NANOVESICLES

Abstract

A process for the preparation of unilamellar vesicles, wherein a unilamellar vesicle comprises an amphiphilic membrane enclosing an aqueous core; and compositions comprising unilamellar vesicles.

The process comprises providing a primary emulsion comprising an amphiphilic membrane forming component, a first aqueous phase W1 and a first oil phase O1. The first aqueous phase W1 is dispersed as droplets in the oil phase O1 such that the primary emulsion is a water-in-oil emulsion. The primary emulsion comprises droplets having a mean diameter of less than 1000 nm.

The process comprises combining the primary emulsion with a second aqueous phase W2 to produce a secondary emulsion that is a water-in-oil-in-water emulsion; dewetting to yield unilamellar vesicles in the secondary emulsion; and isolating the unilamellar vesicles.

Claims

1. A process for the preparation of unilamellar vesicles, a unilamellar vesicle comprising an amphiphilic membrane enclosing an aqueous core, the process comprising providing a primary emulsion comprising an amphiphilic membrane forming component, a first aqueous phase W1 and a first oil phase O1, the first aqueous phase W1 being dispersed as droplets in the oil phase O1 such that the primary emulsion is a water-in-oil emulsion and wherein the primary emulsion comprises droplets having a mean diameter of less than 1000 nm; combining the primary emulsion with a second aqueous phase W2 to produce a secondary emulsion that is a water-in-oil-in-water emulsion; dewetting to yield unilamellar vesicles in the secondary emulsion; and isolating the unilamellar vesicles.

2. The process of claim 1, wherein isolating the unilamellar vesicles comprises separating the secondary emulsion into layers of different densities, at least one layer comprising the first oil phase; at least one layer comprising the second aqueous phase; and at least one layer comprising the unilamellar vesicles.

3. The process of claim 1, wherein isolating the unilamellar vesicles comprises (i) filtration; (ii) chromatography; (iii) the use of a salt gradient; and/or (iv) field flow fractionation.

4. The process off claim 1, wherein the first aqueous phase comprises a nanoparticle, such that at least one unilamellar vesicle encapsulates a nanoparticle.

5. The process of claim 4, wherein the nanoparticle is selected from a bacteriophage, a plasmid, an endolysin or a gene vector.

6. The process of claim 1, wherein providing the primary emulsion comprises preparing the primary emulsion by means of a low shear emulsification process.

7. The process of claim 6, wherein the low shear emulsification process employs a nanoporous membrane or nanochannels.

8. The process of claim 1, wherein dewetting to yield unilamellar vesicles in the secondary emulsion does not comprise evaporation of the oil phase.

9. The process of claim 2, wherein a portion of the secondary emulsion comprising the unilamellar vesicles is transferred to a centrifuge tube.

10. The process of claim 2, wherein a density gradient is employed to separate the secondary emulsion into layers of different densities.

11. The process of claim 2, wherein separating the secondary emulsion into layers of different densities comprises centrifuging the secondary emulsion.

12. The process of claim 1, wherein the second aqueous phase comprises PVA (poly(vinyl) alcohol) or a poloxamer.

13. The process of claim 1, wherein the amphiphilic membrane forming component comprises (i) a lipid, such as a phospholipid; or (ii) a di-block copolymer, such as a PEG di-block copolymer.

14. The process of claim 1, wherein the first oil phase comprises a first organic solvent and a second organic solvent.

15. (canceled)

16. (canceled)

17. The process of claim 1, wherein the unilamellar vesicles are further processed to generate multilamellar vesicles.

18. The process of claim 17, wherein said further processing comprises emulsifying a third aqueous phase comprising the unilamellar vesicles with a second oil phase to form a water-in-oil emulsion and subsequently emulsifying with a fourth aqueous phase to form a water-in-oil-in-water emulsion.

19. A composition comprising layers of different densities; at least one layer comprising an oil phase (O1); at least one layer comprising an aqueous phase (W2); and at least one layer comprising a plurality of unilamellar vesicles, the unilamellar vesicles each comprising an amphiphilic membrane enclosing an aqueous core and having a mean diameter of less than 1000 nm.

20. The composition of claim 19, comprising two or more layers comprising a plurality of unilamellar vesicles.

21. The composition of claim 19, wherein at least one unilamellar vesicle has a diameter of less than 1000 nm and encapsulates a nanoparticle having a mean diameter of 10 to 200 nm.

22. (canceled)

23. (canceled)

24. A multilamellar vesicle comprising two or more amphiphilic membranes successively enclosing an aqueous core, the multilamellar vesicle having a diameter of less than 1000 nm and encapsulating a nanoparticle having a mean diameter of 10 to 200 nm.

25. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0152] FIG. 1: Schematic diagram showing the preparation of a W1/O1/W2 double emulsion by pouring the primary W1/O1 nanoemulsion in an excess of an aqueous phase containing emulsifier to stabilise the vesicle bilayer membrane upon subsequent spontaneous dewetting.

[0153] FIG. 2: Schematic diagram showing the preparation of a W1/O1/W2 double emulsion by contacting the primary W1/O1 nanoemulsion with an aqueous phase (W2) in a microcapillary.

[0154] FIG. 3: Schematic diagram showing assembly of a unilamellar vesicle from dewetting of the double nanoemulsion.

[0155] FIG. 4: Schematic diagram showing the preparation of a W1/O1 nanoemulsion by injecting the inner water phase (W1) through nanopores of a hydrophobic membrane.

[0156] FIG. 5 and FIG. 6: Schematic diagrams showing methods to separate the secondary emulsion into layers having different densities.

[0157] FIG. 7: Schematic diagram showing a multilamellar vesicle (left) and a multivesicular vesicle (right).

[0158] FIG. 8: NTA size analysis of unilamellar liposomes produced using a low shear membrane emulsification process (0.5 μm and 1.1 μm pore glass membranes used to make the W1/O1 primary emulsion).

[0159] FIG. 9: CryoTEM image showing unilamellar liposomes (bar 200 nm).

[0160] FIG. 10: CryoTEM image showing multivesicular liposomes (bar 200 nm).

[0161] FIG. 11: CryoTEM image showing encapsulated phage Mycobacterium D29 in unilamellar liposomes (bar 200 nm).

[0162] FIG. 12: CryoTEM image showing unilamellar polymersomes (bar 200 nm).

[0163] FIG. 13: CryoTEM image showing multilamellar vesicles (bar 200 nm).

[0164] FIG. 14: CryoTEM image showing magnetic nanoparticle encapsulation in liposomes (bar 200 nm).

[0165] FIG. 15: CryoTEM image showing a multivesicular liposome (bar 200 nm).

[0166] FIG. 16: Confocal images of macrophages exposed to phage K encapsulated in DOPE-CHEMS liposomes. After growth, macrophages were exposed to phage K encapsulated in DOPE-CHEMS liposomes, then permealised and stained. Phage K was pre-stained with SYBR gold (left, green in original image). Nuclei are stained with DAPI (left centre, blue in original image). Actin filaments are stained with phalloidin/CFR680 Cy5 fluorophore conjugate (right centre, red in original image). Image on the right is the merged image. Error bar 10 μm.

[0167] FIG. 17: Confocal imaging of macrophages exposed to S. aureus and then treated with CHAP.sub.K and LLO encapsulated in DOPE-CHEMS liposomes. After growth, macrophages were exposed to S. aureus and then treated with DOPE-CHEMS liposomes loaded with CHAP.sub.K and LLO, then permealised and stained. Nuclei are stained with DAPI (left, blue in original image). Actin filaments are stained with phalloidin/CFR680 Cy5 fluorophore conjugate (centre, red in original image). Image on the right is the merged image. Top row=control, bottom row=treated sample. Error bar 10 μm.

[0168] Referring to FIG. 1, there is shown a schematic diagram of a process in accordance with an embodiment of the invention. A container 10 holds a primary emulsion 12 therein. The primary emulsion 12 comprises droplets of a first aqueous phase W1 dispersed in a continuous oil phase O1. The primary emulsion 12 is poured into an excess of a second aqueous phase W2 with stirring provided by a magnetic bar 14. A secondary emulsion 16 is formed, which is a water-in-oil-water emulsion. The secondary emulsion 16 comprises droplets of the first emulsion 12 dispersed in the second aqueous phase W2. It is notable that the droplets of the primary emulsion contain one or more droplets of the first aqueous phase W1.

[0169] Referring to FIG. 2, there is shown a schematic diagram of another process to prepare the secondary emulsion 16. As before, a primary emulsion 12 comprises droplets of the first aqueous phase W1 dispersed in a continuous oil phase O1. The primary emulsion is pumped through a microcapillary channel 20 and the second aqueous phase W2 flows perpendicular to the primary emulsion, through another channel 22, thereby forming an X junction. The primary emulsion 12 and the second aqueous phase W2 combine to form the secondary emulsion 16.

[0170] Referring to FIG. 3, there is shown a schematic diagram showing assembly of a unilamellar vesicle 30 from dewetting of a double nanoemulsion.

[0171] On the left there is shown a droplet of a first aqueous phase W1, which is enclosed within a droplet of a first oil phase O1, which is dispersed in a continuous second aqueous phase W2. An amphiphilic membrane forming component 32 is present, which tends to aggregate at the interface between the oil and water phases.

[0172] In the middle, there is shown partial dewetting. Without being bound by theory, a portion of the oil phase, e.g. a water-soluble organic solvent, is believed to migrate out of the oil phase and this depletion leads the remaining oil phase components to separate, perhaps through a difference in density or due to Brownian thermal motion.

[0173] On the right there is shown a unilamellar liposome 30 comprising an amphiphilic membrane 34 enclosing an aqueous core W1. There is also shown an oil droplet 36 comprising oil phase O1 and excess amphiphilic membrane forming component 32.

EXAMPLES

Example 1: Generation of W1/O1 Nanoemulsion Using a Low Shear Primary Emulsification Process, for the Subsequent Preparation of Liposomes

[0174] 1.5 g of phosphatidylcholine (amphiphilic membrane forming component) was dissolved in 20 ml of a solvent mixture (chloroform/hexane volume ratio 2:3) and used as the organic phase (O1). PVA (2% w/v), PEG-8000 (6% w/v) and a Myovirus Staphylococcus aureus phage K were solubilized in Tris-HCl buffer (W1) to form the first aqueous phase.

[0175] Production of the W1/O1 nanoemulsion was carried out using a nanoporous glass membrane 40 (i.e. membrane emulsification) as illustrated in FIG. 4. The water phase was passed through the porous membrane to form nanodroplets at the membrane surface where they then detach under low shear and were dispersed in the oil phase.

[0176] The nanoporous glass membrane (SPG Technology Co., Ltd, Japan) 40 comprises pores 42 having a pore size of 500 nm. The nanoporous membrane 40 was immersed in the oil phase (O1) and kept under constant gentle stirring to provide gentle shear across the membrane surface. 5 ml of the inner aqueous phase (W1) was transferred to 20 ml of the oil phase (O1) by pushing the water phase W1 under pressure (1 to 10 times greater than the critical pressure) across a 2 cm length of the tubular hydrophobic nanoporous glass membrane. This W1/O1 nanoemulsion was kept stirring throughout.

Example 2: Generation of W1/O1 Nanoemulsion Using a Low Shear Primary Emulsification Process, for the Subsequent Preparation of Polymerosomes

[0177] Example 1 was repeated with 0.5 g PEG(5000)-b-PLA(5000) in place of the 1.5 g of phosphatidylcholine as the amphiphilic membrane forming component. 1 ml of the inner aqueous phase (W1) transferred to 20 ml of the oil phase (O).

Example 3: Generation of W1/O1 Nanoemulsion by Primary Emulsification Step

[0178] 1.5 g of phosphatidylcholine was dissolved in 20 ml of a solvent mixture (chloroform/hexane 2/3) and used as the organic phase (O1). 5 ml of the first aqueous phase (W1; PVA 2% w/v, PEG-8000 6% w/v and calcein in Tris-HCl buffer) was added to the oil phase in a beaker with a paddle mixer to form a premix microemulsion of W1 droplets dispersed in the oil phase.

[0179] The premix was subsequently emulsified at 1200 psi using a single pass homogenization cycle (Microfluidizer LV1) yielding a primary emulsion W1/O1 with droplets between 50 nm-500 nm in size measured using dynamic light scattering (Malvern Zetasizer Nano ZS).

Example 4: Generation of W1/O1 Nanoemulsion by Primary Emulsification Step

[0180] 1.5 g of phosphatidylcholine was dissolved in 20 ml of a solvent mixture (chloroform/hexane 2/3) and used as the organic phase (O). 5 ml of the first aqueous phase (W1; PVA 2% w/v, PEG-8000 6% w/v and calcein in Tris-HCl buffer) were added to the oil phase in a beaker. Production of the W1/O1 primary emulsion was carried out using an ultrasonic probe for emulsification (Hielscher UP200St) yielding a nanoemulsion W1/O1 with droplets between 50 nm-500 nm in size (measured using dynamic light scattering (Malvern Zetasizer Nano ZS).

Example 5: Generation of W1/O/W2 Double Emulsion and Separation

[0181] As illustrated in FIG. 1, 25 ml of the primary W1/O1 emulsion prepared using one of the methods described above (examples 1-4) was poured into 75 ml of the second aqueous phase (the outer phase W2; PVA 10% w/v, Poloxamer-188 5% w/v) under gentle stirring to form the secondary emulsion, which is a water-in-oil-in-water emulsion, i.e. a double emulsion. De-wetting of the double emulsion occurs over a period of several minutes at room temperature. Poloxamer-188 is used to adjust the interfacial tension between phases and thereby aid dewetting.

[0182] Referring to FIG. 5, 10 ml of the secondary emulsion 50 now containing the nanovesicles was aliquoted in a 50 ml centrifuge tube and layered on a 40 ml sucrose gradient 52 (layers 10 wt %, 20 wt %, 30 wt % and 40 wt %) and centrifuged at 50,000 g (Beckman Coulter, Model Avanti JXN-30) for 12 hours at 4° C. Two vesicle rich bands 54a, 54b were generated. The “empty” nanovesicles, which do not encapsulate a nanoparticle are less dense and are present in the upper layer 54a. The nanovesicles with cargo are denser and present in the lower layer 54b. Each vesicle rich layer 54a, 54b was removed using a syringe to pierce the tube and dialysed several times using a 12-14 kDa dialysis membrane and resuspended in Tris-HCl buffer. Vesicles were stored at 4° C.

[0183] Alternative gradients, such as a CsCl (caesium chloride) gradient, can be used in place of a sucrose gradient.

[0184] Another option is illustrated in FIG. 6 wherein the double emulsion comprising the vesicles is first centrifuged to form three layers of different densities: an oil layer 60, a vesicle rich layer 62 and an aqueous layer 64. A sucrose solution 66 (e.g. 100 mM sucrose) is then added to separate the oil layer 60 from the vesicle rich layer 62. The diagram is provided for illustration only and is not to scale.

Example 6: Generation of W1/O1/W2 Double Emulsion Using Membrane Emulsification, and Subsequent Separation

[0185] The primary W1/O1 emulsion prepared using one of the methods illustrated above (examples 1-4) was passed through a porous membrane (e.g. using a High-Speed Mini Kit, KH-125; SPG Technology Co., Ltd.) mounted with a 5 μm pore size hydrophilic membrane and dispersed in the outer phase (W2; PVA 10% w/v, Poloxamer-188 5% w/v). De-wetting of the double emulsion occurs over a period of several minutes at room temperature and vesicles were isolated as described above.

Example 7: Generation of Vesicles by Preparing a W1/O1/W2 Double Emulsion in a Microfluidic Channel

[0186] As illustrated in FIG. 2, the primary W1/O1 emulsion prepared using one of the methods illustrated above (examples 1-4) was pumped (Vici pump, equipped with silicone tubing) into a quartz droplet X-junction chip (190 m, hydrophilic) and emulsified in a second aqueous phase (outer aqueous phase W2; PVA 10% w/v, Poloxamer-188 5% w/v). De-wetting of the double emulsion occurs over a period of several minutes at room temperature and vesicles were separated as described above.

Example 8: Generation of Bilamellar and Multilamellar Liposomes

[0187] 1.5 g of phosphatidylserine was dissolved in 20 ml of a solvent mixture (chloroform/hexane volume ratio 2:3) and used as a second organic phase (O2). PVA (2% w/v), PEG-8000 (6% w/v) and unilamellar liposomes produced using one of the methods illustrated above were suspended in Tris-HCl buffer (W3). Production of a W3/O2 nanoemulsion was carried out using a 1 μm hydrophobic porous glass SPG membrane. The W3/O2 nanoemulsion was poured into the outer aqueous phase (W4; PVA 10% w/v, Poloxamer-188 5% w/v) under gentle stirring. De-wetting of the double emulsion occurs over a period of several minutes at room temperature. 10 ml of the secondary emulsion (nanovesicle mixture) was aliquoted in a 50 ml centrifuge tube and layered on a 40 ml sucrose gradient (layers 10 wt %, 20 wt %, 30 wt % and 40 wt %) and centrifuged at 50,000 g (Beckman Coulter, Model Avanti JXN-30) for 12 hours at 4° C. The vesicle rich band was removed using a syringe to pierce the tube and dialysed several times using a 12-14 kDa dialysis membrane and resuspended in Tris-HCl buffer. Vesicles were stored at 4° C. Multilamellar vesicles were stored at 4° C.

[0188] FIG. 7 shows schematic diagrams of a multilamellar vesicle 70 (left) and a multivesicular vesicle 84 (right).

[0189] The multilamellar vesicle 70 comprises a central aqueous core 72 surrounded by an inner amphiphilic membrane 74, an intermediate amphiphilic membrane 76 and an outer amphiphilic membrane 78. The region between the inner and intermediate amphiphilic membranes can be described as a first shell 80 and the region between the intermediate and outer amphiphilic membranes can be described as a second shell 82.

[0190] The multivesicular vesicle 84 comprises four aqueous cores 86 with each core being enclosed by an inner amphiphilic membrane 88. Each core 86 and inner amphiphilic membrane 88 can be viewed as a unilamellar vesicle, which is enclosed within an outer amphiphilic membrane 90.

[0191] NTA Size Analysis

[0192] A NanoSight LM10 (Malvern Instruments Ltd, UK) using nanoparticle tracking analysis (NTA) was used to determine the average size and size distribution of liposomes/polymerosomes. NTA measurements were performed in a sample chamber equipped with a 640 nm LASER to track the nanoparticles (NPs).

[0193] Typically, a 10 μl aliquot was taken from each sample and diluted 10.sup.2-10.sup.3 fold in order to achieve a particle concentration of 10.sup.7-10.sup.10 ml.sup.−1. The sample was injected into the sample chamber using a sterile syringe and sample flow was maintained through the chamber until all air bubbles were removed. The temperature was registered with a thermometer (RTD Pt100, OMEGA, UK) and temperature correction was carried out.

[0194] The software used for capturing and analysing the data was NTA 3.0 (Malvern Instruments Ltd, UK). Data for each sample was captured over a period of 60 s and each measurement was repeated five times.

[0195] The focus was set to achieve a uniform perfect spherical particle view. Before capturing the video, the camera had to be set-up to ensure all the particles in the sample were clearly visible with no more than 20% saturation. The single gain mode was used throughout the whole measurement process. An example for unilamellar liposomes is shown in FIG. 8.

[0196] CryoTEM Imaging

[0197] A 8 μl aliquot of sample was pipetted onto a carbon coated copper grid (HC300Cu, Holey Carbon film on Copper 300 mesh, EM Resolutions, UK). Excess liquid was blotted away with filter paper (Whatman number 1) and the grid was plunge-frozen in a liquid mixture of ethane/propane cooled by liquid nitrogen. The sample was then kept at liquid nitrogen temperatures throughout the analysis. TEM images were taken on a JEOL 2200FS TEM at 200 keV using a Gatan K2 Summit and Gatan 914 cryo-holder. A selection of images is shown in FIGS. 9 to 15.

[0198] FIG. 11 demonstrates encapsulation of a phage D29. FIG. 13 clearly shows the concentric ring pattern of a multilamellar vesicle. Referring to FIG. 14, there is shown a CryoTEM image showing encapsulation of a magnetic nanoparticle (MNP, iron oxide) in liposomes. MNPs are used in oncology for drug delivery and as contrast agents for magnetic resonance.

Example 9

[0199] DOPE-CHEMS liposomes are composed of dioleoylphosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS).

[0200] We encapsulated CsCl purified S. aureus myoviridae phage K in DOPE-CHEMS liposomes using the method described in example 5 and imaged phage and macrophage compartments by fluorescent staining and confocal microscopy.

[0201] Briefly, 1.5 g of lipids DOPE-CHEMS (2:1 molar ratio) was dissolved in 20 ml of a solvent mixture (chloroform/hexane volume ratio 2:3) and used as the organic phase (O1). PVA (2% w/v), PEG-8000 (6% w/v) and a Myovirus Staphylococcus aureus phage K were solubilized in Tris-HCl buffer (W1) to form the first aqueous phase.

[0202] Production of the W1/O1 nanoemulsion was carried out using a nanoporous glass membrane 40 (i.e. membrane emulsification) as illustrated in FIG. 4. The water phase was passed through the porous membrane to form nanodroplets at the membrane surface where they then detach under low shear and were dispersed in the oil phase.

[0203] The nanoporous glass membrane (SPG Technology Co., Ltd, Japan) comprises pores having a pore size of 1.1 μm. The nanoporous membrane was immersed in the oil phase (O1) and kept under constant gentle stirring to provide gentle shear across the membrane surface. 5 ml of the inner aqueous phase (W1) was transferred to 20 ml of the oil phase (O1) by pushing the water phase W1 under pressure (1 to 10 times greater than the critical pressure) across a 2 cm length of the tubular hydrophobic nanoporous glass membrane. This W1/O1 nanoemulsion was kept stirring throughout.

[0204] As illustrated in FIG. 1, 25 ml of the primary W1/O1 emulsion was poured into 75 ml of the second aqueous phase (the outer phase W2; PVA 10% w/v, Poloxamer-188 5% w/v) under gentle stirring to form the secondary emulsion. De-wetting of the double emulsion occurs over a period of several minutes at room temperature. Poloxamer-188 was used to adjust the interfacial tension between phases and thereby aid dewetting.

[0205] Analogous to FIG. 5, 10 ml of the secondary emulsion now containing the nanovesicles was aliquoted in a 50 ml centrifuge tube and layered on a 40 ml sucrose gradient (layers 10 wt %, 20 wt %, 30 wt % and 40 wt %) and centrifuged at 50,000 g (Beckman Coulter, Model Avanti JXN-30) for 12 hours at 4° C. Two vesicle rich bands were generated. The “empty” nanovesicles, which do not encapsulate phage K are less dense and are present in the upper layer. The nanovesicles with cargo are denser and present in the lower layer 54b. Each vesicle rich layer was removed using a syringe to pierce the tube and dialysed several times using a 12-14 kDa dialysis membrane and resuspended in Tris-HCl buffer. Vesicles were stored at 4° C.

[0206] DOPE-CHEMS liposomes were internalized to a high level as we imaged a high intracellular concentration of fluorescent phage inside the macrophages. Upon uptake of phage K in DOPE-CHEMS liposomes, macrophage cells (FIG. 16) were still viable; indicating that encapsulated phage K was not cytotoxic to the macrophages.

[0207] FIG. 16 shows intracellular delivery of a large myophage (phage K) encapsulated in liposomes into a human macrophage cell line.

Example 10

[0208] We co-encapsulated LLO and S. aureus specific endolysin CHAP.sub.K in pH-responsive DOPE-CHEMS liposomes using the method described in example 9. Briefly, 1.5 g of lipids DOPE-CHEMS (2:1 molar ratio) was dissolved in 20 ml of a solvent mixture (chloroform/hexane volume ratio 2:3) and used as the organic phase (O1). PVA (2% w/v), PEG-8000 (6% w/v) containing LLO (200 μg ml.sup.−1) and purified CHAP.sub.K (50 μg ml.sup.−1) in SM buffer (W1) to form the first aqueous phase. Production of the W1/O1 nanoemulsion was carried out using a 500 nm nanoporous glass membrane (i.e. membrane emulsification) as per the procedure outlined in example 9.

[0209] Macrophages were infected with a deadly concentration of S. aureus and then treated with either empty liposomes as a control (FIG. 17, top row) or LLO/CHAP.sub.K liposomes (FIG. 17, bottom row).

[0210] In the former sample, we found that only few living cells (FIG. 17, top row) and those that are alive are heavily infected with S. aureus (as shown by cell morphology and presence of intracellular S. aureus). In the endolysin-treated sample, instead, we found a high number of cells with normal morphology, implying recovery from the infection, and decreased occurrence of intracellular S. aureus.

[0211] FIG. 17 shows recovery of macrophages treated with a lethal dose of Staphylococcus aureus and then delivery of encapsulated endolysin and its release from the macrophages using a co-encapsulated haemolysin LLO which breaks the liposome upon acidification of the endosome.

REFERENCES

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