 is C1 to C6 alkylene, L2 is not absent; andwherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O)Rx, - C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl.[000106] In one embodiment of formula (la), if n is more than 1, each B is an independently selected hydrocarbon chain. In another embodiment of formula (la), if n is more than 1, each B is the same hydrocarbon chain.[000107] The hydrocarbon chain is a flexible hydrocarbon chain. The hydrocarbon chain is aliphatic. The hydrocarbon chain suitably lacks rigid conjugated double bonds and lacks rigid planar polycyclic elements (e.g. those characteristic of steroid backbones).[000108] The hydrocarbon chain is a C12 to C80 hydrocarbon chain, e.g. a C to Cso hydrocarbon chain, e.g. a C44 to Cso hydrocarbon chain, e.g. a C12 to C80 hydrocarbon chain, a C12 to C50 hydrocarbon chain, a C12 to C44 hydrocarbon chain, e.g. a C12 to C42 hydrocarbon chain, e.g. a C12 to C40 hydrocarbon chain, e.g. a C12 to Css hydrocarbon chain, e.g. a C12 to C36 hydrocarbon chain, e.g. a C12 to C34 hydrocarbon chain, e.g. a C12 to C32 hydrocarbon chain, e.g. a C12 to C30 hydrocarbon chain, e.g. a C12 to C28 hydrocarbon chain, e.g. a C12 to C26 hydrocarbon chain, e.g. a C12 to C24 hydrocarbon chain, e.g. a C12 to C22 hydrocarbon chain, e.g. a C12 to C20 hydrocarbon chain, e.g. a C12 to C hydrocarbon chain.[000109] In one embodiment, the charged lipid comprises two C12 to C80 hydrocarbon chains, e.g. two C12 to C40 hydrocarbon chains.[000110] In one embodiment, the hydrocarbon chain comprises one or more unconjugated double bonds and comprises a maximum of 1 double bond for every 4 carbon atoms in the longest chain, for example, a maximum of 1 double bond for every 8 carbon atoms in the longest chain, for example, a maximum of 1 double bond for every 12 carbon atoms in the longest chain. In one embodiment the hydrocarbon chain is saturated.[000111] In one embodiment, the hydrocarbon chain is branched and comprises a maximum of 1 branch for each 6 carbon atoms in the longest chain, for example a maximum of 1 branch for each 8 carbon atoms in the longest chain.[000112] In another embodiment, the hydrocarbon chain is unbranched (i.e. linear).[000113] The hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O) Rx, -C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is Ci to Cs alkyl. When more than one R group is present, each R is independently selected. When the hydrocarbon chain is substituted by C5-C6 carbocyclyl and / or 5 or 6 membered heterocyclyl, C5-C6 carbocyclyl and / or 5 or 6 membered heterocyclyl are unsubstituted.[000114] In one embodiment, polyether comprises repeating ethylene oxide units.[000115] In one embodiment, the hydrocarbon chain is optionally substituted by one, two or three substituents, e.g. one or two, e.g. one.[000116] In one embodiment, the hydrocarbon chain is optionally substituted by a maximum of one substituent for every 6 carbon atoms in the hydrocarbon chain, e.g. one substituent for every 8 carbon atoms in the hydrocarbon chain, e.g. one substituent for every 10 atoms in the hydrocarbon chain.[000117] In one embodiment, the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, -OR, -C(O)Rx, -C(O)ORx, -OC(O)Rx, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl.[000118] In another embodiment, the hydrocarbon chain is optionally substituted by one or more substituents independently selected from -OR, -C(O)Rx, -C(O)ORx and -OC(O)Rx, wherein Rx is C1 to C6 alkyl.[000119] In another embodiment, the hydrocarbon chain is unsubstituted.[000120] In one embodiment, L2 is absent and the cationic or anionic moiety is connected to the hydrocarbon chain by a bond.[000121] Advantages of flexible hydrocarbon chains include:Membrane flexibility Greater flexibility, allowing for easier formation of adaptable lipid bilayers and better encapsulation of biologies, including larger molecules like bacteriophages.Encapsulation Interact more efficiently to form a compact, flexible bilayer that efficiency improve the ability to trap and protect biologies.Ease of lipid Promotes easier self-assembly into liposomes or lipid assembly nanoparticles improving formulation simplicity and consistency.Adaptability to More adaptable to the size and structure of different biologies, biologic size such as different types of phages, as the flexible chain can adjust better.Membrane Adaptable across a wide range of delivery systems, including fluidity / versatility gels, powders and creams.across applicationsBarrier properties Flexible yet stable barriers that protect biologies while allowing for controlled release.Controlled release Flexible chains can enhance diffusion and fusion with target cells or tissues.Polymeric chain[000122] In one embodiment, with reference to Formula (I), each occurrence of B is independently selected from a polymeric chain.[000116] Accordingly, in one embodiment, the charged lipid of Formula (I) is represented by Formula (lb):A-L1-(L2-B)n (lb)whereinA is an anionic or cationic moiety;L1 is a bond or C1 to C6 alkylene;L2 is absent or -C(O)O-, -OC(O)-, -C(O)-, -NHC(O)-, -C(O)NH- -NRC(O)-, -C(O)NR- - OP(O)2O-, -O-, -OS(O)2O-, -O-C(O)NH-, -NHC(O)O-, wherein R is Ci to C6alkyl;each occurrence of B is independently selected from a polymeric chain;n is 1 to 3; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5- or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, - NH2, -NRZ2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.[000123] In one embodiment of formula (lb), if n is more than 1, each B is an independently selected polymeric chain. In another embodiment of formula (lb), if n is more than 1, each B is the same polymeric chain.[000124] The polymeric chain is a flexible polymeric chain.[000125] Suitable polymeric chains are biocompatible and biodegradable.[000126] The polymeric chain can be a synthetic polymeric chain or a polysaccharide chain.[000127] In one embodiment, the polymeric chain is a synthetic polymeric chain.[000128] In another embodiment, the polymeric chain is a polysaccharide chain.[000129] The polymeric chain can comprise 6 to 200 monomers, for example, 6 to 100 monomers, or 6 to 60 monomers, or 6 to 40 monomers, or 6 to 36 monomers, or 6 to 32 monomers.[000130] In one embodiment, the polymeric chain may be branched, comprising a maximum of 1 branch for every 6 monomers in the longest chain, for example, 1 branch for every 8 monomers in the longest chain, or 1 branch for every 10 monomers in the longest chain, or 1 branch for every 12 monomers in the longest chain in the longest chain.[000131] In another embodiment, the polymeric chain is unbranched.[000132] The polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5- or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, -NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.[000133] In one embodiment, the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, and C1 to C6 alkyl, wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent and Ry are each independently optionally substituted with one or more substituents selected from -OH, -NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.[000134] In another embodiment, the polymeric chain is optionally substituted by one or more substituents independently from -OH, -ORy, -C(O)Ry, -C(O)ORy, -COOH, C1 to C6 alkyl,wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent is independently optionally substituted with one or more substituents selected from -OH, and -COOH.[000135] In one embodiment, the polymeric chain is optionally substituted by a maximum of one substituent for every monomer in the polymeric chain, e.g. one substituent for every two monomers in the polymeric chain, e.g. one substituent for every four monomers in the polymeric chain.[000136] In another embodiment, the polymer chain is unsubstituted.[000137] Examples of polymeric chains include alginate, chitosan, PLGA, PVP, PVA, and synthetic copolymers derived from esters of acrylic and methacrylic acid. Alginate, chitosan, PLGA, PVP, PVA, and synthetic copolymers derived from esters of acrylic and methacrylic acid are integral components in the formulation of lipid-polymer hybrid nanoparticles, each offering unique structural and functional benefits that enhance the encapsulation, stability, and delivery of biologies. Their biocompatibility, versatility, and ability to form responsive and targeted delivery systems position these polymers as key players in advancing nanomedicine and therapeutic applications.[000138] In one embodiment, the charged lipid according to the invention comprises a cationic or anionic moiety and a polymeric chain. In one embodiment, the polymeric chain is a synthetic polymeric chain or polysaccharide chain. In some embodiments, the polymeric chain is unbranched.[000139] In one embodiment, the charged lipid according to the invention comprises chitosan and an ammonium moiety.[000140] In another embodiment, the the charged lipid according to the invention comprises an alginate and a carboxylate moiety.Synthetic polymeric chain[000141] In one embodiment, with reference to Formula (I), each occurrence of B is independently selected from synthetic polymeric chain.[000142] Accordingly, in one embodiment, the charged lipid of Formula (I) is represented by Formula (Ic):A-L1-(L2-B)n (Ic)whereinA is an anionic or cationic moiety;L1 is a bond or C1 to C6 alkylene;L2 is absent or -C(O)O-, -OC(O)-, -C(O)-, -NHC(O)-, -C(O)NH- -NRC(O)-, -C(O)NR- - OP(O)2O-, -O-, -OS(O)2O-, -O-C(O)NH-, -NHC(O)O-, wherein R is Ci to C6alkyl;each occurrence of B is independently selected from a synthetic polymeric chain;n is 1 to 3; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5- or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, - NH2, -NRZ2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.[000143] In another embodiment of formula (Ic), if n is more than 1, each B is an independently selected synthetic polymeric chain. In another embodiment of formula (Ic), if n is more than 1, each B is the same synthetic polymeric chain.[000144] The polymeric chain is a flexible polymeric chain.[000145] Suitable synthetic polymer chains are biocompatible and biodegradable.[000146] Synthetic polymer chains comprise monomers which are typically derived from synthetic chemical processes and are distinct from naturally occurring polysaccharides. Synthetic polymer chains can comprise a wide range of non-natural building blocks, for example, alkenyl monomers or hydroxy-carboxylic acid monomers. Examples of non-natural building blocks include those found in polymers like poly(lactic-co-glycolic acid) (PLGA), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), synthetic copolymers derived from esters of acrylic and methacrylic acid. Synthetic polymer chains also include other artificial polymers used in biomedical, pharmaceutical, and industrial applications.[000147] Examples of suitable synthetic polymers include polymers of hydroxy-carboxylic acids (such as PLGA), PVP, PVA, and synthetic copolymers derived from esters of acrylic and methacrylic acid and other artificial polymers used in biomedical, pharmaceutical, and industrial applications.[000148] Synthetic polymer chains include but are not limited to polymers such as PLGA, PVP, PVA, and synthetic copolymers derived from esters of acrylic and methacrylic acid.[000149] The synthetic polymeric chain can comprise 6 to 200 monomers, for example, 6 to 100 monomers, or 6 to 60 monomers, or 6 to 40 monomers, or 6 to 36 monomers, or 6 to 32 monomers.[000150] In one embodiment, the synthetic polymeric chain may be branched, comprising a maximum of 1 branch for every 6 monomers in the longest chain, for example, 1 branch for every 8 monomers in the longest chain, or 1 branch for every 10 monomers in the longest chain, or 1 branch for every 12 monomers in the longest chain in the longest chain.[000151] In another embodiment, the synthetic polymeric chain is unbranched.[000152] The synthetic polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, Ci to C6alkyl, and 5- or 6-membered heterocyclyl groups; wherein Ry is Ci to C6alkyl, and wherein the Ci to C6alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, -NH2, -NRZ2, -COOH, and -SH; and wherein Rz is Ci to C6alkyl.[000153] In one embodiment, the synthetic polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, and Ci to C6alkyl, wherein Ry is Ci to C6alkyl, and wherein the Ci to C6alkyl substituent and Ry are each independently optionally substituted with one or more substituents selected from -OH, -NH2, -NRz2, -COOH, and -SH; and wherein Rz is Ci to C6alkyl.[000154] In another embodiment, the synthetic polymeric chain is optionally substituted by one or more substituents independently from -OH, -ORy, -C(O)Ry, -C(O)ORy, -COOH, Ci to C6alkyl, wherein Ry is Ci to C6alkyl, and wherein the Ci to C6alkyl substituent is independently optionally substituted with one or more substituents selected from -OH, and -COOH.[000155] In one embodiment, the synthetic polymeric chain is optionally substituted by a maximum of one substituent for every monomer in the synthetic polymeric chain, e.g. one substituent for every two monomers in the synthetic polymeric chain, e.g. one substituent for every four monomers in the synthetic polymeric chain.[000156] In another embodiment, the synthetic polymer chain is unsubstituted.[000157] Examples of synthetic polymeric chains include PLGA, PVP, PVA, and synthetic copolymers derived from esters of acrylic and methacrylic acid.[000158] Suitable synthetic polymers, which are commonly used as excipients, include:Poly(lactic-co-glycolic acid) A biodegradable copolymer derived from the monomers lactic (PLGA): acid and glycolic acid, known for its controlled degradation rate and use in sustained-release drug delivery systemsPolyvinylpyrrolidone (PVP) A water-soluble polymer synthesized from the monomer N- vinyl-2-pyrrolidone, characterized by its excellent film-forming, binding, and stabilizing propertiesPolyvinyl Alcohol (PVA) A synthetic polymer produced by the hydrolysis of polyvinyl acetate, featuring multiple hydroxyl groups (-OH) that confer film-forming and emulsifying capabilitiesSynthetic copolymers Monomers are derived from methacrylic acid and methacrylate derived from esters of acrylic esters, such as methyl methacrylate and ethyl methacrylate and methacrylic acid(Eudragit® Polymers)[000159] PLGA, PVP, PVA, and synthetic copolymers derived from esters of acrylic and methacrylic acid are synthetic polymers widely used in biomedical and pharmaceutical applications. They are characterized by their adaptable structures, typically linear and, in some cases, lightly branched, with hydrocarbon backbones that incorporate diverse functional groups. These functional groups confer specific properties such as biodegradability, biocompatibility, and controlled release capabilities, making these polymers ideal for drug delivery and encapsulation systems.[000160] PLGA, PVP, PVA, and synthetic copolymers derived from esters of acrylic and methacrylic acid are integral components in the formulation of lipid-polymer hybrid nanoparticles, each offering unique structural and functional benefits that enhance the encapsulation, stability, and delivery of biologies. Their biocompatibility, versatility, and ability to form responsive and targeted delivery systems position these polymers as key players in advancing nanomedicine and therapeutic applications.Polysaccharide chain[000161] In one embodiment, with reference to Formula (I), each occurrence of B is independently selected from a polysaccharide chain.[000162] Accordingly, in one embodiment, the charged lipid of Formula (I) is represented by Formula (Id):A-L1-(L2-B)n (Id)whereinA is an anionic or cationic moiety;L1 is a bond or C1 to C6 alkylene;L2 is absent or -C(O)O-, -OC(O)-, -C(O)-, -NHC(O)-, -C(O)NH- -NRC(O)-, -C(O)NR- - OP(O)2O-, -O-, -OS(O)2O-, -O-C(O)NH-, -NHC(O)O-, wherein R is Ci to C6alkyl;each occurrence of B is independently selected from a polysaccharide chain;n is 1 to 3; andwherein the polysaccharide chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5- or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, - NH2, -NRZ2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.[000163] In another embodiment of formula (Id), if n is more than 1, each B is an independently selected polysaccharide chain. In another embodiment of formula (Id), if n is more than 1, each B is the same polysaccharide chain.[000164] The polysaccharide chain is a flexible polysaccharide chain.[000165] Suitable polysaccharide chains are biocompatible and biodegradable.[000166] The polysaccharide chain comprises monomers which are monosaccharides.[000167] The polysaccharide chain can comprise 6 to 200 monosaccharide units, e.g. 6 to 100 monosaccharide units, e.g. 6 to 60 monosaccharide units, e.g. 6 to 40 monosaccharide units, e.g. 6 to 36 monosaccharide units, e.g. 6 to 32 monosaccharide units.[000168] In one embodiment, the polysaccharide chain is branched and comprises a maximum of 1 branch for every 6 monosaccharide rings in the longest chain, e.g. 1 branch forevery 8 monosaccharide rings in the longest chain, e.g. 1 branch for every 10 monosaccharide rings in the longest chain, e.g. 1 branch for every 12 monosaccharide rings in the longest chain.[000169] In another embodiment, the polysaccharide chain is unbranched.[000170] The polsaccharide chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5- or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, -NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.[000171] In one embodiment, the polsaccharide chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, and C1 to C6 alkyl, wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent and Ry are each independently optionally substituted with one or more substituents selected from -OH, -NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.[000172] In another embodiment, the polsaccharide chain is optionally substituted by one or more substituents independently from -OH, -ORy, -C(O)Ry, -C(O)ORy, -COOH, C1 to C6 alkyl, wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent is independently optionally substituted with one or more substituents selected from -OH, and -COOH.[000173] In one embodiment, the polysaccharide chain is optionally substituted by a maximum of one substituent for every monosaccharide in the polysaccharide chain, e.g. one substituent for every two monosaccharides in the polysaccharide chain, e.g. one substituent for every four monosaccharides in the polysaccharide chain.[000174] In another embodiment, the polymer chain is unsubstituted.[000175] In one embodiment, the polysaccharide chain is selected from alginates and chitosan.[000176] Alginates and chitosan are both polysaccharides. They are characterized by their linear, unbranched structures, fully saturated hydrocarbon chains, and distinct functional groups that confer specific charges and functionalities essential for their applications in biomedical and pharmaceutical fields.[000177] Alginate and chitosan are integral components in the formulation of lipid-polymer hybrid nanoparticles, offering unique structural and functional benefits that enhance theencapsulation, stability, and delivery of biologies. Their biocompatibility, versatility, and ability to form responsive and targeted delivery systems position them as key players in the advancement of nanomedicine and therapeutic applications.“n”[000178] n is 1 to 3. In one embodiment, n is 1 or 2. In one embodiment, n is 1. In another embodiment, n is 2. In another embodiment, n is 3.[000179] If n is 2 or 3 and L1 is alkylene, the L2 moieties may be bonded to the same carbon atom of L1 or different carbon atoms of L1.Phage[000180] In some embodiments, the bacteriophage is engineered to have a surface charge that can be externally modulated, allowing for controlled encapsulation and targeted release within the lipid nanoparticles.[000181] In some embodiments, the phage is selected from the group consisting of long tail and short tail phages. In some embodiments, the phage is selected from long tail phages. In some embodiments, the phage is selected from short tail phages.[000182] Long tail phages are bacteriophages characterized by having a long tail (100-150 nm) structure attached to their capsid (head). The tail can be either contractile or non-contractile and serves as a conduit for injecting the phage's genetic material into the host bacterium.[000183] Short tail phages are bacteriophages that possess a short tail (10-15 nm) structure. The tail is generally non-contractile and shorter in length compared to long tail phages.[000184] As used herein, “long tail phages” refer to bacteriophages with long tail structures, including those belonging to the families Myoviridae and Siphoviridae.[000185] “Short tail phages” refer to bacteriophages with short tail structures, particularly those in the family Podoviridae.[000186] In one embodiment, the phage is a Pseudomonas- targeting phage.[000187] In one embodiment, the phage is selected from the group consisting of PEV1 (Myoviridae), PEV40 (Myoviridae), PEV31 (Podoviridae) and PEV2 (Podoviridae).[000188] In one embodiment, the phage is PEV1.[000189] In one embodiment, the phage is PEV40. PEV40 is characterised by a long and contractile tail, similar to PEV1 belongs to Myoviridae.[000190] In one embodiment, the phage is PEV31.[000191] In one embodiment, the phage is PEV2. PEV2 is characterised by a short and non-contractile tail, similar to PEV31 belongs to Podoviridae.Other components[000192] The use, method or composition of the present invention optionally comprises, or comprises use of, one or more additional components, for example, a zwitterionic lipid and / or a neutral lipid.Zwitterioinic lipid[000193] In one embodiment, the use, method or composition of the present invention optionally comprises, or comprises use of, a zwitterioinic lipid (e.g. one or more one, two or three, e.g. one or two, e.g. one).[000194] In one embodiment, the zwitterionic lipid is a phospholipid, such as phosphatidylcholine (PC), DPPC, DSPC.[000195] PC (or potentially interchangeable with DSPC and DPPC) are zwitterionic lipids which are commonly utilized lipids in lipid nanoparticle (LNP) and liposome formulations, contributing to the stability and efficient delivery of mRNA (e.g., COVID vaccines) and other drugs into host cells, ensuring robust immune reponses, while maintaining biocompatibility.[000196] Zwitterionic lipids can stabilise lipid bilayers, ensuring structural integrity and enhancing the overall stability of the nanoparticle of liposome. Furthermore, PC is a major component of natural cell membranes, by mimicking endogenous lipid structures, these formulations are better tolerated by the body, reducing the likelihood of adverse immune response, and contributing to the biocompatibility of lipid-based delivery systems. The inclusion of PC aids in optimising the interaction between LNP / liposomes and target cells, enhancing the uptake and delivery of the therapeutic payload.[000197] In some embodiments, zwitterionic lipids play a role in encapsulation efficiency. Zwitterionic lipids facilitate effective encapsulation and delivery of biologies by creating a protective barrier around encapsulated biologies such as proteins and phages. The barrier shields the agents from degradation, denaturation, and nonspecific interactions with other components.[000198] In some embodiments, zwitterionic lipids assist in providing optimal characteristics. Zwitterionic lipids contribute to stability, reduced immunogenicity, and efficient therapeutic delivery.[000199] Zwitterionic lipids are compatible with functional excipients: The zwitterionic lipids synergise with other components (i.e. stearylamine, DOTAP) to fine-tune nanoparticle properties, including fluidity and flexibility.[000200] In some embodiments, zwitterionic lipids are useful for therapeutic reliability, reproducibility and scalability. PC is well-characterized and their predictable characteristics during formulations ensures consistency. These lipids are widely available and can be produced at large scales.Neutral lipid[000201] In one embodiment, the use, method or composition of the present invention optionally comprises, or comprises use of, a neutral lipid (e.g. one or more, e.g. one, two or three, e.g. one or two, e.g. one).[000202] In one embodiment the lipid particles comprise a neutral lipid.[000203] Examples of neutral lipids include fatty acid esters (e.g. fatty acid-glycerol esters) and steroids.[000204] In one embodiment, the neutral lipid is cholesterol.[000205] In some embodiments, the neutral lipid (e.g. cholesterol) plays a role in enhancing lipid membrane stability, biocompatibility, and encapsulation efficiency, as it has been widely used in real-world applications including vaccine and liposomal drug development.[000206] In some embodiments, the neutral lipid (e.g. cholesterol) enhances membrane rigidity and structural integrity, preventing premature leakage of encapsulated phages. It contributes to the formation of liquid-ordered phases and enhancing bilayer thickness and fluidity.[000207] In some embodiments, the neutral lipid (e.g. cholesterol) assists with enhancement of circulation and biocompatibility by reducing surface-bound proteins, minimising immunogenicity and extending the circulation half-life of LNP / liposomes.[000208] In some embodiments, the neutral lipid (e.g. cholesterol) assists with encapsulation efficiency and controlled release by increasing membrane rigidity, which helps in reducing drug leakage from the liposome / LNP core, thereby enhancing encapsulation efficiency.[000209] In some embodiments a zwitterionic lipid (e.g. phosphatidylcholine (PC)) and a neutral lipid (e.g. cholesterol) establish the skeletal structural framework for lipid nanoparticles (LNPs). This robust lipid architecture is further enhanced by incorporating charged excipients, which attract and encapsulate charged phages within the lipid matrix.[000210] PC can be interchanged with other phospholipids such as DPPC, DSPC, and DOPC, allowing for fine-tuning of membrane fluidity and phase behavior to meet specific therapeutic needs. Additionally, the cholesterol ratio can be adjusted to optimize the stability, rigidity, and permeability of the lipid bilayer.[000211] The present invention also provides a composition comprising a phage encapsulated in nanosize lipid particles;wherein the lipid particles comprise a charged lipid;wherein the charged lipid comprises a cationic or anionic moiety and a C12 to C80 hydrocarbon chain or a polymeric chain;wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O) Rx, -C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5-or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, -NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.[000212] The composition according to the invention may further comprise a carrier, excipient, diluent, adjuvant, stabilizer and / or buffer depending on its intended use. In some embodiments the composition is a pharmaceutically acceptable composition comprising a phage encapsulated in nanosize lipid particles and one or more pharmaceutically acceptable carriers.[000213] As used herein, the term “pharmaceutically acceptable carrier” preferably includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active agents may also be incorporated into the composition.[000214] A pharmaceutical composition is generally formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g. intravenous, intradermal, subcutaneous), oral (e.g. tablets, pills, capsules, troches, inhalation), transdermal, (topical), transmucosal, rectal and vaginal administration. Solutions or suspensions for parenteral, intradermal or subcutaneous administration can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens, antioxidants such as ascorbic acid or sodium bisulfate, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates or phosphates and agents for adjustment of tonicity such as sodium chloride o dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide.Stablilizer[000215] In one embodiment, composition of the present invention optionally comprises a stabilizer (e.g. one or more, e.g. one, two or three, e.g. one or two, e.g. one).[000216] Examples of stabilizers include nonionic surfactants, ionic surfactants and sugars.[000217] Nonionic surfactant[000218] In one embodiment, composition of the present invention optionally comprises a nonioinic surfactant (e.g. one or more, e.g. one, two or three, e.g. one or two, e.g. one).[000219] In one embodiment, the lipid nanoparticles are surface-modified with nonionic surfactants to enhance steric stabilization, reduce aggregation and extend circulation time of lipid nanoparticles[000220] Examples of nonionic surfactants include, but are not limited to, glycoside-based nonionic surfactants, ester-based nonionic surfactants, ether-based nonionic surfactants, natural nonionic surfactants, and block copolymer nonionic surfactants.[000221] Glycoside-Based Nonionic Surfactants:Alkyl polyglucosides selected from Decyl Glucoside and Lauryl Glucoside;Lecithin derivatives selected from Modified Phosphatidylcholine and Nonionic Glycolipids;[000222] Ester-Based Nonionic Surfactants:• Sorbitan esters e.g. selected from Span® 20, Span® 60, Span® 80;• Polyglycerol esters e.g. selected from Polyglycerol Ester of Fatty Acids;• Isostearic acid esters selected from Isostearic Acid Ethyl Ester;[000223] Ether-Based Nonionic Surfactants:• Polyoxyethylene fatty ethers e.g. selected from Brij® 35 and Brij® 78;• Alkyl ethoxylates e.g. selected from Tween® 20 and Tween® 80;• Fatty alcohol ethoxylates e.g. selected from Brij® 58 and Brij® 700;• Brij® series variants e.g. selected from Brij® 35 and Brij® 78;[000224] Natural Nonionic Surfactants:• Saponins e.g. selected from Quillaja Saponin;[000225] Block Copolymer Nonionic Surfactants:• Poloxamers e.g. selected from Pluronic® F68 and Pluronic® F127;sorbitan esters, saponins, polyoxyethylene fatty ethers, poloxamers, alkyl polyglucosides• polyethylene glycol sorbitan monooleate[000226] Alkyl ethoxylates (e.g. Tween 80) are best described as a nonionic surfactant, the structure of which consists of hydrophilic polyoxyethylene chains and a lipophilic oleate ester, making it effective in stabilising formulations and preventing aggregation of lipid particles.[000227] Applications in formulations: alkyl ethoxylates (e.g. Tween 80) are useful for the stability of dispersion of nanoparticles in aqueous environments. It helps achieve a consistent and uniform particle size distribution (contributing to the nanosize encapsulation of the phages), which is also important for reproducible therapeutic efficacy. Additionally, Alkyl ethoxylates (e.g. Tween 80) assists in the efficient encapsulation and delivery of encapsulated phages by maintaining structural integrity of the lipid particles.[000228] Enhanced cellular uptake and penetration: alkyl ethoxylates (e.g. Tween 80) potentially facilitate better penetration of LNP / liposome (e.g., wound tissues), increasing their interaction with skin cell, which enhances cellular uptake.Ionic surfactant[000229] In one embodiment, the composition of the present invention optionally comprises an ionic surfactant (e.g. one or more, e.g. one, two or three, e.g. one or two, e.g. one).[000230] Examples include di-alkyl sulfosuccinate, alkylbenzene sulfonates, lauryl sulfate sodium stearate and benzalkonium chloride.Sugar[000231] In one embodiment, the composition of the present invention optionally comprises a sugar (e.g. one or more, e.g. one, two or three, e.g. one or two, e.g. one). The sugar can act as as a stabilizer to enhance encapsulated phage stability.[000232] The sugar is optionally selected from the group consisting of lactose, sucrose and trehalose.Buffer[000233] In one embodiment, the composition comprises a buffer.[000234] An example of a buffer is a phosphate buffer, e.g. saline phosphate. Other buffers include salt-magnesium buffer (SMB).Concentrations[000235] Exemplary concentrations include the following:• 1 x PBS (pH 7.5): 137 mM NaCI, 2.7 mM KOI, 10 mM Na2HPO4·12H2O, and 1.8 mM KH2PO4• 1 x SMB (pH 7.5): 50mM Tris-HCL, 100mM Sodium Chloride, 8mM Magnesium Sulfate.• 1 x saline phosphate buffer (pH 7.5): 150 mM NaCI, 20 mM Na2HPO4·12H2O, and 10mM KH2PO4.Ratios[000236] In one embodiment, the molar ratio of zwitterionic lipid: neutral lipid: surfactant: charged lipid (e.g. PC, cholesterol, Tween 80 and DOTAP) is about 7:3:1:2.[000237] In one embodiment, the molar ratio of zwitterionic lipid: charged lipid (e.g. PC and DOTAP) is from about 10:1 to about 2:1, e.g. about 7: about 2.[000238] In one embodiment, the molar ratio of neutral lipid: charged lipid (e.g. cholesterol and DOTAP) is from about 3: 1 to about 1:1, e.g. about 3: about 2.Encapsulation[000239] In one embodiment, the phage capsid is encapsulated in lipid particles.[000240] In one embodiment, the phage is completely encapsulated in lipid particles.[000241] In one embodiment, the encapsulation efficiency of the phage is >90% (e.g. >95%, e.g. >98%).[000242] In one embodiment, the lipid nanoparticles are engineered to provide controlled release of the phages over 12 hours at temperatures of 37°C.Method of encapsulating phages[000243] The present invention provides a method of encapsulating phages, the method comprising mixing a charged lipid formulation with a phage formulation.[000244] In one embodiment, the pH of the phage formulation is from 7 to 9.5, e.g. 7, e.g. 7.5, e.g. 8, e.g. 8.5, e.g. 9, e.g. 9.5. The preferred pH of the phage formulation for encapsulation of the phages is influenced by the characteristics of the lipid.[000245] In one embodiment, the phage formulation comprises a buffer (e.g. a phosphate buffer).[000246] In one embodiment, the phage formulation is an aqueous suspension [e.g. PBS (phosphate-buffered saline)].[000247] In one embodiment, the cationic charged lipid formulation comprises a surfactant. In one embodiment, the surfactant is polyethylene glycol sorbitan monooleate.[000248] In one embodiment, the lipid formulation is an organic phase (e.g. ethanol).[000249] In one embodiment, the method further comprises the step of preparing a cationic charged lipid formulation by dissolving lipid and a cationic lipid inducer [e.g. octadecylamine] in a solvent [e.g. EtOH], optionally at RT, optionally sonicating (e.g. 10 mins).[000250] Preferably the organic phase comprises the lipid and the aqueous phase comprises the phage to be encapsulated. Preferably the mixing ratio between organic: aqueous phase is in the range from about 1: about 3 - about 1: about 1 (preferably about 1: about 3, about 1: about 2, about 2: about 3, about 1: about 1).Method of preparing a composition for delivery[000251] The present invention provides a composition comprising phage encapsulated in nanosize lipid particles. The nanosize lipid particles comprise a charged lipid as described herein.[000252] The composition may be prepared using methods including spray drying, freeze drying, and nebulization techniques.[000253] Spray drying, freeze drying, and nebulization techniques provide versatility and potential for various delivery methods. These formulation approaches are applicable to other non-respiratory infections requiring phage-based therapy, such as wound care and skin management. Liposomal phages can be developed into semi-liquid formulations for topical application to infected wounds, leveraging the moisture-retentive properties of liposomes to aid in wound healing. The nanosize and lipid material of liposomes enhances skin penetration, ensuring effective treatment of skin infections. Moreover, liposomal phages offer an eco-friendly alternative to chemical pesticides for treating bacterial infections in plants and animals. They are safe and effective across a range of animal species and environmental conditions, making them highly versatile for both agricultural and veterinary applications.Medical uses[000254] The compositions of the present invention are useful in medicine (including human medicine and veterinary medicine).[000255] In particular, the compositions of the present invention are useful for phage therapy.In one aspect, the present invention provides a method of treating or preventing a condition or disease, comprising administering to a subject in need thereof an effective amount of a composition comprising a phage encapsulated in nanosize lipid particles;wherein the lipid particles comprise a charged lipid;wherein the charged lipid comprises a cationic or anionic moiety and a C12 to C80 hydrocarbon chain or a polymeric chain;wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O) Rx, -C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5-or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, -NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.[000256] In another aspect, the present invention provides a method of treating a bacterial infection in the respiratory system, comprising administering to a subject in need thereof an effective amount of a composition comprising a phage encapsulated in nanosize lipid particles;wherein the lipid particles comprise a charged lipid;wherein the charged lipid comprises a cationic or anionic moiety and a C12 to C80 hydrocarbon chain or a polymeric chain;wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O)Rx, -C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5-or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, -NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.[000257] The present invention further provides use of a composition comprising a phage encapsulated in nanosize lipid particles in the manufacture of a medicament for treating or preventing a condition or disease;wherein the lipid particles comprise a charged lipid;wherein the charged lipid comprises a cationic or anionic moiety and a C12 to C80 hydrocarbon chain or a polymeric chain;wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O) Rx, -C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5-or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, -NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.[000258] In a further aspect, the present invention provides use of a composition comprising a phage encapsulated in nanosize lipid particles in the manufacture of a medicament for treating a bacterial infection in the respiratory system;wherein the lipid particles comprise a charged lipid;wherein the charged lipid comprises a cationic or anionic moiety and a C12 to C80 hydrocarbon chain or a polymeric chain;wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O)Rx, -C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5-or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, -NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.[000259] In yet a further aspect, the present invention provides a composition comprising a phage encapsulated in nanosize lipid particles for use in treating or preventing a condition or disease;wherein the lipid particles comprise a charged lipid;wherein the charged lipid comprises a cationic or anionic moiety and a C12 to C80 hydrocarbon chain or a polymeric chain;wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O) Rx, -C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5-or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, -NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.In another aspect, the present invention provides a composition comprising a phage encapsulated in nanosize lipid particles for use in treating a bacterial infection in the respiratory system;wherein the lipid particles comprise a charged lipid;wherein the charged lipid comprises a cationic or anionic moiety and a C12 to C80 hydrocarbon chain or a polymeric chain;wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(0)Rx, -C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5-or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, -NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.[000260] The compositions of the present invention may be useful for:• treating a bacterial infection in the respiratory system (administration by inhalation) - for example, both chronic lung infections (e.g., cystic fibrosis- associated infections) and acute lung infections (e.g., pneumonia)• wound care and skin management (topical application)• treatment infected wounds (topical application)• treatment of skin infections (topical application)• treating bacterial infections in animals (including mammals e.g. humans)• treating gastrointestinal infections caused by pathogenic bacteria (oral administration)[000261] Examples of delivery methods include, but are not limited to, topical application and inhalation.[000262] Delivery by topical application may involve formulation as an ointment or a cream using suitable excipients.[000263] Delivery by inhalation may involve a nebuliser, such as a jet nebuliser or a vibrating mesh nebuliser.Aspect Jet Nebulisers Vibrating Mesh NebulisersUse compressed air to create Use a vibrating mesh to Mechanism ofaerosol droplets; higher shear generate aerosol droplets; Actionforces. minimal shear stress.Suitable for less sensitiveFormulation Ideal for sensitive formulations medications like bronchodilatorsSuitability such as proteins, peptides.and corticosteroids.Preferred for advanced Commonly used in clinical settings therapies; suitable for home use Applicationand hospitals for general and portable applications due to Settingsrespiratory treatments. their compact size and quiet operation.Higher shear forces may degrade Gentle aerosolization preserves Formulationsensitive medications, limiting use the integrity of delicate Stabilitywith fragile formulations. formulations during delivery.Advanced therapies requiring Standard respiratory medicationsprecise dosing and particle size Typical Uses where precise particle size is lesscontrol, such as inhaled critical.vaccines and gene therapies.[000264] As used herein, the terms “treatment” (or "treating") and prevention" (or preventing") are to be considered in their broadest contexts. For example, the term "treatment” does not necessarily imply that a patient is treated until full recovery. The term "treatment” includes amelioration of the symptoms of a disease disorder or condition or reducing the severity of a disease disorder or condition. Similarly, "prevention" does not necessarily imply that a subject will never contract a disease disorder or condition. " Prevention” may be considered as reducing the likelihood of onset of a disease disorder or condition or preventing or otherwise reducing the risk of developing a disease disorder or condition.[000265] As used herein, the term “subject” or “individual” or “patient” may refer to any subject, particularly a vertebrate subject, and more particularly a mammalian subject, for whom therapy is desired. Suitable vertebrate animals include, but are not limited to primates, avians, livestock animals (e.g. sheep, cows, horses, pigs, donkeys, etc.), laboratory test animals (e.g. rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g. cats, dogs) and captive wild animals (e.g. foxes, deer, dingoes). A preferred subject is a human.[000266] As used herein, and “effective amount” will be understood to mean an amount of the active phage which, when administered according to a desired dosage regimen, provides the desired therapeutic activity. Dosing may occur once, or at intervals of minutes or hours, or continuously over 10 any one of these periods. Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage. A typical dosage is in the range of 1 tg to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage may be in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage may 15 be in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage may be in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per body weight per dosage.[000267] Nebulisation: The aerosol droplets generated have a median diameter (D50) of ≤5 μm and a 90th percentile diameter (D90) of ≤ 10 μm, making them suitable for lung (pulmonary) delivery.Additional uses[000268] The composition of the invention is also useful for treating bacterial infections in plants.[000269] The composition of the invention is also useful for coatings or treatment solutions for surfaces in diverse situations, including industrial equipment, water treatment systems, marine vessels, household appliancesEXAMPLES[000270] The present invention will now be described with reference to the following examples which should be considered in all respects as illustrative and non-restrictive.Example 1[000271] The use, methods and composition according to the invention is useful for incorporating lipid nanoparticles (LNPs) into coatings or treatment solutions for surfaces in diverse situations, including industrial equipment, water treatment systems, marine vessels, household appliances, and healthcare application (both human and animal). The LNPs are composed of charged lipids, to which a non-ionic surfactant such as Tween 80 may be incorporated to stabilize the nanoparticles during formulation and storage. Encapsulated phages (e.g., PEV1, PEV2, PEV31, PEV40) are for their bacteria killing efficacy against Pseudomonas aeruginosa as well as other common pathogens. The use, methods and composition according to the invention is useful for encapsulating other phage types targeting other common biofilmforming pathogens responsible for persistent contamination, infections and equipment fouling.[000272] Broad-Spectrum Biofilm Control: The LNP-phage composition is effective against a variety of biofilm-forming bacteria, making it versatile for multiple applications across different industries.[000273] Enhanced Biofilm Penetration and Disruption: The lipid encapsulation facilitates deeper penetration into established biofilms, enhancing the disruption and eradication of resilient bacterial colonies. A cationic lipid may be used to impart positive charge to LNPs, enhancing interaction with negatively charged bacterial membranes. Gradual phage release from LNPs ensures prolonged antimicrobial activity, reducing the need for frequent reapplications and maintenance.[000274] Improved Stability of Phages: Encapsulation within LNPs protects phages from environmental stresses such as temperature fluctuations, UV exposure, and chemical disinfectants, maintaining their bioactivity over extended periods.[000275] Versatile Application Methods: The composition can be adapted for various application techniques, including surface coatings, sprays, and immersion treatments, catering to the specific needs of different environments.[000276] Environmentally Friendly Alternative: Offers a non-toxic, biodegradable solution for biofilm control, minimizing environmental impact compared to traditional chemical disinfectants.[000277] Reduced Development of Resistance: Utilizing phages as biocontrol agents lowers the risk of developing resistance mechanisms commonly associated with chemical antimicrobials.[000278] Cost-Effective Maintenance: Prolonged efficacy and reduced application frequency translate to lower maintenance costs for industries relying on biofilm-prone equipment and systems.Example 2[000279] Charged liposome to deliver phages into host cells infected by intracellular pathogens, including antibiotic-resistant bacteria (superbugs) such as Mycobacterium tuberculosis (Mtb). The composition comprises a specific mixture of charged lipids. Encapsulated phages are selected for their efficacy against bacteria responsible for persistent and hard-to-treat infections. Liposomal encapsulation enhances the ability of phages to penetrate host cells, including macrophages, which phages alone cannot efficiently infiltrate and thereby reach the bacteria inside the cell.[000280] Facilitated Cellular Penetration: LNP encapsulation enables phages to penetrate host cells, including macrophages, overcoming the inherent limitation of phages being unable to efficiently enter cells on their own.[000281] Enhanced Therapeutic Efficacy: Targeted delivery ensures higher concentrations of phages at the infection site, improving treatment outcomes.[000282] Stimuli-Responsive LNPs: Designing LNPs that release phages in response to intracellular signals, enhancing targeted delivery and ensuring phages are released precisely where needed within the host cells.[000283] Enhanced Stability of Phages: The lipid encapsulation protects phages from degradation within the biological environment, maintaining their bioactivity longer and ensuring consistent therapeutic effects.[000284] Biocompatibility and Safety: The lipid composition is biocompatible and non-toxic, ensuring safe interaction with host cells without eliciting adverse immune responses.Example 3[000285] The liposome encapsulated phages are adapted for inhalation delivery to treat respiratory infections caused by Pseudomonas aeruginosa. The formulation comprises charged lipids with the addition of Tween 80 to stabilize the nanoparticles during nebulization and during spray drying to form inhalable particles. The encapsulated phages (e.g., PEV31, PEV1, PEV2, PEV40) target Pseudomonas aeruginosa as well as other common pathogens that cause severe respiratory infections, particularly in individuals with compromised lung function such as those with cystic fibrosis.[000286] Encapsulation within LNPs safeguards phages from shear forces and other stresses encountered during nebulization and during spray drying to form inhalable particles, ensuring their viability upon delivery.[000287] The LNPs are engineered to be aerosolized into fine particles, facilitating deep lung penetration and effective delivery of phages to the respiratory tract.[000288] Prolonged Therapeutic Action: The specific lipid composition maintains phage stability and bioactivity, promoting sustained antimicrobial effects against Pseudomonas aeruginosa over extended periods.[000289] The formulation reduces recognition and clearance by the immune system, allowing for longer circulation times and enhanced therapeutic efficacy.Example 4[000290] The liposome encapsulated phages can be incorporate into a topical cream, gel or shampoo for application to various skin conditions, including wounds, acne, and other skin infections (both treatment and cosmetic applications). The lipid nanoparticles (LNPs) are composed of charged lipids and phages. Encapsulated phages (e.g., PEV31, PEV1) are selected for their bacteria killing efficacy against Pseudomonas aeruginosa and other common skin pathogens responsible for delayed wound healing, persistent infections, and inflammatory skin conditions like acne. Additionally, the formulation is suitable for encapsulating other phagesthat target a diverse range of skin pathogens, thereby enhancing its versatility and applicability across various types of skin-related infections.[000291] Controlled Phage Release: LNP encapsulation allows for gradual phage release, maintaining therapeutic concentrations at the application site over extended periods. The specific lipid ratios allow for fine-tuning the release rate of phages, optimizing therapeutic outcomes based on the severity of the skin condition and infection load.[000292] Enhanced Penetration of Biofilms: The lipid formulation aids in the penetration and disruption of bacterial biofilms, facilitating the eradication of resistant bacterial colonies.[000293] Improved Phage Skin Penetration: Encapsulation within LNPs enhances the ability of phages to penetrate skin layers, ensuring better access to bacterial pathogens residing deeper in tissues.[000294] Enhanced Stability of Phages: The lipid encapsulation protects phages from external environmental degradation, maintaining their bioactivity longer and ensuring consistent therapeutic effects.[000295] Biocompatibility and Safety: the lipid composition is biocompatible and non-toxic, making the formulation safe for use on sensitive and damaged skin areas.[000296] Minimized Systemic Exposure: Topical application confines the treatment to the affected skin area, reducing the risk of systemic side effects associated with other administration routes.[000297] Versatility in Phage Selection: The formulation can encapsulate various phages targeting different skin pathogens, allowing for customized treatments based on the specific bacterial profile of the infection.Example 5[000298] Adapting the liposomal encapsulating phages for oral administration to treat gastrointestinal infections caused by pathogenic bacteria. The formulation comprises a specific mixture of charged lipids. Encapsulated phages are protected from the acidic environment of the stomach by the lipid encapsulation, ensuring their survival and subsequent release in the intestinal tract. Additionally, a polymeric coating layer, utilizing biocompatible polymers such as chitosan, alginate, or poly(lactic-co-glycolic acid) (PLGA), is applied on top of the LNPs to provide an extra barrier against gastric acids and enzymatic degradation. This dual-encapsulation strategy enhances the stability of phages during transit through the stomach and facilitates targeted delivery to the site of infection within the gastrointestinal mucosa.[000299] Protection from Gastric Acids: LNP encapsulation safeguards phages from the harsh acidic conditions of the stomach, enhancing their survival and therapeutic efficacy.[000300] Polymeric Coating for Enhanced Protection: The additional polymeric coating (e.g., chitosan, alginate, Eudragit, PLGA etc) provides an extra layer of protection against gastric acids and enzymes, further ensuring phage viability during oral transit.a. Poly(lactic-co-glycolic acid) (PLGA), PVP, PVA and Eudragit: Biodegradable and biocompatible polymers commonly used in drug delivery systems for controlled and sustained release of therapeutic agents.b. Chitosan: A natural polysaccharide derived from chitin, used for its mucoadhesive properties and ability to enhance drug delivery across mucosal surfaces.c. Alginate: A natural polymer derived from brown seaweed, used for its gel-forming ability and pH-responsive properties in drug delivery applications.[000301] Enhanced Bioavailability: Facilitates better dispersion and retention of phages in the gut, improving treatment outcomes.[000302] Controlled Release Kinetics: The combination of LNP encapsulation and polymeric coating allows for precise control over phage release rates, optimizing therapeutic efficacy based on the severity and location of the infection. Ensures phages are delivered directly to the site of infection within the gastrointestinal tract, maximizing antimicrobial action.[000303] Versatility in Formulation: The dual-encapsulation approach can be tailored to encapsulate a variety of phages targeting different gastrointestinal pathogens, enhancing the formulation's adaptability.[000304] Biocompatibility and Safety: Both lipid and polymeric components are biocompatible and non-toxic, ensuring safe interaction with the gastrointestinal environment without eliciting adverse immune responses.[000305] Minimized Systemic Exposure: Oral delivery confines the treatment to the gastrointestinal tract, reducing the risk of systemic side effects associated with other administration routes.Example 6[000306] The application of lipid-encapsulated phages can be extended to veterinary medicine, providing treatments for infections in livestock caused by Pseudomonas aeruginosa and other pathogenic bacteria. The formulation offers a versatile approach to managing infections in animals, enhancing animal health, and reducing the reliance on antibiotics in agriculture. The formulation can be incorporated into different administration routes, including oral for passive ingestion, direct injectable administration and topical localised administration.[000307] Alternative to Antibiotics: Provides an effective treatment option that mitigates the development of antibiotic-resistant bacteria in livestock, addressing a significant concern in agriculture and public health.[000308] Versatile Administration Routes: Adaptable to various administration methods (oral, injectable, topical) based on the specific needs of different animal species and types of infections, offering flexibility in treatment protocols.[000309] Improved Animal Health: Enhances the overall health and productivity of livestock by effectively controlling bacterial infections, leading to reduced morbidity and mortality rates.[000310] Reduced Antibiotic Use: Lowers the reliance on traditional antibiotics in agriculture, contributing to the global effort to combat antibiotic resistance.[000311] Enhanced Phage Stability and Viability: LNP encapsulation protects phages from environmental degradation and ensures their viability until they reach the target site, maintaining their lytic activity against pathogens.[000312] Biocompatibility and Safety: The lipid composition is biocompatible and non-toxic, ensuring safe interaction with animal tissues without eliciting adverse immune responses.[000313] Cost-Effective Treatment: Potentially reduces treatment costs by decreasing the need for multiple antibiotic courses and minimizing losses due to infections.[000314] Environmentally Friendly: Utilizes biodegradable lipids and non-toxic emulsifiers, ensuring that the formulation breaks down naturally without causing environmental harm. The formulation may reduce antibiotic runoff. By decreasing the use of traditional antibiotics, the formulation minimizes antibiotic residues entering the environment, thus protecting ecosystems and reducing the spread of antibiotic resistance. The formulation promotes sustainable farming practices by providing a safe and effective alternative to chemical antimicrobials, aligning with environmental conservation goals.Example 7[000315] This example extends the application of lipid-encapsulated phages to both environmental management and agricultural practices, providing effective treatments for bacterial contamination in water systems and soil-borne plant pathogens. The formulation offers a versatile approach to managing infections in diverse settings, enhancing environmental health, promoting sustainable agriculture, and reducing the reliance on chemical disinfectants and antibiotics.[000316] The LNP-phage formulation is introduced into water treatment facilities where it facilitates the stable delivery and sustained release of phages, effectively reducing bacterial loads and preventing waterborne diseases. LNP-phage formulations can be incorporated into existing water filtration systems for continuous bacterial control in irrigation water.[000317] The formulation can be applied directly to the soil or used as seed coatings to manage soil-borne plant pathogens, ensuring robust plant growth, reducing disease incidence and enhancing crop health and yield.[000318] Versatility and Adaptability: Suitable for various environmental and agricultural settings, including industrial water treatment, agricultural soil management, and integrated farming systems. Capable of targeting multiple bacterial strains through customizable phage cocktails, enhancing overall antimicrobial efficacy.[000319] The lipid formulation protects phages from environmental degradation, enhancing their stability and viability in aquatic and soil environments.[000320] Biodegradable Components: Utilizes biodegradable lipids and non-toxic emulsifiers, ensuring that the formulation breaks down naturally without causing environmental harm.[000321] Reduced Antibiotic and Chemical Use: Offers an environmentally safe alternative to chemical disinfectants and antibiotics, minimizing ecological impact and promoting sustainable practices.[000322] Reduced Antibiotic Runoff: By decreasing the use of traditional antibiotics, the formulation minimizes antibiotic residues entering the environment, protecting ecosystems and reducing the spread of antibiotic resistance.Example 8[000323] The encapsulation of two phages (Pseudomonas-targeting phages PEV1 (Myoviridae) and PEV31 (Podoviridae)) using neutral lipid and charged lipid was conducted using various micro-mixing processes, including (i) AXF-Pathfinder mini (AXF mini), (ii) confined impinging jet mixer (Cl J), and (iii) microfluidic chip. The resulting particles were characterised by their size, morphology and encapsulation efficiency.[000324] The size of liposomes encapsulating PEV31 (401-651 nm) was smaller than those encapsulating the Myoviridae PEV1 (744-837 nm), which are consistent with the size difference of the two studied phages.[000325] Transmission electron microscopy (TEM) confirmed the encapsulation of phages in liposomes.[000326] The charged liposomal encapsulation showed maximum encapsulation efficiency of 90% for PEV31 and 82% for PEV1.[000327] Regardless of the micro-mixing technique used, the encapsulation efficiency using charged lipids was generally higher than that obtained from the neutral lipids.[000328] In summary, the new approach of using charged lipid is capable of encapsulating phages in nanosize lipid particles with high encapsulation efficiency and minimal titre reduction. As different types of charged lipids can be used, such versatility can significantly advance phage encapsulation development by providing a more robust and flexible approach, as well as reducing dependency on specific equipment, and potentially improving scalability and efficiency in various production settings.Materials[000329] Two Pseudomonas lytic phages of different morphologies, a Podovirus (PEV31, 2 x 109pfu / mL stock titre) and a Myovirus (PEV1, 3 x 109pfu / mL), were used as model phages for the proposed test. They were isolated from the sewage treatment plant in Olympia (WA, USA) by the Kutter Lab (Evergreen Phage Lab) using P. aeruginosa dog-ear strain PAV237, which was also used as the host bacterial strain to assess phage titre. Amplified phages were filtered using a 0.22 μm polyethersulfone (PES) syringe filter (Merck, Germany). Filtered lysates were treated with 10 pg / mL DNase and RNase (Merck, Germany) and incubated at 37 °C for 30 min. DNase and RNase were physically removed by ultrafiltration at 4000 x g, at 4 °C using the Allegra X-30R benchtop centrifuge (Beckman Coulter, USA) and samples resuspended inphosphate-buffered saline (PBS) (0.01 M phosphate buffer, 0.0027 M KCI, and 0.137 M NaCI), with pH adjusted to 7.2.[000330] EndoTrapOHD (Lionex GmbH, Germany) was used to initially clear the phages of endotoxins prior to purification using the anion-exchange (AEX) column. The optimised AEX process and buffer concentrations in which the purest fraction of phages eluted were determined previously. The entrapped phages were loaded onto the CIMmultus™ diethylamine (DEAE) 2 pm column (BIA Separations, Slovenia) and eluted according to the manufacturer’s protocol, using varying NaCI concentration, unique to each phage. The purest fraction was then cleaned up of endotoxins by a final run with the EndoTrapOHD. The endotoxin levels were quantified by ToxinSensor™ chromogenic Limulus amebocyte lysate assay kit (GenScript, USA).[000331] The final phage preparations, both PEV31 and PEV1 (~1.5x1O10pfu / mL) were stored in PBS with the pH adjusted to either 7.5 or 9.5. In the Applicant’s experiments, the two pH conditions were intentionally selected for comparative studies: phages at pH 7.5 were intended for encapsulation with non-charged lipids, whereas those at pH 9.5 were combined with charged lipids. The Applicant’s results have shown phages in pH 9.5 demonstrated enhanced encapsulation efficiency by charged lipids, attributed to the increased negative surface charge of phages under basic conditions.[000332] 1,2-Distearoyl-sn-glycero-3-phosphorylcholine (DSPC) was from Avanti Polar Lipids, Inc (AL, USA) and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) was purchased from Cayman Chemical (Ml, USA).[000333] Soybean phosphatidylcholine (PC), cholesterol, Tween 80, octadecylamine, and bile salts were purchased form Sigma-Aldrich (NSW, Australia). Ethanol (EtOH) was obtained from Chem-Supply (NSW, Australia).Liposome preparation[000334] To prepare cationic charged lipid formulation, a mixture of PC, cholesterol, octadecylamine and Tween 80 (7:3:2: 1 molar ratio) was dissolved in absolute EtOH with a total solid content of 20 mg / mL with bath sonication and room temperature for 10 min.[000335] Further cationic liposomes were prepared by dissolving DSPC, cholesterol, DOTAP, and Tween 80 (7:3:2: 1 molar ratio) in absolute EtOH at a total solid content of 20 mg / mL. The solution was sonicated in a water bath for 10 minutes to ensure complete solubilization.[000336] Neutral lipid formulation was prepared in the same way with only octadecylamine excluded, having PC, cholesterol and Tween 80 with 9:3:1 molar ratio.1. AXF mini• Two dual syringe pumps (PHD 2000, Harvard Apparatus Inc., Holliston, MA) which were employed to introduce the lipid mixture and phage suspension with a fixed total flow rate (TFR) of 3 mL / min.• The lipid mixture was injected through the continuous phase. Phage suspension stock or PBS was injected into the dispersed phase. The produced liposome was collected into a scintillation vial, placed with a small magnetic stir bar in a 2 mL deionised water quench bath.• The effects of phage / lipid flow rate ratio (FRR, 3:1, 2:1, 3:2) on the liposome size, polydispersity index (pdi) and encapsulation efficiency were investigated. All experiments were performed at room temperature and triplicated.2. CIJ mixer• The aqueous phase was phage suspension stock or PBS, and the organic phase contained the charged or neutral lipid with same preparation as above. The aqueous and organic phases were injected into the CIJ mixer over a range of TFR (35, 17.5, 8.75, 5, 2.5 mL / min) with nonadjustable 1:1 FRR.• The formed liposomes were collected in a same quench bath set up.3. Microfluidic chip• The microfluidic design consists of three-way junction, where one inner aqueous (IA) phase and two lipid-carrying organic (LO) phase and branch out channel.• The liposome preparation is conducted by injecting the lipid formulation solution through the two LO channels and the phage stock into the IA phase. The produced liposome carried out through the branch out channel and was collected for characterisation. The FRR of phage / lipid is fixed with 2:1 with TFR of 3 mL / min.All formulations above were performed in triplicate at room temperature.Ethanol removal[000337] Ethanol-free liposomal phage suspensions were prepared by ultracentrifugation at 10000 x g for 30 min at 4 °C. After each spin, the supernatant was removed, and the pellet was resuspended in PBS. This process was repeated two times to minimise residual ethanol content.Biological assay[000338] The Applicant used the Miles-Misra surface droplet technique (Carlson, K. Working with bacteriophages: common techniques and methodological approaches. Bacteriophages: biology and applications 2005, 1, 437-494) to assess the number of viable phages in original and lysed liposome samples. Serial dilutions (1:10), were performed by adding 20 pL samples to 180 pL PBS. A volume of 200 pL host bacteria containing ~2 x 109 colony forming units (cfu) was mixed with 5 mL molten soft agar (0.4% Amyl agar, 48 °C). The mixture was then overlayed onto a solidified nutrient agar composed of 1.5% Amyl agar and nutrient broth. 10 pL of diluted phage samples were deposited in triplicate on the agar surface, air-dried, and then incubated overnight at 37 °C. Samples yielding 3-30 plaques were used for phage viability calculation.The encapsulation efficiency of the phages[000339] The phage encapsulation efficiency (EE) was calculated using the equation EE (%) = 100 – (Cfree / Ctotal) x 100, where Ctotalrepresent the total phage concentration, and Cfreeis the free phages concentration.[000340] Cfree was determined directly conducting plaque assay on the liposome-encapsulated phage suspension, and Ctotai was obtained by lysing 0.5 mL of liposome-encapsulated phage suspension with 0.5 mL of bile salts (50 mM) before the plaque assay, which previously confirmed that this bile salts concentration has no effect on the stability of phages.Liposomal Size Measurement[000341] The size and PDI of the liposomes were determined using dynamic light scattering (DLS) on a Zetasizer Nano-ZS (Malvern Instruments, UK). The instrument utilized a 633 nm He / Ne laser with a power of 4.0 mW to calculate the intensity-weighted mean diameter (Z-average). Liposomal phage suspensions were diluted at a 1:10 ratio in PBS for size distribution analysis. All measurements were carried out in triplicate at 25 °C.Zeta Potential[000342] The zeta potential of the liposomes was assessed using Laser Doppler Electrophoresis on the Zetasizer Nano-ZS (Malvern Instruments, UK). The values were computed based on the Helmholtz-Smoluchowski equation. For analysis, samples were diluted in PBS to achieve a lipid concentration of 0.2 mg / mL and measured in a folded capillary cell. Each sample was analyzed in triplicate at 25 °C.Transmission electron microscopy (TEM)[000343] TEM images assessed the structure and morphology of original phage and liposome-phage samples using an FEI Tecnai T12 microscope at 120 kV with a 4k x 4k CCD camera. Negative staining using 2% w / v uranyl acetate was applied to all samples.Atomic Force Microscopy Infrared Spectroscopy (AFM-IR)[000344] Nanoscale IR measurements were conducted using an AFM-IR instrument (nanoIR, Anasys Instruments) following an established protocol (Khanal, D., Chang, R. Y. K., Morales, S., Chan, H. K., Chrzanowski, W., 2019. High Resolution Nanoscale Probing of Bacteriophages in an Inhalable Dry Powder Formulation for Pulmonary Infections. Anal Chem 91, 12760-12767). Samples were prepared by diluting 1:10 in ultrapure water, depositing 5 pL onto an argon plasma-treated zinc selenide prism for 3 minutes, and air-drying for 30 minutes after removing excess liquid. IR background spectra (1000 -1800 cm-1) were averaged and normalized for signal calibration. The cantilever ringdown signal was optimized at 75 kHz with 50 kHz window, and the infrared laser hotspot was fine-tuned at specific wavenumbers to maximize the signal-to-noise ratio. For each sample, at least 10 AFM-IR spectra were collected (1000 -1800 cm-1, 2 cm-1intervals, 0.1 Hz scan rate, 16 coaverages) using a silicon nitride cantilever (EXC450 tips, AppNano) with a spring constant 0.5 N m-1. Data analysis was performed with Analysis Studio software, applying Savitzky-Golay smoothing (polynomial order 2, 10 points).ResultsCharacteristics of phage-encapsulated liposomesAXF mini:[000345] Liposomes consisting of soybean and cholesterol were formed at higher encapsulation efficiency with the additional of cationic lipid inducer octadecylamine.1. Liposome morphology[000346] The morphology of free phage suspensions and the liposome-encapsulated phages were imaged by TEM. The complex liposome and phages of unsuccessful encapsulation were also included for comparison.[000347] The TEM images confirmed that the PEV31 phage is a Podoviridae containing an isometric hexagonal head (~65 nm) and a short tail, whereas the PEV1 phage is a Myoviridae with an isometric hexagonal head (~65 nm) and a long contractile tail of -120 nm (Fig. 1a and b).[000348] Phages were completely or only capsid encapsulated in liposomal particles. In general, only one phage can be encapsulated in a single liposome due to the comparable size of liposomes and phages (Fig. 2a and b).[000349] Empty liposomes (Fig. 1c and d) and non-encapsulated phages (Fig. 1e and f), which were mostly attached to the external surface or with other phages, were also observed. The sizes of the liposomes as determined by TEM were consistent with those estimated by DLS measurement.2. Phage stability and encapsulation efficiency[000350] Fig. 3 demonstrates from another perspective that using charged lipids can lead to higher encapsulation efficiency of phages. It illustrates the logarithmic difference (log10difference) in phage titers — measured in plaque-forming units per milliliter (pfu / mL) — of liposome-encapsulated bacteriophages before and after immediate release induced by bile salts. The graph compares formulations utilizing charged lipids versus non-charged lipids.[000351] The phage encapsulation efficiency (EE) calculation described above can be correlated with Fig. 3. Specifically, Fig. 3 depicts the logarithm of the difference between the phage titer after immediate release (equivalent to the description of the total phage concentration, Ctotai) and before immediate release (equivalent to the description of free phage concentration, Cfree), expressed as log10(Ctotal– Cfree).[000352] Bile salts were used to disrupt the lipid bilayer structure encapsulating the phages, leading to their release.[000353] By analyzing the values of log10(Ctotal– Cfree), Fig. 3 showed the charged lipid formulation exhibits a greater log difference in phage titer compared to the non-charged lipid formulation. This indicates that a higher concentration of phages was successfully encapsulated within the charged lipid liposomes before their release by bile salts.[000354] The encapsulation efficiencies for PEV31 and PEV1 produced by the neutral lipid were <15% (Fig. 4). Incorporating octadecylamine to cationise the lipid shows significant improvement for encapsulating PEV31 (highest EE 90.32±0.67%) and PEV1 (highest EE 82.32±0.96%).[000355] Using charged lipid, both phages show highest encapsulation efficiency at 2:1 FRR, followed by 3:1 and 3:2. There are no specific trends noted with the flow conditions.[000356] However, the trend of encapsulation efficiency was improved by the addition of cationic charge in the lipid. As phages in pH 9.5 would still be stable and likely carry negative charge, electrostatic interaction between the charged lipid and the phage would allow the lipid to bond stronger to phages while encapsulation happens, leading to increased encapsulation efficiency.3. Size, pdi and zeta potential[000357] Liposomal encapsulated phages with charged and neutral lipids had comparable sizes (Fig. 5). For PEV31, phages with neutral lipids were larger, while the opposite for PEV1. All formulations were larger than blank liposomes. PEV31 liposomes with charged lipids had pdi of 0.08-0.291, while PEV1 had higher pdi (0.13-0.442) (Fig. 6). PEV1 with neutral lipids had similar homogeneity, but PEV31 was less polydisperse (pdi: 0.11-0.42). Charged lipids shifted zeta potential from positive to neutral (<20 mV), indicating better encapsulation (Fig. 7 and 8) compared to using neutral lipids.CIJ mixer:[000358] Liposomal phages prepared using the CIJ mixer utilised a faster flow rate, resulting in quicker mixing of phages and lipids with reduced contact time. Despite this, using charged lipid formulations for encapsulation consistently achieved promising encapsulation efficiencies of up to 84.15±1.82% for PEV1 and 72.63±2.77% for PEV31 (Table 1a and b). This suggests that special lipid formulations are effective for phage encapsulation across different devices, not tying to a specific micro-mixing method.Table 1a (charged lipid) PEV 1Group Total flow rate Flow rate ratio EE (%)(phage: lipid)1 35 mL / min 1:1 59.63±2.822 17.5 mL / min 1:1 41.13±3.163 8.75 mL / min 1:1 84.15±1.824 5 mL / min 1:1 83.10±1.235 2.5 mL / min 1:1 78.57±1.05Table 1b Test 2 (neutral lipid) PEV 1Group Total flow rate Flow rate ratio EE (%)(phage: lipid)1 35 mL / min 1:1 11.64±1.162 17.5 mL / min 1:1 14.35±1.733 8.75 mL / min 1:1 15.30±1.444 5 mL / min 1:1 10.39±0.655 2.5 mL / min 1:1 8.80±2.85[000359] Similar to AXF mini, the liposomal phages produced using the CIJ mixer exhibited increased size and a shift to a more positive charge with cationic lipid formulation compared to neutral lipid formulations (Table 2a, b and 3a, b).Table 2a (charged lipid) PEV 31Group Total flow rate Flow rate ratio EE (%)(phage: lipid)1 35 mL / min 1:1 72.63±2.772 17.5 mL / min 1:1 57.04±2.733 8.75 mL / min 1:1 56.69±2.634 5 mL / min 1:1 39.32±1.505 2.5 mL / min 1:1 36.00±3.27Table 2b (neutral lipid) PEV 31Group Total flow rate Flow rate ratio EE (%)(phage: lipid)1 35 mL / min 1:1 16.56±1.822 17.5 mL / min 1:1 8.77±1.443 8.75 mL / min 1:1 23.89±1.984 5 mL / min 1:1 14.76±3.455 2.5 mL / min 1:1 15.88±1.12Table 3a (charged lipid) PEV 1Group Total Flow rate ratio Size (nm) pdi Zeta flow rate (phage: lipid) potential (mL / min) (mV)1 35 1:1 960.53±22.98 0.191±0.052 12.00±1.412 17.5 1:1 869.07±5.99 0.223±0.028 13.47±0.753 8.75 1:1 1013.43±31.66 0.864±0.033 20.47±1.434 5 1:1 1543.33±75.30 0.539±0.053 13.87±1.335 2.5 1:1 1695.33±55.72 0.268±0.110 16.93±1.55Table 3b (neutral lipid) PEV 1Group Total Flow rate Size (nm) pdi Zetaflow rate ratio potential (mL / min) (phage: lipid) (mV)1 35 1:1 169.33±3.06 0.158±0.024 -18.17±1.432 17.5 1:1 176.57±5.30 0.151±0.039 -17.80±0.983 8.75 1:1 206.37±5.27 0.283±0.021 -16.23±1.364 5 1:1 307.77±7.20 0.138±0.130 -12.87±0.985 2.5 1:1 219.80±8.75 0.137±0.053 -12.30±1.20[000360] Compared to blank liposomes, charged liposomal phages showed zeta potentials shifting towards neutral, indicating that encapsulation leads to charge neutralisation.Microfluidic mixer:[000361] The preparation of liposomal phage using a microfluidic mixer employs three streams but utilizes a slower flow rate. Despite this, the use of charged lipid formulations with adjusted pH for phages consistently produces higher encapsulation efficiencies, achieving up to 63.96±1.43%for PEV1 and 70.96±3.59% for PEV31 (Tables 6a, b and 7a, b). These results further confirm the versatility of the formulations, indicating their general applicability to different micro-mixing devices.Table 6a (charged lipid) PEV 1Group Total flow rate Flow rate ratio EE (%)(phage: lipid)1 3 mL / min 2:1 64.292 3 mL / min 2:1 65.523 3 mL / min 2:1 62.07Mean 63.96 ± 1.43Table 6b (neutral lipid) PEV 1Group Total flow rate Flow rate ratio EE (%) (phage: lipid)1 3 mL / min 2:1 11.112 3 mL / min 2:1 20.693 3 mL / min 2:1 17.86Mean 16.55 ± 4.02Table 7a (charged lipid) PEV 31Group Total flow rate Flow rate ratio EE (%) (phage: lipid)1 3 mL / min 2:1 67.52 3 mL / min 2:1 75.913 3 mL / min 2:1 69.47Mean 70.96 ± 3.59Table 7b (neutral lipid) PEV 31Group Total flow rate Flow rate ratio EE (%) (phage: lipid)1 3 mL / min 2:1 18.752 3 mL / min 2:1 22.223 3 mL / min 2:1 16.65Mean 19.21 ± 2.30Testing other phage strains using SA lipid formulationPhage type Total flow rate Flow rate ratio EE (%)(phage: lipid)PEV1 3 mL / min 2:1 80.82 ± 2.28PEV31 3 mL / min 2:1 88.23 ± 1.10Dobby 3 mL / min 2:1 67.67 ± 1.52NQBD 3mL / min 2:1 68.08 ± 2.67DOTAP and dicetyl phosphate (DCP) charged lipid formulationsLipid formulations• Cationic lipid formulation:• Phages: PEV31 and PEV1 suspended in pH 9.5 MQ water. The original stock titre is ~109pfu / mL.• Charged lipids in etOH, concentration 20 mg / mL.PC, cholesterol, Tween 80 and DOTAP with molar ratio 7:3:1:2.• Anionic lipid formulation:• Phages: PEV1 suspended in pH 5 MQ water. The original stock titre is ~109pfu / mL.• Charged lipids in etOH, concentration 20 mg / mL.PC, cholesterol, Tween 80 and diacetyl phosphate (DCP) with molar ratio 7:3: 1:2.(DOTAP cationic charged lipid) PEV 1Group Total flow rate Flow rate ratio EE (%)(phage: lipid)1 3 mL / min 3:1 91.58 ± 1.762 3 mL / min 2:1 90.49 ± 1.683 3 mL / min 3:2 90.44 ± 0.58(DOTAP cationic charged lipid) PEV 31Group Total flow rate Flow rate ratio EE (%)(phage: lipid)1 3 mL / min 3:1 87.86 ± 1.272 3 mL / min 2:1 90.18 ± 1.53 3 mL / min 3:2 83.10 ± 0.68(DOTAP cationic charged lipid) encapsulating PEV1Group Total Flow rate Size (nm) pdi Zeta flow rate ratio potential (phage: (mV) lipid)1 3 mL / min 3:1 703.5 ± 0.21 ± 0.02 -4.19 ± 0.652.852 3 mL / min 2:1 693.3 ± 9.7 0.19 ± 0.03 -1.99 ± 0.993 3 mL / min 3:2 735.4 ± 9.8 0.22 ± 0.02 -5.41 ± 0.69(DOTAP cationic charged lipid) encapsulating PEV31Group Total flow Flow rate Size (nm) pdi Zeta rate ratio potential (phage: (mV) lipid)1 3 mL / min 3:1 130.3 ± 2.3 0.18 ± 0.01 -4.57 ± 1.902 3 mL / min 2:1 142.3 ± 5.0 0.19 ± 0.01 -5.50 ± 2.013 3 mL / min 3:2 169.2 ± 8.5 0.21 ± 0.01 -3.55 ± 1.09(DOTAP cationic charged empty lipid particle) no phageGroup Total flow Flow rate Size (nm) pdi Zeta rate ratio potential (phage: (mV) lipid)1 3 mL / min 3:1 71.00 ± 0.155 ± 46.0 ± 4.01.57 0.0172 3 mL / min 2:1 75.05 ± 0.16 ± 0.03 48.7 ± 5.11.053 3 mL / min 3:2 72.63 ± 0.16 ± 0.03 47.8 ± 7.41.22(DCP anionic charged lipid) PEV 1Group Total flow Flow rate ratio (phage: EE (%)rate lipid)1 3 mL / min 3:1 56.10 ± 3.282 3 mL / min 2:1 59.83 ± 2.713 3 mL / min 3:2 55.69 ± 4.49(neutral lipid) PEV 1Group Total flow Flow rate ratio (phage: EE (%)rate lipid)1 3 mL / min 3:1 11.64 ± 1.162 3 mL / min 2:1 14.35 ± 1.733 3 mL / min 3:2 15.30 ± 1.44(DCP anionic charged) encapsulating PEV1Group Total flow Flow rate ratio Size (nm) pdi Zeta rate (phage: lipid) potential (mV) 1 3 mL / min 3:1 996.0 ± 2.00 0.671 ± 0.028 -6.5 ±2.02 3 mL / min 2:1 710.2 ± 7.56 0.246 ± 0.035 -7.9 ± 1.13 3 mL / min 3:2 819.4 ± 0.079 ± 0.027 -9.2 ± 0.815.50(DCP anionic charged) no phageGroup Total flow Flow rate ratio Size (nm) pdi Zeta rate (phage: lipid) potential (mV)1 3 mL / min 3:1 397.8 ± 7.5 0.283 ± 0.013 -72.8 ±2.52 3 mL / min 2:1 109.0 ± 1.1 0.378 ± 0.018 -82.6 ± 5.03 3 mL / min 3:2 463.6 ± 14.5 0.371 ± 0.033 -65.0 ±4.5Temperature trigger release• To determine the release profile of the DOTAP lipid formulation encapsulating phages over 24 hours at 37°C.MethodIncubation conditions:• Temperature: place the vials containing sample and pH 7.4 PBS (1:5) in a water bath with 37°C on a heat plate.• Ensure the stirrer is set at a gentle speed to avoid disrupting the liposome.Sampling at defined time points:• Sample at defined time intervals (0, 1, 2, 4, 6, 12, and 24 hours).• At each time point, use pipette to carefully withdraw a small amount of the release medium from the vial (0.2 mL) for plaque assay.• After sampling, replace the volume of the release medium with fresh PBS to maintain the total volume and prevent concentration effects from altering the release profile (i.e., maintain sink conditions).Control experiments:• Free phage control: prepare a separate vial with the free phage (not encapsulated) in PBS under the same conditions (including magnetic stirring) to compare.• Complete release control: at the end of the experiment, treat a vial with bile salt to disrupt the liposomes and release the remaining encapsulated phage.Results are shown in Fig. 8.Results indicate that PEV1 achieved a cumulative release of 100% at 12 hours, while PEV31 reached this milestone at 8 hours.Results: DOTAPCharacteristics of phage-encapsulated liposomesAXF-mini:[000362] Liposomes formulated with DOTAP were larger when encapsulating phages compared to those with neutral lipids. The average size across different FRR was 250 nm for PEV31 and 710 nm for PEV1, whereas neutral lipids measured -182 nm and -232 nm for PEV31 and PEV1, respectively (Fig. 12a). Charged lipid formulations produced liposomes with narrower PDI values compared to their neutral counterparts. PDIs ranged from 0.17 to 0.22 for PEV31 and 0.16 to 0.24 for PEV1 in charged lipid formulations, while neutral lipid formulations showed broader ranges of 0.17 to 0.47 for PEV31 and 0.23 to 0.55 for PEV1 (Fig. 12d). Zeta potential analysis revealed that neutral liposomal phage samples had a negative charge of approximately -20 mV. DOTAP-based liposomal samples showed slightly positive values of -6 mV for PEV31 and -4 mV for PEV1 (Fig. 13a).CIJ mixer:[000363] For DOTAP formulations, particle sizes ranged from 176 to 786 nm for PEV31 and 463 to 1000 nm for PEV1. In comparison, neutral lipid formulations were smaller, with sizes of 156 to 233 nm for PEV31 and 166 to 315 nm for PEV1 (Fig. 12b). The PDIs varied across formulations, with values of 0.08 to 0.47 for PEV31 and 0.14 to 0.59 for PEV1, reflectingrelatively uniform size distributions (Fig. 12e). Zeta potentials showed that DOTAP liposomal phage samples were slightly positive, measuring 18.2 ± 2.70 mV for PEV31 and 15.7 ± 3.59 mV for PEV1. In contrast, neutral lipid formulations encapsulating phages exhibited negative zeta potential values of approximately -15 mV (Fig. 13b).Microfluidic chip:[000364] Under fixed TFR and FRR conditions, DOTAP-based liposomal phage samples had particle sizes of 225 ± 44.1 nm for PEV31 and 688 ± 138.8 nm for PEV1, which were larger than those produced with neutral lipid formulation (Fig. 12c). The PDIs were consistent and narrow, measuring 0.24 ± 0.01 for PEV31 and 0.21 ± 0.07 for PEV1 (Fig. 12f). Zeta potential measurements indicated slightly positive surface charges for DOTAP encapsulated phage formulation, with values of 14.0 ± 1.33 mV for PEV31 and 13.7 ± 2.40 mV for PEV1, compared to the negative values of -12.26 ± 4.38 mV (PEV31) and -19.74 ± 1.10 mV (PEV1) recorded for neutral lipid formulation (Fig. 13c).Encapsulation efficiency and phage viability[000365] Using AXF-mini, neutral lipid formulation showed EE% below 15% for both PEV31 and PEV1 (Fig. 14a). In contrast, DOTAP-based formulations significantly improved encapsulation, reaching maximum values of 90.2 + 1.50% for PEV31 and 91.6 ± 1.76% for PEV1. The highest EE% was achieved at a 2:1 FRR, followed by 3:1 and 3:2, with no consistent trend observed. Titer reductions remained minimal, below 0.1 log₁₀ for both phages (Fig. 15a). In the CIJ mixer system, DOTAP formulations achieved EE% of up to 84.2 + 2.24% for PEV31 and 76.6 ± 2.77% for PEV1, while neutral lipid resulted in efficiencies of ≤ 18% (Fig. 14b). Phage viability was maintained, with titer reductions below 0.1 log₁₀ for PEV31 and approximately 0.2 log₁₀ for PEV1 (Fig. 15b). Similarly, in the microfluidic system, DOTAP-based liposomes demonstrated EE% of 84.4 + 4.39% for PEV31 and 76.3 ± 4.66% for PEV1, whereas neutral lipid resulted in significantly lower efficiencies of 19.2 + 2.81% and 16.6 + 4.92% for PEV31 and PEV1, respectively (Fig. 14c). Titer reductions were minimal (~0.05 log10) for both phages (Fig.15c).Phage morphology[000366] TEM images showed PEV31, a podovirus having an isometric hexagonal head (~65 nm) with a short tail, while PEV1, from the myovirus family, featuring a similar head (~65 nm) and a long contractile tail (-120 nm) Fig. 1 and 2). In the lipid-encapsulated samples, phages were observed to be either fully or partially encapsulated within liposomes, typically with one phage per liposome due to their comparable sizes. Empty liposomes (Fig. 1c) and non-encapsulated phages were also visible (Fig. 1e and Fig. 1f), with the latter often attached to liposome surfaces or forming aggregates with other phages. Contact mode AFM imaging (Fig.11) of a drop-casted liposome phage suspension further confirmed phage encapsulation within liposome vesicle. AFM-IR spectra acquired from the encapsulated phages showed characteristic peaks for amide I and amide II bands at 1650 and 1550 cm-1, respectively. Peaks related to DSPC were identified at 1734 and 1468 cm-1(Crea, F., Vorkas, A., Redlich, A., Cruz, R., Shi, C., Trauner, D., Lange, A., Schlesinger, R., Heberle, J., 2022. Photoactivation of a Mechanosensitive Channel. Front Mol Biosci 9, 905306), while signals for Tween 80 appeared at 1173 and 1085 cm-1(Fu, X., Kong, W., Zhang, Y., Jiang, L., Wang, J., Lei, J., 2015. Novel solid-solid phase change materials with biodegradable trihydroxy surfactants for thermal energy storage. RSC Adv. 5, 68881-68889). DOTAP was associated with peaks at 1740, 1489 and 1220 cm-1(Miatmoko, A., Asmoro, F. H., Azhari, A. A., Rosita, N., Huang, C. S., 2023. The effect of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) Addition on the physical characteristics of beta-ionone liposomes. Sci Rep 13, 4324), and cholesterol signals at 1378 and 1438 cm-1(Gupta, U., Singh, V. K., Kumar, V., Khajuria, Y., 2014. Spectroscopic studies of cholesterol: fourier transform infra-red and vibrational frequency analysis. Mater. Focus 3, 211-217) (Fig.16a-c).Discussion[000367] This study presents a versatile liposomal encapsulation approach designed to accommodate diverse phage morphotypes while ensuring compatibility across multiple manufacturing systems. The formulation strategy was evaluated using two P. aeruginosa phages with distinct morphologies: short-tailed podovirus PEV31, and a long-tailed myovirus PEV1. Using a cationic lipid formulation containing DSPC:cholesterol: DOTAP: Tween 80 (7:3:2: 1 molar ratio), high encapsulation efficiencies of up to 90% were achieved across three micromixing systems — AXF-mini Pathfinder, CM, and microfluidic chip — significantly exceeding the -50% values typically reported for encapsulating phages using neutral lipids (Cinquerrui, S., Mancuso, F., Vladisavljevic, G. T., Bakker, S. E., Malik, D. J., 2018. Nanoencapsulation of Bacteriophages in Liposomes Prepared Using Microfluidic Hydrodynamic Flow Focusing. Front Microbiol 9, Leung, S. S. Y., Morales, S., Britton, W., Kutter, E., Chan, H. K., 2018. Microfluidic-assisted bacteriophage encapsulation into liposomes. Int J Pharm 545, 176-182). Importantly, the method preserved phage viability with negligible titer loss (<0.2 log ) (Fig.15) and consistently produced nanosized liposomes (<1 pm) (Fig. 12), addressing key challenges in phage encapsulation and demonstrating the robustness of the formulation.[000368] Visual Demonstration and Electrostatic Interactions. TEM images provided clear visual evidence of successful phage encapsulation within liposomal nanoparticles. For PEV1,two encapsulation states were observed: (1) complete encapsulation of both the capsid and tail within the lipid bilayer vesicular structure, and (2) partial encapsulation where only the capsid was enveloped. The short tail PEV31 exhibited consistent full encapsulation due to its compact architecture. The majority of liposomes contained a single phage, attributed to the comparable sizes of the phages and liposome carriers.[000369] The success of the Applicant’s encapsulation strategy stems from the electrostatic interactions between phages and the liposomal carriers. Zeta potential measurements showed that empty charged liposomes displayed a strongly positive zeta potential (56.39 ± 5.52 mV) (Fig. 7a), confirming the incorporation of cationic lipids (DOTAP) into the lipid bilayer. In contrast, pure phages at pH 9.5 exhibited negative surface charge (PEV31: -28.83 ± 0.31 mV; PEV1: -27.37 ± 0.86 mV) (Fig. 7b). This is probably due to the exposure of acidic amino acid residues on their capsid surfaces under alkaline conditions, which aligns with previous reports on pH-dependent charge distributions on phage surfaces (Nap, R. J., Bozic, A. L., Szleifer, I., Podgornik, R., 2014. The role of solution conditions in the bacteriophage PP7 capsid charge regulation. Biophys J 107, 1970-1979.).[000370] The significant charge disparity between the negatively charged phages and positively charged liposomes created strong electrostatic attraction that facilitates encapsulation (Tenchov, R., Bird, R., Curtze, A. E., Zhou, Q., 2021. Lipid Nanoparticles horizontal line From Liposomes to mRNA Vaccine Delivery, a Landscape of Research Diversity and Advancement. ACS Nano 15, 16982-17015). This mechanism contrasts with traditional neutral lipid formulations, which rely on passive entrapment and often result in lower encapsulation efficiencies (Cinquerrui, S., Mancuso, F., Vladisavljevic, G. T., Bakker, S. E., Malik, D. J., 2018. Nanoencapsulation of Bacteriophages in Liposomes Prepared Using Microfluidic Hydrodynamic Flow Focusing. Front Microbiol 9, Leung, S. S. Y., Morales, S., Britton, W., Kutter, E., Chan, H. K., 2018. Microfluidic-assisted bacteriophage encapsulation into liposomes. Int J Pharm 545, 176-182). Notably, the similar zeta potentials observed for PEV31 and PEV1 suggest a uniform charge profile across distinct morphotypes, which could have broader implications for developing universal encapsulation strategies.[000371] Encapsulation Efficiency, Phage Viability, and Morphotype Compatibility. The Applicant’s electrostatic-driven strategy significantly improved encapsulation efficiency (EE%) across various mixing systems and phage morphotypes. This is the first report on using AXF system for phage encapsulation, and the Applicant’s DOTAP formulations achieved maximum EE% values >90% for both PEV31 and PEV1, compared to <15% for neutral lipids. The Applicant observed a peak in encapsulation efficiency at a 2:1 FRR, which highlights the importance of precise control over AXF system parameters in maximizing phage encapsulation(Hussain, M., Binici, B., O'Connor, L., Perrie, Y., 2024. Production of mRNA lipid nanoparticles using advanced crossflow micromixing. J Pharm Pharmacol). The CIJ mixer and microfluidic system also showed marked improvements with charge driven encapsulation, reaching EE% of up to 84.15 ± 2.24% and 84.41 ± 4.39%, respectively, significantly outperforming neutral lipid formulations (< 20%). This consistency across membrane-based (AXF), turbulent (CIJ) and laminar (microfluidics) mixing regimes demonstrates the versatility of the Applicant’s charge-driven encapsulation strategy. Critically, phage viability was well preserved in all systems (<0.1 logic titer reduction). These results support the Applicant’s formulation-centric approach, which leverages electrostatic assembly under ambient conditions while avoiding harsh processing steps (e.g., heat, extrusion, sonication) typical of traditional encapsulation methods (Colom, J., Cano-Sarabia, M., Otero, J., Cortes, P., Maspoch, D., Llagostera, M., 2015. Liposome-Encapsulated Bacteriophages for Enhanced Oral Phage Therapy against Salmonella spp. Appl Environ Microbiol 81, 4841-4849, Leung, S. S. Y., Morales, S., Britton, W., Kutter, E., Chan, H. K., 2018. Microfluidic-assisted bacteriophage encapsulation into liposomes. Int J Pharm 545, 176-182). By efficiently encapsulating diverse phages with different tail lengths, the Applicant’s electrostatic interaction driven strategy can potentially simplify the liposomal formulation process for therapeutic applications (Chan et al., 2013; Li et al., 2021).[000372] Nanoparticle Characteristics and Advanced Analytical Confirmation. In the AXF-mini system, DOTAP formulations produced larger liposomes when encapsulating phages compared to neutral lipid formulations and blank liposomes. The average size was -250 nm for PEV31 -710 nm for PEV1, with the size difference likely attributed to the longer tail structure of PEV1. Liposomes with charged lipids exhibited slightly lower PDIs (0.17-0.24) compared to neutral lipids (0.17-0.55), indicating improved size uniformity. Similarly, under fixed conditions in the microfluidic chip (total flow rate of 3mL / min and flow rate ratio of 2:1), DOTAP-based encapsulated phages consistently maintained nanoscale liposomes with narrow PDIs.[000373] In the CIJ mixer, which operates at a fixed flow rate ratio (FRR) of 1:1, the Applicant evaluated the effect of four different total flow rate (TFR) values on particle characteristics. For DOTAP formulations, particle sizes ranged from 176 to 786 nm for PEV31 and from 463 to 1000 nm for PEV1. Higher TFR produced smaller particles, likely due to increased shear forces during mixing — a finding consistent with a previous study showing that higher TFR enhances mixing efficiency and promotes rapid assembly of lipid bilayer into smaller liposomes (Zheng, H., Tao, H., Wan, J., Lee, K. Y., Zheng, Z., Leung, S. S. Y., 2022. Preparation of Drug-Loaded Liposomes with Multi-Inlet Vortex Mixers. Pharmaceutics 14). PDIs ranged from 0.08-0.59, with lower values observed at higher TFRs, a trend that aligns with previous observation (Kulkarni, J. A., Tam, Y. Y. C., Chen, S., Tam, Y. K., Zaifman, J., Cullis, P. R., Biswas, S., 2017. Rapid synthesis of lipid nanoparticles containing hydrophobic inorganic nanoparticles.Nanoscale 9, 13600-13609). Notably, the highest TFR did not result in the greatest encapsulation efficiency: while PEV1 achieved peak encapsulation efficiency at 17.5 mL / min, PEV31 exhibited high efficiencies at both 8 mL / min and 17.5 mL / min. These results suggest that conditions favoring the formation of smaller, more uniform particles do not necessarily optimize phage encapsulation, highlighting the need for fine-tune multiple parameters in turbulent mixing system (He, H., Lu, Y., Qi, J., Zhu, Q., Chen, Z., Wu, W., 2019. Adapting liposomes for oral drug delivery. Acta Pharm Sin B 9, 36-48.)[000374] Zeta potential measurements further confirmed phage encapsulation across all systems. Phage-encapsulated liposomes displayed slightly positive values (~5 mV in AXF; ~18.2 mV in CIJ and ~15 mV in microfluidic system), reflecting effective neutralization of the phages' negative charge by cationic lipids. This slight positive charge may also promote mucoadhesion and enhance cellular uptake by promoting interactions with negatively charged cell membranes (Dabbagh, A., Abu Kasim, N. H., Yeong, C. H., Wong, T. W., Abdul Rahman, N., 2018. Critical Parameters for Particle- Based Pulmonary Delivery of Chemotherapeutics. J Aerosol Med Pulm Drug Deliv 31, 139-154, Hu, H., Yang, C., Li, M., Shao, D., Mao, H. Q., Leong, K. W., 2021. Flash Technology-Based Self-Assembly in Nanoformulation: From Fabrication to Biomedical Applications. Mater Today (Kidlington) 42, 99-116, Yang, B., Geng, S. Y., Liu, X. M., Wang, J. T., Chen, Y. K., Wang, Y. L., Wang, J. Y., 2012. Positively charged cholesterol derivative combined with liposomes as an efficient drug delivery system, in vitro and in vivo study. J. Soft Matter s, 518-525).[000375] Advanced nanochemical analysis using AFM-IR further corroborated phage encapsulation within lipid vesicles. Consistent with the Applicant’s TEM observations, the AFM images visually confirmed phages encapsulated within liposomes, while the corresponding IR spectra provided molecular-level evidence of lipid-phage colocalization. The colour grids indicate the regions from which IR signals were collected. Specifically, the detection of amide I and II bands (1650 and 1550 cm-1) confirms the presence of phage proteins (Cao, Y., Khanal, D., Kim, J., Chang, R. Y. K., Byun, A. S., Morales, S., Banaszak Holl, M. M., Chan, H. K., 2023. Stability of bacteriophages in organic solvents for formulations. Int J Pharm 646, 123505). Since neither PEV31 nor PEV1 phages contain lipid components, the peaks at 1734 and 1468 cm-1are attributed to DSPC. Additionally, signals at 1740, 1489, and 1220 cm-1indicate the presence of DOTAP, while peaks corresponding to Tween 80 (1173 and 1085 cm-1) and cholesterol (1378 and 1438 cm-1) further validate the integrity of the lipid formulation (Crea, F., Vorkas, A., Redlich, A., Cruz, R., Shi, C., Trauner, D., Lange, A., Schlesinger, R., Heberle, J., 2022. Photoactivation of a Mechanosensitive Channel. Front Mol Biosci 9, 905306, Fu, X., Kong, W., Zhang, Y., Jiang, L., Wang, J., Lei, J., 2015. Novel solid-solid phase change materials with biodegradable trihydroxy surfactants for thermal energy storage. RSC Adv. 5, 68881-68889, Gupta, U., Singh,V. K., Kumar, V., Khajuria, Y., 2014. Spectroscopic studies of cholesterol: fourier transform infrared and vibrational frequency analysis. Mater. Focus 3, 211-217, Miatmoko, A., Asmoro, F. H., Azhari, A. A., Rosita, N., Huang, C. S., 2023. The effect of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) Addition on the physical characteristics of beta-ionone liposomes. Sci Rep 13, 4324).[000376] Scalability and Equipment-Independency. A key strength of the Applicant’s charge-driven encapsulation strategy is its independence from specific equipment, enabling consistent and high-quality output across platforms. The platforms the Applicant employed in its study all played a distinct role in the production pipeline. For example, while microfluidic chips can be engineered to GMP-scale, they are best suited for small-batch applications that demands precise control over particle size and uniformity, making them ideal for proof-of-concept studies (Webb, C., Forbes, N., Roces, C. B., Anderluzzi, G., Lou, G., Abraham, S., Ingalls, L., Marshall, K., Leaver, T. J., Watts, J. A., Aylott, J. W., Perrie, Y., 2020. Using microfluidics for scalable manufacturing of nanomedicines from bench to GMP: A case study using protein-loaded liposomes. Int J Pharm 582, 119266.). In contrast, the AXF system, which employs crossflow membrane micromixing, is optimized for high-volume batch production and well-suited for large-scale manufacturing (Hussain, M., Binici, B., O'Connor, L., Perrie, Y., 2024. Production of mRNA lipid nanoparticles using advanced crossflow micromixing. J Pharm Pharmacol). The CIJ mixer, based on flash nanoprecipitation (FNP) principle, supports continuous workflows and is valuable for scaling up production at intermediate volumes (Subraveti, S. N., Wilson, B. K., Bizmark, N., Liu, J., Prud'homme, R. K., 2024. Synthesizing Lipid Nanoparticles by Turbulent Flow in Confined Impinging Jet Mixers. J Vis Exp). Although the CIJ mixer itself may not be directly applicable to industrial-scale manufacturing, it shares the same FNP principle with larger systems like multiinlet vortex mixer (MIVM). This commonality allows the Applicant’s charge-assisted encapsulation strategy to be seamlessly adapted to the MIVM, thereby facilitating efficient scale-up to industrial volumes while maintaining consistent nanoparticle characteristics (Feng, J., Markwaiter, C. E., Tian, C., Armstrong, M., Prud'homme, R. K., 2019. Translational formulation of nanoparticle therapeutics from laboratory discovery to clinical scale. J Transl Med 17, 200).[000377] This study presents a versatile, electrostatically driven liposomal formulation for phage encapsulation that is compatible with multiple manufacturing systems. By employing the cationic lipid (DOTAP) and tuning phage surface charge under alkaline condition, the Applicant’s strategy achieved high encapsulation efficiencies (up to 90% for PEV31 and 91% for PEV1) across AXF-mini, CIJ, and microfluidic systems — significantly outperforming conventional neutral lipid-based methods (< 50%) while preserving excellent phage viability (<0.2 Iog10 titer loss). Moreover, the formulation effectively encapsulated structurally distinct phages — PEV31 (short-tailed podovirus) and PEV1 (long-tailed myovirus) — producing nanosized liposomalphages (< 1000 nm) with slightly positive zeta potentials (5-18 mV). TEM and AFM-IR analyses provided strong evidence for phage encapsulation and lipid-phage colocalization. This charge-assisted encapsulation strategy addresses key challenges in phage therapy by achieving high encapsulation efficiency, preserving phage viability, and accommodating diverse morphotypes, while also demonstrating promising scalability for industrial applications.Summary[000378] Liposome-encapsulated bacteriophages offer promising potential for targeted antimicrobial therapy against multidrug-resistant infections, by enhancing phage stability and delivery. Current encapsulation methods face challenges due to low efficiency (<50%), phage inactivation, and limited scalability for industrial production.[000379] To overcome these challenges, this study presents an electrostatically driven encapsulation method to produce phage liposomal formulations using cationic lipids (DOTAP) in various scalable micromixing systems (AXF™mini, confined impinging jet (CM), microfluidic chip). Encapsulation was evaluated by mixing a lipid-carrying organic stream (ethanol) with a phage-containing aqueous stream at flow rate ratios (FRR) of 3:1, 2:1, and 3:2 in AXF™mini, total flow rates (TFR) of 5-35 mL / min in Cl J, and a fixed FRR of 2:1 with a TFR of 3 mL / min in the microfluidic chip.[000380] The electrostatic interactions between the positively charged lipids and negatively charged phages significantly improved the encapsulation efficiency to 90-91% for podovirus PEV31 (short-tailed) and myovirus PEV1 (long-tailed) while maintaining phage viability with <0.2 log reduction in titre. This encapsulation approach suited both phage morphotypes, producing uniformly sized liposomal phages (< 1000 nm, PDI <0.3) with a slightly positive zeta potential. Structural observation using transmission electron microscopy and atomic force microscopy infrared spectroscopy confirmed phage encapsulation within intact vesicles and lipid-phage colocalization.[000381] This versatile formulation strategy addresses key challenges in phage therapy by achieving high encapsulation efficiency, accommodating diverse phage morphotypes, and preserving viability while offering potential scalability for industrial applications.[000382] This study presents a versatile, electrostatically driven liposomal formulation for phage encapsulation that is compatible with multiple manufacturing systems. By employing the cationic lipid (DOTAP) and tuning phage surface charge under alkaline condition, the Applicant’s strategy achieved high encapsulation efficiencies (up to 90% for PEV31 and 91% for PEV1) across AXF-mini, CM, and microfluidic systems — significantly outperforming conventionalneutral lipid-based methods (< 50%) while preserving excellent phage viability (<0.2 log titer loss). Moreover, the formulation effectively encapsulated structurally distinct phages — PEV31 (short-tailed podovirus) and PEV1 (long-tailed myovirus) — producing nanosized liposomal phages (< 1000 nm) with slightly positive zeta potentials (5-18 mV). TEM and AFM-IR analyses provided strong evidence for phage encapsulation and lipid-phage colocalization. This charge-assisted encapsulation strategy addresses key challenges in phage therapy by achieving high encapsulation efficiency, preserving phage viability, and accommodating diverse morphotypes, while also demonstrating promising scalability for industrial applications.Example 9Objectives[000383] Using PEV2 and PEV40 (fragile phages)1. Evaluate the protective effect of lipid encapsulation during nebulization:[000384] Assess the viability and stability of lipid-encapsulated phages after jet and mesh nebulization.[000385] Compare the survival rates of encapsulated phages to non-encapsulated (free) phages post-nebulization.2. Assess aerosolisation characteristics of the lipo-phage formulations:[000386] Measure aerosol (droplet size distribution, respirable fraction etc) of the nebulised formulations.[000387] Ensure that the aerosolised particles are within the optimal size range for pulmonary delivery.Materials and methodsBacteriophages[000388] Two types of Pseudomonas lytic phage, a podovirus (PEV2, short-tailed) and a myovirus (PEV40, long-tailed) were originally isolated from the sewage treatment plant in Olympia (WA, USA) by the Kutter Lab (Evergreen Phage Lab). Phage isolation utilized Pseudomonas aeruginosa dog-ear strain PA 237, which also served as the host for titer assessment. Phage purification was conducted as described Carlson, K., 2005. Working with bacteriophages: common techniques and methodological approaches. Bacteriophages: biologyand applications 1, 437-494. Briefly, amplified phages were filtered using a 0.22 μm PES syringe filter, treated with 10 pg / mL DNase and RNase at 37 °C for 30 min, and purified via ultrafiltration at 4000 x g at 4 °C. Samples were resuspended in phosphate buffered saline (PBS) at pH 7.2, and endotoxins removed with EndoTrapOHD prior to anion-exchange (AEX) chromatography using a CIMmultus™ DEAE column. Phages were eluted with NaCI buffers optimized for purity, followed by endotoxin cleanup with EndoTrapOHD. Endotoxin levels were quantified with the ToxinSensor™ LAL assay, and purified phages PEV2 and PEV40 (1 x 1010pfu / mL) were stored in PBS.Lipid encapsulated phages[000389] A mixture of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, Tween 80, and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) at molar ratio of 7:3: 1:2 was dissolved in absolute ethanol with a total solid content of 20 mg / mL. The solution was bath-sonicated for 10 min under ambient condition. For lipid-encapsulated phage preparation, a confined impinging jet mixer (Cl J) — previously previously described for nanoparticle synthesis (Chiou, H., Chan, H. K., Prud'homme, R. K., Raper, J. A., 2008. Evaluation on the use of confined liquid impinging jets for the synthesis of nanodrug particles. Drug Dev Ind Pharm 34, 59-64) — was employed. The lipid mixture was injected into one inlet channel of the Cl J, while the phage suspension was introduced into the second inlet channel. The resulting liposome was collected via the outlet tube into a scintillation vial. The total flow rate (TFR) was maintained at 17.5 mL / min. All experiments were conducted at room temperature and triplicated.Ethanol removal[000390] Ethanol-free liposome-phage suspensions were obtained by ultracentrifugation at 10000xg for 30 min at 4 °C. The supernatant was decanted, and the pellet was resuspended in PBS. This process was repeated two times to minimise residual ethanol content.Liposomal size measurement[000391] The size and PDI of the liposomes were measured using dynamic light scattering (DLS) on a Zetasizer Nano-ZS (Malvern Instruments, UK) with a 633 nm He / Ne laser and 4.0 mW power to report the intensity mean diameter (Z-average). Liposomal phage suspensions were diluted 1:10 in PBS for analysis of size distribution. Measurements were conducted in triplicate at 25 °C.Zeta potential[000392] The zeta potential of liposomes was measured by Laser Doppler Electrophoresis using the Zetasizer Nano ZS (Malvern Instruments, UK) and calculated with the Helmholtz-Smoluchowski equation. Samples were diluted in PBS to a lipid concentration of 0.2 mg / mL and analyzed in a folded capillary cell. Measurements were performed in triplicate at 25 °C.Transmission electron microscopy (TEM)[000393] Morphology and structure of phage-loaded liposomes before and after nebulisation were examined by transmission electron microscopy (TEM). Similar to the previous protocol (Cao, Y., Khanal, D., Kim, J., Chang, R. Y. K., Byun, A. S., Morales, S., Banaszak Holl, M. M., Chan, H. K., 2023. Stability of bacteriophages in organic solvents for formulations. Int J Pharm 646), the Applicant employed an FEI Tecnai T12 microscope (FEI, USA) with a 4k x 4k CCD camera. Samples were negatively stained on glow-discharged carbon-coated copper grids (GSCU150CC-50, Proscitech, Australia) to reduce hydrophobicity. For staining, 10 pL drops of the sample, 2% w / v uranyl acetate, and Milli-Q water were placed on parafilm. Grids were immersed in the sample for 2 min, blotted, stained for 10 sec, rinsed in water for 10 sec, and airdried for 3 min before imaging.Plaque assay[000394] The Miles-Misra surface droplet technique (Carlson, K., 2005. Working with bacteriophages: common techniques and methodological approaches. Bacteriophages: biology and applications 1, 437-494) was used to quantify viable phages in original and lysed liposome samples before and after nebulisation. Serial 1:10 dilutions were prepared by mixing 20 pL of the sample with 180 pL PBS. A suspension of ~2 x 109CFU host bacteria in 5 mL molten soft agar (0.4% Amyl agar, 48 °C) was overlaid onto a nutrient agar plate (1.5% Amyl agar and nutrient broth). Diluted phage samples (10 pL) were spotted onto the agar in triplicate, air-dried, and incubated at 37 °C overnight. Plaques within the number range of 3-30 were counted to calculate phage viability.The encapsulation efficiency of the phages[000395] Phage encapsulation efficiency (EE) was calculated using the formula: EE (%) = 100 - (Cfree / Ctotai)x100, where Ctotai is the total phage titer, and Cfree is the free phage concentration. Cfree was measured via plaque assay on the liposome-encapsulated phage suspension, while Ctotai was determined by lysing 0.5 mL of the suspension with 0.5 mL bile salts (Sigma-Aldrich, NSW, Australia) (50 mM) before the plaque assay. Control tests confirmed that this bile salt concentration did not affect phage infectivity across titers ranging from 101to 101° pfu / mL.Phage nebulisation[000396] A 3 mL phage stock (free phage or liposome encapsulated phage) suspension was nebulized using two types of nebulizers: a jet nebulizer (PARI LC Sprint, Pari Respiratory Equipment, USA) and a vibrating mesh nebulizer (eFlow Rapid, Pari Respiratory Equipment, USA). Nebulized aerosols were collected using a method adapted from previous studies (Astudillo, A., Leung, S. S. Y., Kutter, E., Morales, S., Chan, H. K., 2018. Nebulization effects on structural stability of bacteriophage PEV 44. Eur J Pharm Biopharm 125, 124-130, Cipolla, D. C. G., I., 1994. Method for Collection of Nebulized Proteins, in Formulation and Delivery of Proteins and Peptides. American Chemical Society, 343-352), designed for nebulized protein collection (Fig. 1). The aerosol was drawn into an ice-cooled test tube inside an aspiration flask via Tygon tubing connected to a 2.0 mL plastic pipette, operating at 8 L / min. For the mesh nebulizers, the apparatus functioned without compressed air. Samples collected in the test tube were analyzed via TEM and plaque assay. All nebulization runs were performed in triplicate. Aerosol collection efficiency was assessed by the volume recovered in the test tube, with aerosols deposited in tubing washed and included. Recovery was >80% for the air-jet nebulizer and >90% for the mesh nebulizers. The lower efficiency of the air-jet nebulizer was attributed to fine aerosol droplets escaping in the airstream. Despite this, the apparatus effectively captured the majority of aerosols generated by all nebulizers.Statistical analysis[000397] Statistical analysis was conducted using one-way analysis of variance (ANOVA) and unpaired two-sample t-tests at a 95% confidence level to assess differences in liposome size, zeta potential, titer reduction, and encapsulation efficiency. Statistical significance was determined for p values <0.05.Aerosol droplet size distribution[000398] To assess the particle size distribution (PSD) of nebulized phage aerosols produced from the nebulizer devices, laser diffraction was employed using a Spraytec system (Malvern Instruments Ltd., UK). The Applicant conducted continuous nebulization for 60 seconds, taking measurements at one-second intervals. The data was then analyzed to determine the median diameter (D50) and span. The span was calculated using the formula (D90-D10) / D50, where D10, D50, and D90 represent the particle diameters at the 10th, 50th, and 90th percentiles of the distribution, respectively.Viable phage respirable fraction[000399] A multi-stage liquid impinger (MSLI, Copley, Nottingham, UK) running at 30 L / min was used to measure the total viable phage in aerosol droplets (<4.5 pm ) (Astudillo, A., Leung, S. S. Y., Kutter, E., Morales, S., Chan, H. K., 2018. Nebulization effects on structural stability of bacteriophage PEV 44. Eur J Pharm Biopharm 125, 124-130). The MSLI stages had cut-off diameters of 16.9 pm (stage 1), 9.3 pm (stage 2), 4.5 pm (stage 3), and 2.5 pm (stage 4), with the filter (stage 5) collecting particles <2.5 pm. During measurement, each impinger stage was pre-filled with 10 mL PBS, and the nebulizer was connected to the MSLI using a silicone adapter and a United States Pharmacopeia (USP) induction port. A 3 mL phage suspension was nebulized into the MSLI, and the adapter, USP throat, and filter stage were washed separately with 10 mL PBS to ensure complete recovery. Viable phages at each stage were quantified using plaque assays. The titers from stage 3, 4 and the filter stage were summed to determine the viable phage content in the respirable fraction (RF). The viable respirable fraction (VRF) was defined as the titers recovered from these two stages relative to the total titer initially loaded into the nebulizer.ResultsLipo_phage characterisationPhage viability and encapsulation efficiencyThe encapsulation efficiencies for PEV2 and PEV40 produced by the CIJ mixer were 77.9 ± 1.48%, 83.9 ± 4.23%, respectively:Total flow rate Flow rate ratio EE (%)EncapsulatedmL / minPhage8.75 1:1 77.9 ± 1.48PEV28.75 1:1 83.93 ±4.23PEV40Liposomal size distribution and zeta potential[000400] The liposomes entrapping short-tail phage PEV2 averaged 301 ±35.8 nm with a polydispersity index (PDI) of 0.28 ± 0.06, while those encapsulating long-tail phage PEV40 were larger at 651 ± 14.3 nm with a PDI of 0.37 ±0.12 (see table below). The z-average values were 70.2 ± 2.73 for PEV2, and 185 ± 6.94 for PEV40. The surface charges of the phage-encapsulated liposomes were near-neutral, with zeta potentials of 0.17 ± 0.04 (encapsulatedPEV2) and 0.90 ± 0.72 (encapsulated PEV40). In contrast, free phages showed negative zeta potentials of -19.3 ± 3.03 (PEV2) and -25.5 ± 2.22 (PEV40) (see table below).Phage samples Size (nm) Pdi Zeta potential (mV)70.2 ± 2.73 0.11 ± 0.03 -19.3 ± 3.03PEV2185 ± 6.94 0.19 ± 0.03 -25.5 ± 2.22PEV40Encapsulated Total Flow Size (nm) Pdi ZetaPhage samples flow rate rate potential mL / min ratio (mV)8.75 1:1 300.87 ±35.78 0.55 ±0.04 0.17±0.04 Lipo_PEV28.75 1:1 651.33 ± 14.26 0.79 ±0.07 0.90 ±0.72Lipo_PEV40[000401] Both phages PEV40 (long tail) and PEV2 (short tail) are efficiently encapsulated within lipid nanoparticles at an optimal nano size.[000402] Similar to our previous formulation, the lipid comprised of DSPC:cholesterol: DOTAP: Tween 80 with molar ratio 7:3:2: 1, respectively.Phage morphology[000403] Fig. 17 illustrates the intact and damaged morphologies of the phages. PEV2 had a short, non-contractile tail (~10nm) and a ~60nm icosahedral head, while PEV40 possessed a contractile tail (-140 nm) with a similarly sized head (-60 nm). Encapsulated phages were shown located within the larger size white vesicles along with smaller empty liposomes nearby.Effect of nebulisationStability comparison / Titer reduction[000404] Phage viability analysis (Fig. 9) showed that non-encapsulated PEV40 experienced mild titer loss of 0.67 logic in the vibrating mesh nebulization process, but it was substantially inactivated in the air jet nebulization (1.23 logic titer loss). In contrast, liposome-encapsulated PEV40 remained practically unaffected by both nebulization processes, with titer loss being less than 0.4 logic. Similarly, for liposome-encapsulated PEV2, both types of nebulizers had a negligible effect with titer reduction of <0.07 logic. Like PEV40, non¬ encapsulated PEV2 was significantly inactivated (1.1 logic titer loss) by jet nebulization, but with only little titer loss by vibrating mesh nebulization (-0.26 logic).[000405] Fig. 9 compares titer loss between lipid nanoparticle-encapsulated (LNP-encap) and non-encapsulated phages (Non-encap) PEV40 and PEV2, using jet and vibrating mesh nebulizers.[000406] Encapsulated phages show significantly less titer reduction, indicating effective protection during both nebulization methods. These suggest lipid nanoparticles may improve phage stability for respiratory delivery using nebulisation.Aerosol droplet size distribution / Particle size distribution of nebulised aerosols[000407] The geometrical sizes of aerosols generated by the two nebulizers are shown in the table below. The D50 of liposome-encapsulated PEV2 and PEV40 droplets produced by the jet nebulizer were 3.10 ± 0.05 pm and 2.98 ± 0.04 pm, respectively. The vibrating mesh nebulizer generated larger droplets of liposomal PEV2 (5.60 ± 0.13 pm) and PEV40 (5.83 ± 0.21 pm), which are still suitable for inhalation (Astudillo, A., Leung, S. S. Y., Kutter, E., Morales, S., Chan, H. K., 2018. Nebulization effects on structural stability of bacteriophage PEV 44. Eur J Pharm Biopharm 125, 124-130).Nebulisers Encapsulated D10 (pm) D50(pm) D90(pm)samplesAir-Jet Lipo_PEV2 1.29 ± 0.01 3.10 ± 0.05 7.61 ± 0.20Lipo_PEV40 1.27 ± 0.0.2 2.98 ± 0.04 7.08 ± 0.21Vibrating Lipo_PEV2 3.08 ± 0.08 5.60 ± 0.13 10.43 ± 0.54meshLipo_PEV40 3.11 ± 0.08 5.83 ± 0.21 11.23 ± 0.74Results are average values of triplication measurement. Variations between the three measurements are <5%[000408] Figs 9 and 10 present the viable phage respirable fraction, assessed via multi-stage liquid impinger (MSLI), and aerosol droplet size distribution, measured with Spraytec.In vitro aerosolization performance[000409] The deposition profiles, recovery, and VRF from the two nebulizers are shown in Fig. 18 and Fig. 19. At the same loading volume (3 mL suspension sample), the amount of viable encapsulated phages retained in the device was lower in the vibrating mesh nebulizer than with the jet nebulizer. The vibrating mesh-nebulized aerosols had significantly higher deposition at the lower stages (stage 2-4 and filter) of the impactor, compared with the jet-nebulized ones. As a result, the vibrating mesh nebulizer demonstrated superior performance, yielding high recoveries for both PEV2 (94.3±3.60%) and PEV40 (96.2±2.34%). In comparison, the jet nebulizer showed comparable recovery for PEV2(95.1±3.65%) but significantly lower recovery for PEV40 (48.9±0.84%). Regarding the VRF, which indicates the proportion of phages likely to reach the deep lungs, the vibrating mesh nebulizer produced 70.3±1.78% (PEV2) and 74.8±1.96% (PEV40), which are substantially higher than those from the jet nebulizer, 44.0±2.6% (PEV2) and (28.2±0.99% (PEV40).Discussion[000410] This study examined the potential of liposomal encapsulation to overcome phage inactivation during nebulization for pulmonary delivery. The Applicant investigated two P. aeruginosa phages with distinct morphologies: PEV2 (a short-tailed podovirus) and PEV40 (a long-tailed myovirus). The results demonstrated that liposome encapsulation effectively protected both types of phage during jet and vibrating mesh nebulization, maintaining their viability while producing aerosols suitable for pulmonary delivery. This approach addresses the critical challenge of preserving phage infectivity during the aerosolization process, a key hurdle in developing inhaled phage therapies.[000411] Liposome-encapsulated Phage Formulation. The Applicant first achieved reasonably high encapsulation efficiencies of approximately 80% for PEV2 and 90% for PEV40 using the Cl J mixing method. TEM images (Fig. 17) confirmed that each phage particle was encapsulated within a larger liposome. The liposome-encapsulated PEV2 had a smaller mean diameter (-300 nm) compared to PEV40 (-650 nm), reflecting their structural differences in tail length (Table 2.). The shift from negative to near-neutral zeta potentials is consistent with theshielding of the negative charge of the phages inside liposomes due to effective phage-lipid interactions, further supporting successful encapsulation of phages into the liposomal structure.[000412] Comparison of Titre Reduction: Encapsulated vs. Free Phages. Notably, titer reductions for liposome-encapsulated phages were minimal after each kind of nebulization, with <0.07 logic for PEV2 and <0.4 logic for PEV40, even in the high-shear environment of the jet nebulizer. In contrast, biological assays revealed that non-encapsulated phages experienced moderate titer reductions under vibrating mesh nebulization, but significant losses following jet nebulisation (up to 1.23 logic loss) (Fig. 9). TEM images (Fig. 17) corroborated these findings, showing different morphological alterations in non-encapsulated phages after nebulization, including capsid damage, tail detachment, and tail contraction in these unprotected phages. These results are consistent with our previous findings that phage structure and nebulization method play critical roles in infectivity loss (Carrigy, N. B., Chang, R. Y., Leung, S. S. Y., Harrison, M., Petrova, Z., Pope, W. H., Hatfull, G. F., Britton, W. J., Chan, H. K., Sauvageau, D., Finlay, W. H., Vehring, R., 2017. Anti-Tuberculosis Bacteriophage D29 Delivery with a Vibrating Mesh Nebulizer, Jet Nebulizer, and Soft Mist Inhaler. Pharm Res 34, 2084-2096, Cipolla, D., Shekunov, B., Blanchard, J., Hickey, A., 2014. Lipid-based carriers for pulmonary products: preclinical development and case studies in humans. Adv Drug Deliv Rev 75, 53-80, Fister, S., Robben, C., Witte, A. K., Schoder, D., Wagner, M., Rossmanith, P., 2016. Influence of Environmental Factors on Phage-Bacteria Interaction and on the Efficacy and Infectivity of Phage P100. Front Microbiol 7, 1152). Liposomal encapsulation could effectively protect the vulnerable structures of phages against mechanical damage during nebulization.[000413] Analysis of Protective Mechanism. Our liposome formulation is composed of DSPC, cholesterol, DOTAP and Tween 80 (molar ratio 7:3:2: 1). The lipid bilayer acts as a protective shell, which buffers the mechanical forces that would otherwise directly impact on the phage structure. This protection mechanism can be understood by interpreting the role of each component and comparing our approach to existing literature on liposome stability during nebulization.[000414] DSPC, a saturated phospholipid with a relatively high phase transition temperature (~55°C), that exceeds physiological conditions, combined with cholesterol, provides structural rigidity to the liposome bilayer. This enhanced rigidity improves resistance against mechanical disruption inherent to pressurized airflow and droplet formation during atomization (Anderson, M., Omri, A., 2004. The effect of different lipid components on the in vitro stability and release kinetics of liposome formulations. Drug Deliv 11, 33-39, Briuglia, M. L., Rotella, C., McFarlane, A., Lamprou, D. A., 2015. Influence of cholesterol on liposome stability and on in vitro drug release. Drug Deliv Transl Res 5, 231-242). The importance of cholesterolin preserving membrane properties of liposomes during nebulization has been well-documented. Szabova et al. (Szabova, J. M., O.; Havlikova, M.; Lfzal, F.; Mravec, F., 2021. Influence of liposomes composition on their stability during the nebulization process by vibrating mesh nebulizer. Colloids Surf B Biointerfaces) demonstrated that plain DPPC liposomes lacking cholesterol broke apart under vibrating mesh nebulization and then re-aggregated into larger vesicles with high polydispersity, as evidenced by the zeta potential having shifted from positive to negative values. Incorporating cholesterol (20-40 mol%) significantly improved membrane rigidity, preserving the original size distribution even after nebulization (Szabova, J. M., O.; Havlikova, M.; Lizal, F.; Mravec, F., 2021. Influence of liposomes composition on their stability during the nebulization process by vibrating mesh nebulizer. Colloids Surf B Biointerfaces). Several studies suggested at least 30 mol% is usually required to minimize liposomes from content leakage (Briuglia, M. L., Rotella, C., McFarlane, A., Lamprou, D. A., 2015. Influence of cholesterol on liposome stability and on in vitro drug release. Drug Deliv Transl Res 5, 231-242, Soto-Arriaza, M. A., Olivares-Ortega, C., Quina, F. H., Aguilar, L. F., Sotomayor, C. P., 2013. Effect of cholesterol content on the structural and dynamic membrane properties of DMPC / DSPC large unilamellar bilayers. Biochim Biophys Acta 1828, 2763-2769). However, our formulation achieves stability with a slightly lower cholesterol content (~23 mol% of total lipids), which is likely due to the high proportion of DSPC in keeping the membrane rigidity (Hac-Wydro, K. J., K.; Dynarowicz-Latka, P., 2009. Effect of saturation degree on the interactions between fatty acids and phosphatidylcholines in binary and ternary Langmuir monolayers. Colloids Surf B Biointerfaces 72, 101-111).[000415] In its formulation, The Applicant uses the DOTAP, which is a cationic lipid to introduce a net positive charge, thus promoting electrostatic interactions with the negatively charged capsid and / or tail of phages (Cuervo, A., Fabrega-Ferrer, M., Machon, C., Conesa, J. J., Fernandez, F. J., Perez-Luque, R., Perez-Ruiz, M., Pous, J., Vega, M. C., Carrascosa, J. L., Coll, M., 2019. Structures of T7 bacteriophage portal and tail suggest a viral DNA retention and ejection mechanism. Nat Commun 10, 3746, Nap, R. J., Bozic, A. L., Szleifer, I., Podgornik, R., 2014. The role of solution conditions in the bacteriophage PP7 capsid charge regulation. Biophys J 107, 1970-1979) to enhance entrapment of the phages within vesicles. Additionally, DOTAP may improve liposome vesicle stability by inducing electrostatic repulsion between liposomes and minimising aggregation during nebulization. Tween 80 as a non-ionic surfactant which by reducing surface tension at air-liquid interfaces (Montefusco-Pereira, C. V., 2023. Steps toward nebulization in-use studies to understand the stability of new biological entities. Drug Discov Today 28, 103461, Tai, K., He, X., Yuan, X., Meng, K., Gao, Y., Yuan, F., 2017. A comparison of physicochemical and functional properties of icaritin-loaded liposomes based on different surfactants. Colloids Surf. A: Physicochem. Eng. Asp. 518, 218-231), inhibitingexcessive vesicle aggregation (Hertel, S. P., Winter, G., Friess, W., 2015. Protein stability in pulmonary drug delivery via nebulization. Adv Drug Deliv Rev 93, 79-94). Additionally, Tween 80 form a foaming layer during nebulization, potentially cushioning liposomes and by adsorbing and dissipating the mechanical stress (Israelachvili, J. N., 2011. Intermolecular and surface forces, 3rd ed. Academic Press, Burlington, MA, Rosen, M. J., 2004. Surfactants and interfacial phenomena, 3rd ed. Wiley-lnterscience, Hoboken, N. J.)[000416] Similar strategies have also been employed to preserve the aerosol stability of numerous drugs and biologies with promising outcomes (Ehsan, Z., Clancy, J. P., 2015. Management of Pseudomonas aeruginosa infection in cystic fibrosis patients using inhaled antibiotics with a focus on nebulized liposomal amikacin. Future Microbiol 10, 1901-1912, Lokugamage, M. P., Vanover, D., Beyersdorf, J., Hatit, M. Z. C., Rotolo, L., Echeverri, E. S., Peck, H. E., Ni, H., Yoon, J. K., Kim, Y., Santangelo, P. J., Dahlman, J. E., 2021. Optimization of lipid nanoparticles for the delivery of nebulized therapeutic mRNA to the lungs. Nat Biomed Eng 5, 1059-1068), with the impact of lipid composition on liposome performance comprehensively reported (Anderson, M., Omri, A., 2004. The effect of different lipid components on the in vitro stability and release kinetics of liposome formulations. Drug Deliv 11, 33-39, Briuglia, M. L., Rotella, C., McFarlane, A., Lamprou, D. A., 2015. Influence of cholesterol on liposome stability and on in vitro drug release. Drug Deliv Transl Res 5, 231-242, Szabova, J. M., O.; Havlikova, M.; Lfzal, F.; Mravec, F., 2021. Influence of liposomes composition on their stability during the nebulization process by vibrating mesh nebulizer. Colloids Surf B Biointerfaces). Our results align with these approaches, emphasizing that careful optimization of lipid compositions and stabilizers like Tween 80 can protect phages against the mechanical insults of nebulization, thereby preserving infectivity.[000417] Aerosolization Characteristics. The particle sizing data (see table above summarising D10 (pm), D50 (pm) and D90 (pm) data) indicate that most droplets were under 5 pm, which are suitable for lung delivery. Both liposome-encapsulated PEV2 and PEV40 phages maintained their final titers of approximately 109PFU / mL post-nebulization, a concentration shown to control P. aeruginosa infections in animal models (Debarbieux, L. L., D.; Maura, D.; Morello, E.; Criscuolo, A.; Grossi, O.; Balloy, V.; Touqui, L.. 2010. Bacteriophages can treat and prevent Pseudomonas aeruginosa lung infections. J Infect Dis 201, 1096-1104, Morello, E. S., E.; Maura, D.; Huerre, M.; Touqui, L.; & Debarbieux, L., 2011. Pulmonary delivery of bacteriophages targeting Pseudomonas aeruginosa: potential treatment of acute lung infections. PloS one 6, e16963).[000418] In Vitro Aerosolization Performance. The respirable fraction of viable phages was measured using MSLI at a flow rate 30 L / min. The jet nebulizer produced lower values of VRF(28.15 ± 0.99%) and recovery (48.89 ± 0.84%) for PEV40 compared with PEV2 (VRF 44.03 ± 2.60% and >90% recovery). The vibrating mesh nebulizer achieved significantly higher VRF values (>70%) for both encapsulated phage types with a high viable phage recovery (>90%), which are most likely attributed to the milder mechanical stress to the phages during operation. Jet nebulization, which uses a high-velocity gas stream to atomize the liposomal-phage suspension into droplets which repeatedly impact on a baffle system, typically subjects particles to intense mechanical stress and air-liquid interfaces. Vibrating mesh nebulizers operate by pushing the liposomal-phage suspension through a mesh (containing -1000 micro-sized apertures of -3 pm) via piezoelectric vibration, generally considered a gentler process (Carrigy, N. B., Chang, R. Y., Leung, S. S. Y., Harrison, M., Petrova, Z., Pope, W. H., Hatfull, G. F., Britton, W. J., Chan, H. K., Sauvageau, D., Finlay, W. H., Vehring, R., 2017. Anti-Tuberculosis Bacteriophage D29 Delivery with a Vibrating Mesh Nebulizer, Jet Nebulizer, and Soft Mist Inhaler. Pharm Res 34, 2084-2096.). Our liposomal formulation demonstrated robust protection in both nebulization systems, regardless of their distinctly different atomization mechanisms, thus broadening the range of nebulizer devices to be used for inhaled phage therapy. This is particularly noteworthy in view of the mixed findings in the literature: while mesh nebulization is generally shown to be less damaging to phages than jet nebulization (Astudillo, A., Leung, S. S. Y., Kutter, E., Morales, S., Chan, H. K., 2018. Nebulization effects on structural stability of bacteriophage PEV44. Eur J Pharm Biopharm 125, 124-130, Carrigy, N. B., Chang, R. Y., Leung, S. S. Y., Harrison, M., Petrova, Z., Pope, W. H., Hatfull, G. F., Britton, W. J., Chan, H. K., Sauvageau, D., Finlay, W. H., Vehring, R., 2017. Anti-Tuberculosis Bacteriophage D29 Delivery with a Vibrating Mesh Nebulizer, Jet Nebulizer, and Soft Mist Inhaler. Pharm Res 34, 2084-2096, Leung, S. S. Y., Carrigy, N. B., Vehring, R., Finlay, W. H., Morales, S., Carter, E. A., Britton, W. J., Kutter, E., Chan, H. K., 2019. Jet nebulization of bacteriophages with different tail morphologies - Structural effects. Int J Pharm 554, 322-326), contradictory findings have also been reported (Flint, R., Laucirica, D. R., Chan, H. K., Chang, B. J., Stick, S. M., Kicic, A., 2023. Stability Considerations for Bacteriophages in Liquid Formulations Designed for Nebulization. Cells 12). The consistency of the liposome formulation in maintaining the bioactivity of phages is clinically valuable, as it accommodates variability in clinical equipment and patient-specific delivery requirements (Ari, A., 2014. Jet, Ultrasonic, and Mesh Nebulizers: An Evaluation of Nebulizers for Better Clinical Outcomes. Eurasian J. Pulmonol 16, 1-7). When compared to the low VRF values (<15%) observed in nebulizing free phages (Astudillo, A., Leung, S. S. Y., Kutter, E., Morales, S., Chan, H. K., 2018. Nebulization effects on structural stability of bacteriophage PEV 44. Eur J Pharm Biopharm 125, 124-130), the high VRF values of our liposomal formulations show they can significantly improve delivery to the lungs.[000419] Results indicate that lipid nanoparticle-encapsulated phages achieve an optimal size range for pulmonary delivery. These findings highlight the potential suitability of lipid-encapsulated phages for effective respiratory administration.Summary[000420] Inhaled bacteriophage (phage) therapy is emerging as a promising approach to combat multidrug-resistant (MDR) respiratory pathogens such as Pseudomonas aeruginosa. Aerosol delivery by nebulization poses challenges for maintaining phage stability, often resulting in titer losses due to mechanical stresses.[000421] This study evaluated the use of liposomal encapsulation to protect phages during nebulization. Two P. aeruginosa phages, PEV2 (short-tail) and PEV40 (long-tail), were selected for this work. Liposomes were prepared using DSPC, cholesterol, Tween 80, and cationic lipid DOTAP. Encapsulation efficiencies were 78% for PEV2 and 90% for PEV40, with mean particle sizes of 300 nm and 650 nm, respectively.[000422] Nebulization by jet and vibrating mesh devices showed that the liposome-encapsulated phages were able to preserve viability, with titer losses below 0.4 logi0(PEV40) and 0.07 logi0(PEV2). In contrast, non-encapsulated phages experienced titer reductions of up to 1.23 logic, especially by jet nebulization. Vibrating mesh nebulization generated slightly larger droplets (~5.6 pm) but with better phage recovery (> 90%) and respirable fractions (> 70%) for both types of phages encapsulated in liposomes.[000423] These results demonstrate that the approach of lipid encapsulation effectively protects phages from mechanical damage during nebulization, maintaining bioactivity for aerosol delivery to enhance the success of inhaled phage therapy.[000424] Lipid-based encapsulation using DSPC, cholesterol, Tween 80, and DOTAP effectively protected phages during nebulization, addressing a critical challenge in inhaled phage therapy. The inventors reached reasonably high encapsulation efficiencies (77.9% for short-tail PEV2 and 89.93% for long-tail PEV40) of phages in the lipids, which helped preserve phage structure and viability in both jet and vibrating mesh nebulization. Encapsulated PEV2 and PEV40 showed negligible titer reduction (<0.4 logw), whereas free phages suffered significant inactivation (highest titer loss at 1.23 logw). The vibrating mesh nebulization achieved higher phage recovery (-95%) and a respirable fraction of -70-75% for both PEV2 and PEV40 phages, which are desirable for lung deposition. These findings highlight how carefully optimized liposome encapsulation enhances phage stability and delivery to the lungs, thereby offering a promising approach for inhaled phage therapy against MDR pathogens and broadeningtherapeutic possibilities for challenging pulmonary diseases, including those complicated by biofilm formation.Example 10Enhanced Eradication of Pseudomonas aeruginosa (PA07) Biofilms using Liposomal Encapsulated Phages: A Live Cell Imaging Study1. Introduction[000425] Bacterial biofilms represent a significant challenge in antimicrobial therapy due to the dense extracellular polymeric substance (EPS) matrix that acts as a physical barrier, preventing therapeutic agents from reaching embedded bacteria. While bacteriophages (phages) offer a promising alternative to antibiotics, their efficacy is often limited by their inability to penetrate deep into the biofilm structure.[000426] This study evaluates a liposomal encapsulation strategy designed to enhance the penetration of long-tail (PEV1) and short-tail (PEV2) phages. To assess the efficacy of this formulation, we utilised the 3D Cell Explorer (Nanolive, Switzerland), a label-free holotomographic microscopy system. This technology allows for the real-time visualisation of biofilm disruption without the artifacts introduced by chemical staining, providing a qualitative comparison between free phage and liposomal encapsulated phage treatments.2. Materials and methods2.1. Preparation of liposomal encapsulated phages[000427] Liposomal phages were prepared via a crossflow micromixing approach as described above. The lipid phase consisted of DSPC, cholesterol, stearylamine, and Tween 80 in a molar ratio of 7:3:2:1, respectively. The aqueous phase contained bacteriophages (PEV1 or PEV2, titre ~107PFU / mL) suspended in PBS with the pH adjusted to 9.5 to optimise encapsulation efficiency. Encapsulation was performed using the AXFTM-mini-Pathfinder system. The total flow rate (TFR) was maintained at 3 mL / min, with a flow rate ratio (FRR) between the lipid and aqueous phases set at 1:2. This formulation resulted in an encapsulation efficiency of approximately 90% across both PEV1 and PEV2 liposomal preparations.2.2. Biofilm cultivation[000428] A clinical strain of Pseudomonas aeruginosa (PA07) bacterial suspension in the early log phase was inoculated into a micro-dish (Ibidi p-Dish 35 mm, low ibiTreat). The culture was incubated at 37 °C for 2 hours in a static incubator to allow initial cell attachment. Followingthis adhesion period, 500 pL of nutrient broth was added. The dishes were further incubated for 48 hours at 37 °C under static conditions to trigger mature biofilm formation.[000429] After the 48-hour growth period, the spent medium was carefully removed from the vehicle control and treatment groups’ micro-dishes. Subsequently, 250 pL of the respective treatment formulations were added to the biofilms. All samples were then incubated for an additional 24 hours prior to imaging.2.3. Treatment groups[000430] The biofilms were treated with the following preparations:1. Control: Untreated biofilm.2. Vehicle control: Biofilm treated with Phosphate Buffered Saline (PBS).3. Free phage treatment: Biofilm treated with unencapsulated PEV1 or PEV2.4. Physical mixture: Biofilm treated with a physical mix of empty liposomes and free phages (i.e., 1:1 volume ratio + vortex mixing).5. Experimental group: Biofilm treated with Liposomal Encapsulated Phages (Lip- PEV1 or Lip-PEV2).2.4. Live cell imaging and digital staining[000431] The biofilm disruption potential was visualised 24 hours post-treatment using the 3D Cell Explorer (Nanolive, Switzerland).[000432] Digital staining methodology: Unlike traditional fluorescence microscopy, the 3D Cell Explorer utilises holotomography to measure the Refractive Index (Rl) distribution within the sample. " Digital Staining" is achieved by software algorithms that assign specific colours to distinct Rl ranges, allowing for the differentiation of cellular structures (in this case bacteria cells walls) without the use of phototoxic chemical dyes (Lopez-Osorio et al., Parasitol Res. 2022, Apr;121(4):1179-1189).[000433] To ensure valid comparisons between treatment groups, the specific Rl transfer functions (staining parameters) were standardised. The Rl threshold settings, contrast, and colour mapping used to visualise the bacterial density were kept consistent across all acquired images to prevent visual bias during analysis.3. Results and Discussion3.1. Assessment of biofilm disruption[000434] Imaging data obtained via the Nanolive system highlighted distinct differences in biofilm architecture across the treatment groups.• Controls (untreated and PBS): Images of the Untreated and PBS-Treated samples (Fig. 20a, 20b) displayed a robust, highly dense bacterial cell architecture. The PA07 bacteria appeared as tightly aggregated clusters embedded within a continuous EPS matrix. The consistent density across these two controls confirms that the biofilm structure was stable and that the PBS vehicle had no inherent disruptive effect.• Free phage and physical mixture treatment: Biofilms treated with free phages (PEV1 and PEV2) (Fig. 20c, 20f) exhibited signs of partial eradication when compared to the controls. While a visible decrease in overall bacterial density was observed, large aggregates of intact, viable bacteria remained clearly visible in the deeper layers of the biofilm.[000435] Similarly, the physical mixture groups (different phages + empty liposomes) (Fig.20d, 20g) showed results with similar density and limited eradication efficacy compared to the Free Phage treatment only. This crucial finding indicates that the simple co-administration of empty liposomes and phages does not significantly enhance penetration or lytic activity, confirming that the enhanced efficacy is dependent on the actual encapsulation of the phages.[000436] These results suggest that while free phages caused lysis of surface-level bacteria, they were likely unable to penetrate the deeper, protected layers of the biofilm efficiently due to the physical barrier imposed by the EPS matrix.• Liposomal encapsulated phage treatment: The experimental groups treated with Liposomal encapsulated phages (Fig 20e, 20h) showed the most marked reduction in biofilm integrity. Nanolive imaging revealed a high level of bacterial cell eradication, characterised by a sparse distribution of remaining cells and a significant accumulation of cellular debris. The structural breakage of the biofilm matrix was evident, resulting in significantly fewer aggregated clusters compared to the free phage and physical mixture groups. This qualitative data strongly indicates that the liposomal formulation successfully facilitated the transport of the phages through the EPS matrix, enabling them to reach and kill bacteria embedded in the deeper biofilm layers.3.2. Interpretation & limitations[000437] These preliminary imaging data support the hypothesis that liposomal encapsulation protects bacteriophages and facilitates their transport through the EPS matrix. The "digital staining" revealed that the liposomal formulation successfully reached bacteria embedded deep within the biofilm that were otherwise inaccessible to free phages.[000438] While this report provides compelling visual evidence of eradication, traditional quantitative measurements via Optical Density (OD) plate reading were not included in this dataset.[000439] OD measurements rely on the principle of light scattering (absorbance) to estimate bacterial population density. However, liposomes are colloidal suspensions that inherently scatter light, creating turbidity.[000440] Consequently, standard microplate reader analysis yielded confounded results, where the turbidity of the liposomes masked the reduction in optical density caused by bacterial lysis. Therefore, the label-free Nanolive imaging provides a preliminary indication of the biological efficacy in this specific context. Future experiments will require alternative quantitative assays that are not susceptible to lipid-induced turbidity.4. Conclusion[000441] The use of the 3D Cell Explorer provided critical real-time insights into the structural degradation of PA07 biofilms. The results demonstrate that liposomal encapsulated phages possess a superior ability to eradicate biofilm-embedded bacteria compared to free phages. These findings validate the potential of the specified liposomal formulation (DSPC: Chol: Stearylamine: Tween 80) as a delivery vehicle to overcome the physical barriers of bacterial biofilms.[000442] The current formulation is hypothesised to facilitate cellular uptake, allowing the encapsulated phages to reach and kill pathogens residing within host cells.[000443] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms, and in particular features of any one of the various described examples may be provided in any combination in any of the other described examples. Various modifications and alterations to this invention will become apparent to those skilled in the art without departing from the scope and spirit of this invention. It should be understood that this invention is not intended to be unduly limited by the illustrative embodiments and examples set forth herein and that such examples and embodiments are presented by way of example only with the scope of the invention intended to be limited only by the claims set forth herein as follows.

Claims

CLAIMS1. Use of a charged lipid for encapsulating phages in nanosize lipid particles,wherein the charged lipid is cationic or anionic;wherein the charged lipid comprises a cationic or anionic moiety and a C12 to C80 hydrocarbon chain or a polymeric chain;wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O)Rx, - C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5- or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, - NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.

2. Use of claim 1, wherein the charged lipid is a lipid of formula (I)A-L1-(L2-B)nwhereinA is an anionic or cationic moiety e.g. phosphate or ammonium;L1 is a bond or C1 to C6 alkylene;L2 is absent or -C(O)O-, -OC(O)-, -C(O)-, -NHC(O)-, -C(O)NH- -NRC(O)-, -C(O)NR- - OP(O)2O-, -O-, -OS(O)2O-, -O-C(O)NH-, -NHC(O)O-, wherein R is Ci to C6alkyl;B is a C12 to C80 hydrocarbon chain or a polymeric chain;n = 1 to 3;wherein if L1 is C1 to C6 alkylene and B is C12 to C80 hydrocarbon chain, L2 is not absent;wherein if n is more than 1, each B is independently selected,wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O)Rx, - C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5- or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, - NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.

3. Use of claim 1 or claim 2, wherein the charged lipid comprises a C12 to C80 hydrocarbon chain.

4. Use of claim 1 or claim 2, wherein the charged lipid comprises two C12 to C80 hydrocarbon chains, e.g. two C12 to C40 hydrocarbon chains.

5. Use of any one of claims 1 to 4, wherein the hydrocarbon chain comprises one or more unconjugated double bonds and comprises a maximum of 1 double bond for every 4 carbon atoms in the longest chain.

6. Use of any one of claims 1 to 5, wherein the hydrocarbon chain is branched and comprises a maximum of 1 branch for each 6 carbon atoms in the longest chain.

7. Use of any one of claims 1 to 5, wherein the hydrocarbon chain is unbranched.

8. Use of any one of claims 1 to 7, wherein the hydrocarbon chain is unsubstituted.

9. Use of any one of claims 1 to 8, wherein the cationic or anionic moiety is connected to the hydrocarbon chain by a bond.

10. Use of claim 1 or claim 2, wherein the charged lipid comprises a cationic or anionic moiety and a polymeric chain.

11. A method of encapsulating phages, the method comprising mixing a charged lipid formulation with a phage formulation;wherein the charged lipid formulation comprises a charged lipid;wherein the charged lipid is cationic or anionic;wherein the charged lipid comprises a cationic or anionic moiety and a C12 to C80 hydrocarbon chain or a polymeric chain;wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O)Rx, - C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl;wherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5- or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, - NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.

12. The method of claim 11, wherein the pH of the phage formulation is from 7 to 9.5, e.g. 7, e.g. 9.5.

13. The method of claim 11 or claim 12, wherein the phage formulation comprises a buffer.

14. The method of any one of claims 11 to 13, wherein the phage formulation is an aqueous suspension [e.g. PBS (phosphate-buffered saline)].

15. The method of any one of claims 11 to 14 wherein the cationic charged lipid formulation comprises a surfactant, optionally wherein the surfactant is polyethylene glycol sorbitan monooleate.

16. The method of any one of claims 11 to 15, wherein the lipid formulation is an organic phase (e.g. ethanol).

17. The method of any one of claims 11 to 16, wherein the method further comprises the step of preparing a cationic charged lipid formulation by dissolving lipid and a cationic lipid inducer (e.g. octadecylamine) in a solvent (e.g. EtOH), optionally at RT, optionally sonicating (e.g. 10 mins)18. A composition comprising a phage encapsulated in nanosize lipid particles;wherein the lipid particles comprise a charged lipid;wherein the charged lipid comprises a cationic or anionic moiety and a C12 to C80 hydrocarbon chain or a polymeric chain;wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O)Rx, - C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5- or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, - NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.

19. A method of treating or preventing a condition or disease, comprising administering to a subject in need thereof an effective amount of a composition comprising a phage encapsulated in nanosize lipid particles;wherein the lipid particles comprise a charged lipid;wherein the charged lipid comprises a cationic or anionic moiety and a C12 to C80 hydrocarbon chain or a polymeric chain;wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O)Rx, - C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl; andwherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5- or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, - NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.

20. A method of treating a bacterial infection in the respiratory system, comprising administering to a subject in need thereof an effective amount of a composition comprising a phage encapsulated in nanosize lipid particles;wherein the lipid particles comprise a charged lipid;wherein the charged lipid comprises a cationic or anionic moiety and a C12 to C80 hydrocarbon chain or a polymeric chain;wherein the hydrocarbon chain is optionally substituted by one or more substituents independently selected from OH, NH2, NHRx, NRx2, polyether, -OR, -C(O)Rx, -C(O)ORx, -OC(O)Rx, -C(O)NHRx, -C(O)NRx2, C5-C6 carbocyclyl and 5- or 6-membered heterocyclyl, wherein Rx is C1 to C6 alkyl;wherein the polymeric chain is optionally substituted by one or more substituents independently selected from -OH, -NH2, -NRy2, -ORy, -C(O)Ry, -C(O)ORy, -COOH, -SH, C1 to C6 alkyl, and 5- or 6-membered heterocyclyl groups; wherein Ry is C1 to C6 alkyl, and wherein the C1 to C6 alkyl substituent, Ry and the heterocyclyl group are each independently optionally substituted with one or more substituents selected from -OH, -NH2, -NRz2, -COOH, and -SH; and wherein Rz is C1 to C6 alkyl.

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