Method for producing and processing dynamic metastable polymersomes under continuous flow

Field of the Invention

[0001] The present invention relates to a method for producing metastable polymersomes and an apparatus for preparing the same. In particular, the present invention relates to metastable polymersome produced by the method and apparatus, and metastable polymersomes which can be further processed downstream by an external stimulus.

[0002] In particular, the present invention relates to continuous production, and optionally downstream processing of, polymersomes under continuous flow conditions. The present invention can produce near-monodisperse polymersomes in significant quantities (>3 g/h). The polymersomes produced by the present invention are metastable, such that their physical properties including size or shape/morphology can be manipulated downstream after initial polymersome formation by using an external stimulus such as temperature, pH and/or osmosis. The polymersomes of the invention find particular use in a variety of technical fields, such as in pharmaceuticals, agriculture, paint coatings and implantable scaffolds. However, it will be appreciated that the invention is not limited to these particular fields of use.

Background of the Invention

[0003] 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.

[0004] Polymersomes, also known as "polymer vesicles", are hollow polymer (nano)particles comprised of amphiphilic block co-polymers which have the ability to encapsulate hydrophobic and hydrophilic materials such as therapeutic agents and other additives in their membrane structure and hollow core, respectively. Polymersomes have high chemical and physical stability and are considered robust alternatives to liposomes (or "lipid vesicles" generally based on phospholipids rather than polymers). Liposomes are commonly used in the pharmaceutical and cosmetic industry such as a component in some mRNA vaccines and skin care creams, however, are susceptible to degradation as phospholipids have poor long-term stability. [0005] Accordingly, polymers have gained considerable interest as an alternative to liposomes and have been extensively explored in the drug delivery field as nano-sized containers for the delivery of therapeutic agents. Other areas of application of polymersomes include use as artificial cell scaffolds in synthetic biology and solubilizers of insoluble compounds in aqueous environments such as organic dyes or inorganic compounds.

[0006] Polymersomes are most commonly prepared under batch conditions using a selfassembly technique known as precipitation (also known as the co-solvent process), which relies on a change in solvent quality to drive polymersome formation from block co-polymers. This preparation method can also be referred to as nanoprecipitation if the polymersomes formed are in the nanoscale dimension. Precipitation generally involves (i) the dissolution of a block co-polymer in a good solvent, (ii) the addition of a non-solvent for the hydrophobic block, followed by (iii) the removal of the good solvent by evaporation or dialysis. Use of a good solvent ensures that the block co-polymer is molecularly dissolved to effectively carry out selfassembly. Next, the subsequent addition of a non-solvent, typically water, selectively precipitates the hydrophobic block, and initiates polymersome formation. Finally, the third step removes the good solvent, leaving behind an aqueous polymersome solution ready for use in solvent-free applications.

[0007] However, this technique suffers from several drawbacks including poor scalability, batch-to-batch variation, and non-uniform polydispersity. These drawbacks are primarily due to the second step of the precipitation process, during which a non-solvent is added to the system. This is typically performed either slowly using a syringe pump or abruptly in one portion using a pipette. Irrespective of how the non-solvent is introduced, the timescale of mixing is restricted to the order of seconds. In effect, the block co-polymer inevitably experiences an inhomogeneous change in solvent quality throughout the solution during mixing, ultimately leading to inhomogeneous nucleation and self-assembly. This issue can be amplified when the process is carried out on large scales. The preparation process for precipitation is generally also tedious because it requires multiple procedural steps (e.g., polymer dissolution, selfassembly, purification, and post-preparation processing) which cannot be carried out concurrently due to the nature of the batch method.

[0008] To address these drawbacks, researchers have turned their attention to flow-based systems using microfluidics such as double emulsion templating. This top-down technique, which relies on the confined phase separation of block co-polymers in water/oil/water (w/o/w) emulsion droplets, can be used to produce monodisperse polymersomes with high reproducibility. However, drawbacks include size limitations (ranging from tens to hundreds of pm) and poor scalability because microfluidics can only operate at flow rates of several microliters per minute (pL/min). Other microfluidics devices, such as those based on hydrodynamic flow focusing or liquid plugs produce polymersomes with sub-micrometer sizes but suffer from non-uniform polydispersity and again, poor scalability owing to their low tolerable flow rates.

[0009] Another drawback of both the techniques is that the polymersomes produced are kinetically trapped structures (i.e., polymersomes whose physicochemical properties are unalterable after formation). Kinetically trapped polymersomes hinders a user from manipulating polymersome properties in situ via downstream processing. From a continuous flow production perspective, this also limits the full potential because advantages of continuous flow relate to modularity, where one can, for instance, equip additional flow accessories to integrate downstream processes in a plug-and-play fashion.

[0010] Given the limitations of current polymersome formation methods, it is therefore desirable to provide an improved polymersome preparation method.

[0011] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

[0012] Although the invention will be 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.

Summary of the Invention

[0013] Continued research interest and development of polymersomes has driven the desire to develop an improved polymersome preparation processes. In particular, there is a desire to develop improved polymersome preparation processes which are metastable such that the polymersomes can be manipulated in situ via downstream processing to induce for example, morphological changes of the polymersome structures. For example, downstream processing can induce at least one of at least a change in size, change in shape (i.e., morphology) and a change of surface chemistry (e.g., charge).

[0014] According to one aspect, the present invention provides a method for producing metastable polymersomes under continuous flow conditions, the method comprising the steps of: providing a stream (A) of a solution of an amphiphilic block co-polymer in an organic solvent; providing a stream (B) of a non-solvent; and mixing stream (A) and stream (B) under conditions suitable to form metastable polymersomes. [0015] Advantageously, the present inventors have developed a method for producing metastable polymersomes suitable for downstream processing.

[0016] In certain embodiments, the ratio of a flow rate of stream (A) to a total flow rate of stream (A) and (B) is between a range of about 0.2 to about 0.95, preferably between about 0.5 to about 0.8, more preferably between about 0.6 to about 0.8 and yet more preferably between about 0.6 to about 0.7. The present inventors surprisingly found that increasing the ratio of a flow rate of stream (A) to a total flow rate of stream (A) and (B) favours polymersome formation compared to other morphologies such as micelles.

[0017] In certain embodiments, the mixing of stream (A) and stream (B) is under non-laminar flow conditions. In preferred embodiments, the mixing of stream (A) and stream (B) is under turbulent flow conditions. In certain embodiments, the mixing of stream (A) and stream (B) is under laminar flow conditions.

[0018] Advantageously, mixing stream (A) and stream (B) under non-laminar flow conditions and more preferably under turbulent flow conditions provides formation of metastable polymersomes which can be produced at scale unlike alternative continuous fluid methods such as microfluidics. This is because polymersome formation using microfluidics requires laminar flow conditions to ensure the desired polymersome morphology is obtained.

[0019] In some embodiments, the total flow rate of stream (A) and stream (B) is between about 0.5 to about 50 mL/min, about 0.5 to about 25 mL/min, preferably between about 2 to about 20 mL/min, yet more preferably between about 4 to about 20 mL/min. As would be appreciated by a skilled addressee, the total flow rate of stream (A) and stream (B) can depend on the pump used. In industrial settings, significantly higher total flow rates can be used. In these embodiments, an industrial grade mixer can be used together with industrial grade pumps. In these embodiments, the total flow rate of stream (A) and stream (B) can be in the order of litres per minute.

[0020] The present inventors surprisingly found that when the total flow rate of stream (A) and stream (B) is greater than about 4 mL/min, micromixing efficiency is sufficiently high as to provide immediate homogenisation within the mixer.

[0021] Advantageously, the metastable polymersomes can be processed downstream by at least one of thermal annealing, thermal cooling, and/or secondary (downstream) mixing to control at least one of polymersome size, polydispersity and morphology. In some embodiments, the method comprises an annealing step. In some embodiments, the method comprises a cooling step. In some embodiments, the method comprises exposing the metastable polymersomes to an external stimulus. [0022] In certain embodiments, the method comprises a dialysis step. In this embodiment, the substantial removal of the good solvent quenches the polymersomes in a kinetically entrapped state which provides stable polymersomes.

[0023] According to another aspect, the present invention provides an apparatus for producing metastable polymersomes, the apparatus comprising: a first reservoir for receiving a solution of an amphiphilic block co-polymer, and having an outlet adapted to provide a stream (A) of a solution of the amphiphilic block co-polymer in an organic solvent; a second reservoir having an outlet adapted to provide a stream (B) of a non-solvent; a mixer having a first inlet in fluid communication with the outlet of the first reservoir and second inlet in fluid communication with the outlet of the second reservoir, and an outlet to dispense the metastable polymersomes, wherein the mixer is adapted to mix stream (A) and stream (B) under conditions suitable to form metastable polymersomes.

[0024] In certain embodiments, the non-solvent is an aqueous solution, a polar organic solvent and combinations thereof. In some embodiments, the aqueous solution is water. In preferred embodiments, the non-solvent is an aqueous solution.

[0025] In certain embodiments, the mixer is configured to mix the streams under non- laminar flow conditions. In preferred embodiments, the mixer is configured to mix the streams under turbulent flow conditions.

[0026] In certain embodiments, the apparatus further comprises at least one of an equilibration conduit, an annealing conduit and a cooling conduit downstream of the mixer. In certain embodiments, the apparatus further comprises an equilibration conduit in fluid communication with the mixer, preferably, the equilibration conduit is at a temperature of between about 15 to about 35 °C. In certain embodiments, the apparatus further comprises an annealing conduit in fluid communication with the equilibration conduit or with the mixer, preferably, the annealing conduit is at a temperature of between about 15 to about 80 °C. In certain embodiments, the apparatus further comprises a cooling conduit in fluid communication with the annealing conduit, preferably, the cooling conduit is at a temperature of between about 30 to about 50 °C.

[0027] In some embodiments, the apparatus further comprises a downstream mixer having a first inlet in fluid communication with an outlet of the equilibration conduit, annealing conduit, cooling conduit or backpressure regulator and a second inlet in fluid communication with the outlet of a third reservoir for receiving a morphological change inducing additive, and an outlet to dispense downstream processed metastable polymersomes.

[0028] Advantageously, the provision of at least one of the annealing conduit, the cooling conduit and downstream mixer provides downstream processing of the metastable polymersomes in use to control at least one of polymersome size, polydispersity and morphology. This is because the apparatus provides modularity in a plug-and-play fashion and can integrate downstream processes without requiring discrete steps and equipment.

[0029] In preferred embodiments, the apparatus further comprises a backpressure regulator prior to an apparatus outlet. Advantageously, the backpressure regulator can increase the boiling point of the organic phase of the mixed streams (A) and (B) to prevent solvent outgassing and/or evaporation.

[0030] In some embodiments, the apparatus further comprises a dialysis cell in fluid communication with an apparatus outlet. Advantageously, the dialysis cell can provide a downstream purification step to substantially remove the organic solvent from the solution comprising the metastable polymersomes under continuous flow conditions which significantly reduces production time and improves efficiency because traditional dialysis using a dialysis tube or bag is not required after polymersome formation which typically requires at least several hours to days for sufficient organic solvent removal.

[0031] According to yet another aspect, the present invention provides metastable polymersomes produced by the method as described herein.

[0032] According to a further aspect, the present invention provides use of the apparatus as described herein for producing metastable polymersomes.

Definitions

[0033] 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. 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.

[0034] As used herein, the term “metastable polymersome” refers to polymersomes which are in their non-equilibrium state. Metastable polymersomes can be processed after initial formation by external stimuli such as temperature, osmosis, pH and the like to control at least one of polymersome size, polydispersity and morphology. [0035] As used herein, the term “dynamic” refers to polymersomes (either in equilibrium or non-equilibrium state) which can be downstream processed to control at least one of polymersome size, polydispersity and morphology.

[0036] As used herein, the term “kinetically trapped”, “kinetic traps” and the like refers to self-assembled structures such as polymersomes and micelles which are prevented from equilibrating into their ordered free-energy minima. For example, when the metastable polymersomes produced by the present invention are transferred into water, the hydrophobic block of the amphiphilic block-co-polymer transitions into a glassy state in the absence of any plasticising organic solvent which is known as a “quenched state”.

[0037] As used herein, the term “non-solvent” (also known as a non-selective solvent or anti-solvent) refers to a solvent in which the amphiphilic block co-polymer has low solubility at 25 °C at 1 atm. For example, the addition of a non-solvent to a molecularly dissolved solution of an amphiphilic block co-polymer results in precipitation of at least one polymer block of the amphiphilic block co-polymer.

[0038] Unless the context clearly requires otherwise, throughout the description and the claims, the words “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”.

[0039] As used herein, the phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. When the phrase “consists of” (or variations thereof) appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole. As used herein, the phrase “consisting essentially of” limits the scope of a claim to the specified elements or method steps, plus those that do not materially affect the basis and novel characteristic(s) of the claimed subject matter.

[0040] 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’.

[0041] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term “about”. The examples are not intended to limit the scope of the invention. In what follows, or where otherwise indicated, “%” will mean “weight %”, “ratio” will mean “weight ratio” and “parts” will mean “weight parts”.

[0042] The term “substantially” as used herein shall mean comprising more than 50% by weight, where relevant, unless otherwise indicated.

[0043] The recitation of a numerical range using endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0044] 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.

[0045] It must also be noted that, as used in the specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0046] The prior art referred to herein is fully incorporated herein by reference.

[0047] Although exemplary embodiments of the disclosed technology are explained in detail herein, it is to be understood that other embodiments are contemplated. Accordingly, it is not intended that the disclosed technology be limited in its scope to the details of construction and arrangement of components set forth in the following description or illustrated in the drawings. The disclosed technology is capable of other embodiments and of being practiced or carried out in various ways.

Brief Description of the Drawings

[0048] Preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:

[0049] Figure 1. Characterisation data of PEC^-b-PSse- A) ¹ H NMR spectrum (400 MHz, CDCh) and (B) DMF GPC trace. The small shoulder observed at 28.5 min corresponds to unreacted PEO44 left behind during PEO44-DDMAT macroRAFT agent synthesis.

[0050] Figure 2. Photograph showing an embodiment used for continuous flow selfassembly.

[0051] Figure 3. A series of photographs (A-E) outlining a degassing procedure used in an embodiment of the present invention. The process was repeated at least five times to ensure proper degassing. [0052] Figure 4. (A) Schematic of the continuous flow setup and chemical structure of the polymer (PEO44-6-PS86) used in an embodiment of the present invention. (B) DLS particle size distributions obtained at different asymmetric flow rates (Qorganic/Qtotai) . (C) Intensity-averaged hydrodynamic diameters (Dh, intensity) and polydispersity indices (PDI) derived from the data shown in B. The different shades of black in C depict a pseudo-phase diagram. TEM images of (D) micelles obtained at Qorganic/Qtotai = 0.2, (E) a mixture of micelles and polymersomes at Qorganic/Qwater = 0.4 and (F) polymersomes obtained at Qorganic Qtotai = 0.6. All samples were analysed in their respective organic solvent/water mixtures.

[0053] Figure 5. (A) Proposed free energy diagram depicting two accessible continuous flow self-assembly pathways using the method for an embodiment of the present invention. Micelles generate via Pathway 1 are kinetically trapped, while polymersomes generated via Pathway 2 are metastable and grow with age until an equilibrium state is reached. (B) Turbidity changes in a micelle Solution (Qorganic/Qtotai = 0.1) VS a pOlymerSOme Solution (Qorganic/Qtotai = 0.7) monitored over a 14-day period. Both samples were analysed in their respective organic solvent/water mixtures. (C) DLS data showing how polymersomes can be quenched in water via dialysis on different aging days to give different polymersome sizes. TEM images of (D) pristine polymersomes quenched on day 0 (immediately after continuous flow self-assembly) and (E) aged polymersomes quenched after 14 days of aging. Corresponding cryo-TEM images are shown inset in D and E. (F) Box-and-whiskers plots showing the membrane thickness (dmembrane) difference between the two polymersome samples shown in D and E (n = 115 polymersomes based on cryo-TEM images, P<0.0001 determined by paired t-test). (G) Schematic illustrating how small metastable polymersomes spontaneously age into larger equilibrium polymersomes. Despite their equilibrium state, the larger, aged polymersomes remain dynamic, and can be transformed into polymersome stomatocytes by osmotic deformation. (H) (i) TEM and (ii, iii) cryo-TEM images of stomatocytes obtained by subjecting a 14-day aged polymersome sample to osmotic deformation using 50 mM NaCI. (I) DLS data showing polymersomes generated at Qorganic/Qtotai = 0.7 (Pathway 2) increase in size with aging for 7 days before plateauing, while micelles generated at Qorganic/Qtotai = 0.1 (Pathway 1) retain their size even after aging for 14 days. All samples were analysed in their respective organic solvent/water mixtures.

[0054] Figure 6. (A) DLS data showing the effects of polymer concentration (c _po iymer) on polymersome size. (B) DLS data showing the effects of total flow rate (Qtotai) on polymersome size. (C) TEM image of near-monodisperse polymersomes prepared at Qtotai = 8 mL/min and Cpoiymer = 1 mg/mL. (D) TEM image of polymersomes produced at a production rate of 3.02 g of polymersomes/hour (Qtotai = 8 mL/min, c _poiy mer = 9 mg/mL). Shown inset in D is a DLS particle size distribution for the same sample. All experiments in A-D were conducted on quenched aqueous polymersome samples originally prepared at Qorganic/Qtotai = 0.7. (E,F) Chemical structures, TEM images and DLS particle distributions of (E) PE044-b-P4VP2i-b-PS3oo polymersomes and (F) PAA26-6-PS81 polymersomes. Chemical structures of (E(i)) PEO44-6- P4VP2i-b-PS3oo and (F(i)) PAA26-6-PS81, and TEM images of their corresponding polymersome structures (ii) before and (iii) after aging for 7 days. The growth process during aging was monitored for 7 days by (iv) DLS.

[0055] Figure 7. Photograph of solutions obtained using the continuous flow setup in Figure 2 at flow rates of 0.5, 1 , 2, 4 and 8 mL/min. For all flow rates tested, the occurrence of polymersome formation by TEM analysis was confirmed. However, at lower flow rates (0.5-2 mL/min), the polymersome phase, which displays a blue tint due to light scattering, is demixed from the organic phase, presumably due to the peculiar miscibility gap between THF and water. The interface between the two phases is highly diffuse, and if left to sit undisturbed, would gradually mix over time. A gentle swirl or quick flip of the vial is sufficient to homogenise these demixed solutions without significantly compromising polymersome quality. At higher flow rates (4-8 mL/min), the micromixing efficiency is sufficiently high as to allow immediate homogenisation within the mixing chamber of the micromixer.

[0056] Figure 8. (A) ¹ H NMR spectra (400 MHz, CDCI3) and (B) DMF GPC traces of PEO ₄₄ - b-P4VP _2i and PE0 ₄ 4-6-P4VP ₂ i-b-PS3oo.

[0057] Figure 9. (A) ¹ H NMR spectra (400 MHz) of PtBA ₂₆ in CDCI3, PtBA ₂ 6-6-PS _8i in CDCI3, and PAA ₂ 6-£>-PS8i in 1 :5 (v/v) MeOD/CDCh. (B) DMF GPC traces of PtBA ₂ 6 and PtBA ₂ 6-b-PS8i. PAA ₂ 6-£>-PS8i was not analysed by GPC because PAA strongly interacts with the GPC column media.

[0058] Figure 10. (A) Schematic of a continuous flow setup used in an embodiment of the present invention for polymersome self-assembly and downstream annealing to manipulate polymersome size. BPR, back-pressure regulator. (B) DLS particle size distributions of aqueous polymersomes prepared at different annealing temperatures (Tanneaiing). (C) Intensity- averaged hydrodynamic diameters (Dh, intensity) and polydispersity indices (PDI) derived from the data shown in B. TEM images of polymersomes annealed at (D) 20 °C, (E) 50 °C and (F) 70 °C for a residence time under heating (/residence, annealing) of 30 s. Flow conditions used for polymersome formation: Qtotai = 4 mL/min, Qorganic/Qtotai = 0.7 and c _po iymer = 1 mg/mL. All samples in B-F were dialysed against water prior to analysis.

[0059] Figure 11 . Photograph of setup of an embodiment used to perform continuous flow self-assembly and downstream annealing. [0060] Figure 12. (A) Schematic of a continuous flow setup used in an embodiment of the present invention for sequential polymersome self-assembly, downstream polymersome size control and shape transformation. BPR, back-pressure regulator. (B) Turbidity assay monitored at 402 nm revealing that the growth process of 70 °C-annealed polymersomes occurs during cooling. (C) (i) TEM and (ii) cryo-TEM images of stomatocytes obtained by downstream annealing and shape transformation. Flow conditions used for polymersome formation: Qtotai = 4 mL/min, Q _or ganic/Qtotai = 0.7 and c _poiy mer = 1 mg/mL. Downstream manipulation conditions: Tannealing ⁼ 70 , /residence, annealing ⁼ 30 S, 7cooling ^— 40 C, /residence, cooling ^— 7.5 min, CNaci = 5.05 M and QNaci = 0.04 mL/min. C

[0061] Figure 13. Photograph of setup of an embodiment used to perform continuous flow self-assembly, downstream annealing, cooling, and shape transformation.

[0062] Figure 14. (A) Schematic of continuous flow setup in combination with downstream purification using (B) an embodiment of an in-line dialysis cell.

[0063] Figure 15. Schematic showing use of pH stimuli to induce downstream morphological transformation (i.e. , polymersome-to-micelle transition) for PAA26-£>-PSSI.

Detailed Description of the Invention

[0064] 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.

Method for producing metastable polymersomes

[0065] As discussed above, the present invention relates to a method for producing metastable polymersomes under continuous flow conditions, the method comprising the steps of: providing a stream (A) of a solution of an amphiphilic block co-polymer in an organic solvent; providing a stream (B) of a non-solvent; mixing stream (A) and stream (B) under conditions suitable to form metastable polymersomes. In these embodiments, stream (A) is an organic solvent stream comprising a solution of an amphiphilic block co-polymer and stream (B) is a non-solvent stream.

[0066] As would be appreciated by a skilled addressee, any suitable ratio of the flow rate of stream (A) to the total flow rate of streams (A) and (B) can be used to form metastable polymersomes. In certain embodiments, the ratio of the flow rate of stream (A) (Q _or ganic) to the total flow rate of stream (A) and (B) (Qtotai) is between a range of about 0.2 to about 0.95. In certain embodiments, the ratio of the flow rate of stream (A) (Qorganic) to the total flow rate of stream (A) and (B) (Qtotai) is between a range of about 0.2 to about 0.9. In certain embodiments, the ratio of the flow rate of stream (A) (Qorganic) to the total flow rate of stream (A) and (B) (Qtotai) is between a range of about 0.2 to about 0.7. In certain embodiments, the ratio of the flow rate of stream (A) (Qorganic) to the total flow rate of stream (A) and (B) (Qtotai) is between a range of about 0.3 to about 0.9. In certain embodiments, the ratio of the flow rate of stream (A) (Qorganic) to the total flow rate of stream (A) and (B) (Qtotai) is between a range of about 0.4 to about 0.9. In certain embodiments, the ratio of the flow rate of stream (A) (Qorganic) to the total flow rate of stream (A) and (B) (Qtotai) is between a range of about 0.5 to about 0.9. In certain embodiments, the ratio of the flow rate of stream (A) (Qorganic) to the total flow rate of stream (A) and (B) (Qtotai) is between a range of about 0.6 to about 0.9. In certain embodiments, the ratio of the flow rate of stream (A) (Qorganic) to the total flow rate of stream (A) and (B) (Qtotai) is between a range of about 0.5 to about 0.8. In certain embodiments, the ratio of the flow rate of stream (A) (Qorganic) to the total flow rate of stream (A) and (B) (Qtotai) is between a range of about 0.55 to about 0.8. In certain embodiments, the ratio of the flow rate of stream (A) (Qorganic) to the total flow rate of stream (A) and (B) (Qtotai) is between a range of about 0.6 to about 0.8. In certain embodiments, the ratio of the flow rate of stream (A) (Qorganic) to the total flow rate of stream (A) and (B) (Qtotai) is between a range of about 0.55 to about 0.7. In preferred embodiments, the ratio of the flow rate of stream (A) (Qorganic) to the total flow rate of stream (A) and (B) (Qtotai) is between a range of about 0.6 to about 0.7. In certain embodiments, the ratio of the flow rate of stream (A) (Qorganic) to the total flow rate of stream (A) and (B) (Qtotai) is between a range of about 0.85 to about 0.95. In certain embodiments, the ratio of the flow rate of stream (A) (Qorganic) to the total flow rate of stream (A) and (B) (Qtotai) is about 0.6. In certain embodiments, the ratio of the flow rate of stream (A) (Qorganic) to the total flow rate of stream (A) and (B) (Qtotai) is about 0.8. In certain embodiments, the ratio of the flow rate of stream (A) (Qorganic) to the total flow rate of stream (A) and (B) (Qtotai) is about 0.9. In preferred embodiments, the ratio of the flow rate of stream (A) (Qorganic) to the total flow rate of stream (A) and (B) (Qtotai) is about 0.7.

[0067] In certain embodiments, the mixing of stream (A) and stream (B) is under non-laminar flow conditions. In preferred embodiments, the mixing of stream (A) and stream (B) is under turbulent flow conditions. In certain embodiments, the mixing of stream (A) and stream (B) is under laminar flow conditions. Advantageously, the mixing of stream (A) and stream (B) under turbulent flow conditions requires less precision equipment to form the metastable polymersomes which allows for scalability in production and ease of handling.

[0068] As would be appreciated by a skilled addressee, any suitable total flow rate of stream (A) and stream (B) can be used to form metastable polymersomes. In some embodiments, the total flow rate of stream (A) and stream (B) is between about 0.5 to about 50 mL/min. In some embodiments, the total flow rate of stream (A) and stream (B) is between about 0.5 to about 40 mL/min. In some embodiments, the total flow rate of stream (A) and stream (B) is between about 0.5 to about 30 mL/min. In some embodiments, the total flow rate of stream (A) and stream (B) is between about 0.5 to about 25 mL/min. In some embodiments, the total flow rate of stream (A) and stream (B) is between about 1 to about 20 mL/min. In some embodiments, the total flow rate of stream (A) and stream (B) is between about 0.5 to about 15 mL/min. In some embodiments, the total flow rate of stream (A) and stream (B) is between about 2 to about 20 mL/min. In some embodiments, the total flow rate of stream (A) and stream (B) is between about 4 to about 20 mL/min. In some embodiments, the total flow rate of stream (A) and stream (B) is between about 1 to about 15 mL/min. In some embodiments, the total flow rate of stream (A) and stream (B) is between about 1 to about 10 mL/min. In some embodiments, the total flow rate of stream (A) and stream (B) is between about 1 to about 9 mL/min. In some embodiments, the total flow rate of stream (A) and stream (B) is between about 1 to about 8 mL/min. In some embodiments, the total flow rate of stream (A) and stream (B) is between about 2 to about 8 mL/min. In certain embodiments, the total flow rate of stream (A) and stream (B) is between about 4 to about 8 mL/min.

[0069] The present inventors surprisingly found that when the total flow rate of stream (A) and stream (B) is greater than about 4 mL/min, micromixing efficiency is sufficiently high as to provide immediate homogenisation within the mixer. In certain embodiments depending on the solvent(s) used of stream (A), at lower flow rates (for example, 0.5-2 mL/min), the polymersome containing phase (primarily aqueous component) in the collected sample can phase separate from the organic phase. However, lower flow rates can still be used to form metastable polymersomes because if left undisturbed, the two phases would gradually mix over time. Alternatively, mechanical agitation can also readily homogenise and mix the separated phases without significantly compromising polymersome quality.

[0070] As would be appreciated by a skilled addressee, the total flow rate of stream (A) and stream (B) can depend on the pump used. In industrial settings, significantly higher total flow rates can be used. In these embodiments, an industrial grade mixer can be used together with industrial grade pumps. In these embodiments, the total flow rate of stream (A) and stream (B) can be in the order of litres per minute. In certain embodiments, the total flow rate of stream (A) and stream (B) is less than about 70 L/min, less than about 60 L/min, less than about 50 L/min, less than about 40 L/min, less than about 30 L/min, less than about 20 L/min, less than about 10 L/min, less than about 5 L/min, less than about 1 L/min, less than about 0.5 L/min or less than about 0.3 L/min.

[0071] The amount of metastable polymersomes formed by the present invention can be controlled based on the total flow rate. For example, the higher the total flow rate, the greater the amount of metastable polymersomes formed. In certain embodiments, the amount of metastable polymersomes formed is between about 0.2 to about 15 g/h, between about 0.2 to about 12 g/h, between about 0.2 to about 10 g/h, between about 0.2 to about 8 g/h, between about 0.2 to about 6 g/h, between about 0.2 to about 5 g/h, between about 0.5 to about 5 g/h, between about 1 to about 5 g/h, between about 1 to about 4 g/h or about 3 g/h. In certain embodiments, the amount of metastable polymersomes formed is greater than about 0.2 g/h, greater than about 0.5 g/h, greater than about 1 g/h, greater than about 2 g/h or greater than about 3 g/h. In certain embodiments, the amount of metastable polymersomes formed is less than about 15 g/h, less than about 12 g/h, less than about 10 g/h, less than about 8 g/h, less than about 5 g/h, less than about 4 g/h or less than about 3 g/h. Under industrial scale conditions, the amount of metastable polymersomes that are formed can be in the order of kg/h. The amount of metastable polymersomes formed can be dependent on the total flow rate and the concentration of the amphiphilic block co-polymer.

[0072] In some embodiments, the flow rate of stream (A) is between about 0.2 to about 40 mL/min. In some embodiments, the flow rate of stream (A) is between about 0.2 to about 32 mL/min. In some embodiments, the flow rate of stream (A) is between about 0.2 to about 24 mL/min. In some embodiments, the flow rate of stream (A) is between about 0.2 to about 20 mL/min. In some embodiments, the flow rate of stream (A) is between about 0.2 to about 10 mL/min. In some embodiments, the flow rate of stream (A) is between about 0.5 to about 10 mL/min. In some embodiments, the flow rate of stream (A) is between about 1 to about 10 mL/min. In some embodiments, the flow rate of stream (A) is between about 1 to about 8 mL/min. In some embodiments, the flow rate of stream (A) is between about 2 to about 8 mL/min. In some embodiments, the flow rate of stream (A) is between about 2.5 to about 7.5 mL/min. In some embodiments, the flow rate of stream (A) is between about 2 to about 5 mL/min. In some embodiments, the flow rate of stream (A) is between about 2 to about 4 mL/min. In some embodiments, the flow rate of stream (A) is between about 5 to about 8 mL/min. In some embodiments, the flow rate of stream (A) is between about 5.5 to about 7.5 mL/min.

[0073] As would be appreciated by a skilled addressee, the total flow rate of stream (A) can depend on the pump used. In industrial settings, significantly higher total flow rates can be used. In certain embodiments, the total flow rate of stream (A) is less than about 56 L/min, less than about 48 L/min, less than about 40 L/min, less than about 32 L/min, less than about 24 L/min, less than about 16 L/min, less than about 8 L/min, less than about 4 L/min, less than about 0.8 L/min, less than about 0.4 L/min or less than about 0.24 L/min.

[0074] In some embodiments, the flow rate of stream (B) is between about 0.1 to about 20 mL/min. In some embodiments, the flow rate of stream (B) is between about 0.1 to about 18 mL/min. In some embodiments, the flow rate of stream (B) is between about 0.1 to about 15 mL/min. In some embodiments, the flow rate of stream (B) is between about 0.1 to about 10 mL/min. In some embodiments, the flow rate of stream (B) is between about 0.1 to about 8 mL/min. In some embodiments, the flow rate of stream (B) is between about 0.1 to about 5 mL/min. In some embodiments, the flow rate of stream (B) is between about 0.4 to about 5 mL/min. In some embodiments, the flow rate of stream (B) is between about 0.8 to about 5 mL/min. In some embodiments, the flow rate of stream (B) is between about 0.8 to about 2.5 mL/min. In some embodiments, the flow rate of stream (B) is between about 0.8 to about 2.5 mL/min.

[0075] As would be appreciated by a skilled addressee, the total flow rate of stream (B) can depend on the pump used. In industrial settings, significantly higher total flow rates can be used. In certain embodiments, the total flow rate of stream (B) is less than about 14 L/min, less than about 12 L/min, less than about 10 L/min, less than about 8 L/min, less than about6 L/min, less than about 4 L/min, less than about 2 L/min, less than about 1 L/min, less than about 0.2 L/min, less than about 0.1 L/min or less than about 0.06 L/min.

[0076] The solution of the amphiphilic block co-polymer in an organic solvent may be at any suitable concentration. It is to be understood that the concentration should be sufficient to form polymersomes. Typically, the solution of the amphiphilic block co-polymer in an organic solvent is at a concentration of less than 30 mg/mL. In certain embodiments the concentration of the solution of the amphiphilic block co-polymer is between about 0.1 to about 30 mg/mL, between about 0.1 to about 25 mg/mL, between about 0.1 to about 20 mg/mL, between about 0.1 to about 15 mg/mL, between about 0.5 to about 15 mg/mL, between about 1 to about 12 mg/mL, between about 1 to about 10 mg/mL or preferably between about 1 to about 9 mg/mL.

[0077] In some embodiments, the method of the present invention further comprises an equilibration step. In this embodiment, the two streams (A) and (B) are mixed for a sufficient time to provide a substantially homogenised mixture before further downstream processing. Without being bound by any one theory, the present inventors believe that the equilibration step ensures that the pump pressures (or pump pressure profiles) are stable and/or the conduits are sufficiently purged of air bubbles and residual fluid (dead volume) and sufficiently wetted with the fluids of streams (A) and/or (B). In certain embodiments, the equilibration step is performed at a temperature of less than about 40 °C, less than about 35 °C or preferably less than about 30 °C. In certain embodiments, the equilibration step is performed at a temperature between about 10 °C to about 40 °C. In certain embodiments, the equilibration step is performed at a temperature between about 10 °C to about 35 °C. In certain embodiments, the equilibration step is performed at a temperature between about 15 °C to about 35 °C. In certain embodiments, the equilibration step is performed at a temperature between about 20 °C to about 30 °C. In certain embodiments, the equilibration step is performed at a temperature about 25 °C. In certain embodiments, the equilibration step is performed at ambient temperature.

[0078] In some embodiments, the equilibration step is performed for a duration (i.e., residence time) between about 1 to 8 times the volume of the equilibration conduit, between about 2 to 8 times the volume of the equilibration conduit, between about 2 to 6 times the volume of the equilibration conduit, between about 4 to 6 times the volume of the equilibration conduit, between about 4 to 5 times the volume of the equilibration conduit or preferably about 4 times the volume of the equilibration conduit.

[0079] As would be appreciated by a skilled addressee, in certain embodiments of the present invention, the dispensed metastable polymersomes can be allowed to age after initial polymersome formation. In this embodiment, the aging step can increase the size of the metastable polymersomes until an equilibrium state is reached.

[0080] In certain embodiments, the metastable polymersomes are aged for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days or at least 14 days.

[0081] In certain embodiments, the metastable polymersomes are aged between about 1 day to about 14 days, between about 1 day to about 12 days, between about 1 day to about 10 days, between about 1 day to about 8 days, between about 1 day to about 7 days, between about 3 days to about 7 days, or about 7 days.

[0082] Any suitable organic solvent can be used in the present invention to molecularly dissolve the amphiphilic block co-polymer. In preferred embodiments, the organic solvent is a polar organic solvent. In certain embodiments, the organic solvent is selected from the group consisting of dichloromethane, ethyl acetate, acetone, dimethylformamide (DMF), acetonitrile, dimethylacetamide (DMAc), chloroform, tetrahydrofuran (THF), dimethylsulfoxide (DMSO), dioxane, nitromethane, propylene carbonate, glycerol, glycol, /V-methyl-2-pyrrolidone (NMP), alcohols (such as n-butanol, n-propanol, ethanol, methanol, isopropanol) and combinations thereof. In preferred embodiments, the organic solvent is selected from the group consisting of tetra hydrofuran (THF), dimethylsulfoxide (DMSO), dimethylacetamide (DMAc), acetonitrile, dioxane, acetone, dimethylformamide (DMF), glycerol, alcohol and combinations thereof.

[0083] Any suitable non-solvent can be used in the present invention to form the polymersomes. In certain embodiments, the non-solvent is an aqueous solution, a polar organic solvent and combinations thereof. In some embodiments, the aqueous solution is water. In some embodiments, the non-solvent is an aqueous solution, alcohol and combinations thereof. Suitable alcohols include for example glycerol, glycol, n-butanol, n- propanol, ethanol, methanol, isopropanol and combinations thereof. In preferred embodiments, the non-solvent is an aqueous solution.

[0084] As would be appreciated by a skilled addressee, the specific organic solvent and non-solvent used in the present invention can depend on a number of factors including the specific amphiphilic block co-polymers used.

[0085] The polymersomes of the present invention can be used for a number of applications. In particular, the applications include drug delivery, therapeutic treatments, agriculture, paint coatings and diagnostic imaging techniques. In some embodiments, the polymersomes of the present invention are stable in the presence of biological media and therefore stable in biological environments, which allows the polymersomes to be used in biological applications. For example, the polymersomes can be used in intracellular drug delivery systems, extracellular remote-controlled drug delivery systems, closed-loop insulin delivery systems, biological targeting systems, and biomimetic protocells.

[0086] Accordingly, the polymersomes can be loaded with any suitable agent known to a skilled person which will be determined based on the desired use of the polymersome.

[0087] In some embodiments, the agent is a biological agent having a desired biological activity. The agent may be a pharmaceutically active agent or a veterinary active agent. As used herein, the term biological agent includes prodrugs and precursors which become active when administered to a subject in need thereof. In principle any agent that can be used can be delivered by the polymersomes of the present invention. Potential agents may include proteins or protein crystals, peptides, phospholipids, DNA, polymer-drug conjugates, hydrophobic and hydrophilic drugs (and prodrugs thereof), inorganic and organic nanoparticles e.g. metalorganic frameworks (MOFs), micelles, magnetite, and quantum dots. In other embodiments, the agent may have a detectable moiety for imaging such as a fluorescent dye for fluorescent imaging, a near-infrared dye for near-infrared imaging, and metal complexes which can be used as MRI, CT and PET contrast agents.

[0088] In some embodiments, the agent is a pigment (such as an inorganic or organic pigment), a filler, a pesticide, a herbicide, an antifungal agent, growth hormone and combinations thereof.

[0089] In a typical approach, the agent can be encapsulated into the polymersome by addition of the active agent in either of the solution of stream (A) or stream (B) depending on the solubility of the agent. In an alternative approach, the agent can be encapsulated by providing a stream (C) of a solution of an agent in an aqueous or organic solvent and mixed with streams (A) and (B). The agent can be encapsulated in the hollow core or the membrane of the polymersome depending on the hydrophobicity of the agent. For example, a hydrophilic agent is generally encapsulated in the hollow core whereas a hydrophobic agent is generally encapsulated in the membrane of the polymersome.

Downstream processing of method

[0090] As described herein, physical properties of the metastable polymersomes produced by the present invention can be manipulated downstream after initial formation including control of size, polydispersity or shape/morphology by using an external stimulus. Advantageously, the metastable polymersomes can be processed downstream by at least one of thermal annealing, thermal cooling, and/or secondary (downstream) mixing to control at least one of polymersome size, polydispersity and morphology.

[0091] In certain embodiments, the downstream process can include exposing the metastable polymersomes to an external stimulus as described herein. A suitable external stimulus includes pH, osmotic pressure, temperature, radiation, ultrasonication, electric field and combinations thereof. The external stimulus can be provided as part of the continuous flow method and apparatus thereof. Alternatively, the dispensed metastable polymersomes can be collected and exposed to the external stimulus batch-wise such as exposing the collected metastable polymersomes to different temperature environments using an oven, oil bath, water bath, heat lamp and the like.

[0092] For example, temperature as an external stimulus in relation to a continuous flow method and apparatus can be provided using an annealing and/or cooling step via an annealing conduit and/or cooling conduit, respectively.

[0093] In some embodiments, the method of the present invention further comprises an annealing step. The present inventors surprisingly found that the temperature of the annealing step can have an effect on the size of the polymersomes. Without being bound by any one theory, the present inventors believe that annealing reduces the time needed for the polymersomes to reach a global equilibrium state.

[0094] In certain embodiments, increasing the annealing temperature increases the relative size of the metastable polymersomes. In certain embodiments, the annealing step is performed at a temperature between about 20 °C to about 90 °C. In certain embodiments, the annealing step is performed at a temperature between about 15 °C to about 80 °C. In certain embodiments, the annealing step is performed at a temperature between about 20 °C to about 80 °C. In certain embodiments, the annealing step is performed at a temperature between about 20 °C to about 70 °C. In certain embodiments, the annealing step is performed at a temperature between about 30 °C to about 70 °C. In certain embodiments, the annealing step is performed at a temperature between about 40 °C to about 70 °C. In certain embodiments, the annealing step is performed at a temperature between about 50 °C to about 70 °C. In certain embodiments, the annealing step is performed at a temperature between about 60 °C to about 70 °C. In certain embodiments, the annealing step is performed at a temperature of about 70 °C.

[0095] As would be appreciated by a skilled addressee, the duration of the annealing step can be any suitable residence time which is dependent on the flow rate and volume of the annealing conduit chosen. As would be appreciated by a skilled addressee, the duration of the annealing step is the time needed for the metastable polymersomes to reach their equilibrium size. In this embodiment, once the metastable polymersomes have reached their equilibrium size, further annealing would not lead to any size increase (i.e., growth). In some embodiments, the annealing step is performed for a duration (i.e., residence time) between about 5 seconds to about 1 hour, between about 5 seconds to about 50 minutes, between about 5 seconds to about 40 minutes, between about 5 seconds to about 30 minutes, between about 5 seconds to about 20 minutes, between about 5 seconds to about 15 minutes, between about 5 seconds to about 10 minutes, between about 5 seconds to about 5 minutes, between about 5 seconds to about 3 minutes, between about 5 seconds to about 60 seconds, between about 10 seconds to about 50 seconds, between about 10 seconds to about 40 seconds, between about 10 seconds to about 35 seconds, between about 15 seconds to about 30 seconds, or about 15 seconds or about 30 seconds.

[0096] In some embodiments, the method of the present invention further comprises a cooling step. The present inventors surprisingly found that gradual cooling of the metastable polymersomes after the annealing step can increase the size of the metastable polymersomes (i.e., growth). Without being bound by any one theory, the present inventors believe that the cooling step can reduce and/or control the cooling rate sufficiently such that the annealed polymersomes can reach their equilibrium size during the predetermined cooling residence time.

[0097] In certain embodiments, the cooling step is performed at a temperature between about 20 °C to about 60 °C. In certain embodiments, the cooling step is performed at a temperature between about 30 °C to about 50 °C. In certain embodiments, the cooling step is performed at a temperature between about 35 °C to about 45 °C. In certain embodiments, the cooling step is performed at a temperature of about 40 °C. [0098] As would be appreciated by a skilled addressee, the duration of the cooling step can be any suitable residence time which is dependent on the flow rate and volume of the cooling conduit chosen. In some embodiments, the cooling step is performed for a duration (i.e., residence time) between about 1 minute to about 30 minutes, between about 3 minutes to about 25 minutes, between about 3 minutes to about 20 minutes, between about 3 minutes to about 15 minutes, between about 5 minutes to about 15 minutes, between about 5 minutes to about 10 minutes, or about 7.5 minutes.

[0099] In certain embodiments, the external stimulus can be pH. In these embodiments, the metastable polymersomes can be exposed to an acidic or basic environment. For example, the solution comprising metastable polymersomes can be exposed to an acid. Any suitable acid can be used in the present invention. Suitable acids can be selected from the group consisting of sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, boric acid, formic acid, acetic acid, citric acid, hydrobromic acid, perchloric acid, hydroiodic acid, trifluoroacetic acid, polyacids and combinations thereof. Examples of polyacids include polyacrylic acid, polymethacrylic acid and derivatives thereof.

[00100] In some embodiments, the solution comprising metastable polymersomes can be exposed to a base. Any suitable base can be used in the present invention. Suitable bases can be selected from the group consisting of sodium hydroxide, potassium hydroxide, magnesium hydroxide, ammonium hydroxide, calcium hydroxide, sodium bicarbonate, sodium carbonate, potassium carbonate, potassium phosphate, triethylamine, /V,/V-diisopropylethylamine, 1 ,8- diazabicyclo[5.4.0]undec-7-ene (DBU), polybase and combinations thereof. An example of a polybase is a polyamine.

[00101] In certain embodiments, the external stimulus can be osmotic pressure. In this embodiment, an osmotic agent can be used in the present invention to provide the osmotic pressure. In some embodiments, the osmotic agent is a polyol, a salt and combinations thereof. In certain embodiments, the polyol is selected from the group consisting of a sugar alcohol, a polyether polyol, a polyester polyol, a caprolactone polyol and combinations thereof. In some embodiments, the sugar alcohol is a monosaccharide, or a polysaccharide (including disaccharide). In some embodiments, the sugar alcohol is selected from the group consisting of glucose, sucrose, xylose, ethylene glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, mallotriitol, maltotetraitol, polygycitol and combinations thereof.

[00102] In some embodiments, the salt or salt solution can be used to provide an osmotic pressure gradient between the hollow core of the polymersome and the bulk solution. Any salt or salt solution can be used for osmosis. In certain embodiments, the salt forming anion can be selected from a chloride, nitrate, sulfate, permanganate, acetate, fluoride, bromide, phosphate, hydrogen phosphate, carbonate, bicarbonate, thiocyanate and combinations thereof. In certain embodiments, salt forming cation can be selected from ammonium, calcium, iron, magnesium, pyridinium, potassium, sodium, copper, lithium and combinations thereof. In preferred embodiments, the salt is selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium sulfate, potassium sulfate, magnesium sulfate, calcium sulfate, sodium phosphate, potassium phosphate, magnesium phosphate, calcium phosphate and combinations thereof.

[00103] In some embodiments, the osmotic agent is a polyethylene glycol, sucrose, salt or salt solution and combinations thereof.

[00104] In certain embodiments, the external stimulus can be radiation. Any suitable form of radiation can be used such as infrared, microwave, ultraviolet, visible, beta, alpha and combinations thereof.

[00105] As discussed herein, the exposure of external stimuli can control the size and/or shape/morphology of the metastable polymersomes. For example, exposure to salt for osmosis or pH can induce a change in morphology of the metastable polymersomes from polymersomes to stomatocytes, micelles and the like.

[00106] In certain embodiments, the method comprises a dialysis step. In this embodiment, the substantial removal of the good solvent quenches the polymersomes in a kinetically entrapped state (i.e, “quenched”) which provides stable polymersomes. In certain embodiments, the metastable polymersomes can be dialysed against a salt solution or water. For example, dialysis against a salt solution can induce a change in morphology of the polymersomes.

Apparatus

[00107] As discussed above, the present invention provides an apparatus for producing metastable polymersomes, the apparatus comprising: a first reservoir for receiving a solution of an amphiphilic block co-polymer, and having an outlet adapted to provide a stream (A) of a solution of the amphiphilic block co-polymer in an organic solvent; a second reservoir having an outlet adapted to provide a stream (B) of a non-solvent; a mixer having a first inlet in fluid communication with the outlet of the first reservoir and second inlet in fluid communication with the outlet of the second reservoir, and an outlet to dispense the metastable polymersomes, wherein the mixer is adapted to mix stream (A) and stream (B) under conditions suitable to form metastable polymersomes. [00108] In certain embodiments, the mixer is configured to mix the streams under non- laminar flow conditions. In preferred embodiments, the mixer is configured to mix the streams under turbulent flow conditions.

[00109] As described herein, by using a mixer configured to mix the streams (A) and (B) under non-laminar flow conditions and preferably under turbulent flow conditions allows the metastable polymersomes to be produced at scale without requiring the use of microminiaturised devices (i.e., microfluidic chips) and therefore metastable polymersomes can be formed without requiring highly sophisticated and precise devices.

[00110] The mixer can have any suitable configuration to mix the streams (A) and (B) under non-laminar flow conditions and preferably under turbulent flow conditions. In certain embodiments, the mixer is a mixing tee, preferably a high-pressure mixing tee such as a high pressure liquid chromatography (HPLC) mixing tee. In certain embodiments, the mixer is a high-pressure mixing tee, a confined impinging jet mixer (CUM), a multi-inlet vortex mixer (MIVM), a staggered herringbone mixer, a split-and-recombine mixer or a plug flow reactor. In preferred embodiments, the mixer is a high-pressure mixing tee, a confined impinging jet mixer (CUM) or a multi-inlet vortex mixer (MIVM).

[00111] In certain embodiments, the mixer has a first inlet and second inlet configured to be radially spaced between about 10° and about 180° relative to each other. In certain embodiments, the mixer has a first inlet and second inlet configured to be radially spaced greater than about 10° to less than about 180° relative to each other. In certain embodiments, the mixer has a first inlet and second inlet configured to be radially spaced greater than about 10° to less than about 150° relative to each other. In certain embodiments, the mixer has a first inlet and second inlet configured to be radially spaced greater than about 10° to less than about 130° relative to each other. In certain embodiments, the mixer has a first inlet and second inlet configured to be radially spaced greater than about 10° to less than about 110° relative to each other. In certain embodiments, the mixer has a first inlet and second inlet configured to be radially spaced greater than about 10° to less than about 90° relative to each other. In certain embodiments, the mixer has a first inlet and second inlet configured to be radially spaced between about 10° and about 90° relative to each other.

[00112] In certain embodiments, the outlet of the mixer is configured to be radially spaced between the first inlet and second inlet. In certain embodiments, the outlet of the mixer is configured to be radially spaced to less than about 90° relative to the midpoint of the first inlet and second inlet. In certain embodiments, the outlet of the mixer is configured to be radially spaced to less than about 80° relative to the midpoint of the first inlet and second inlet. In certain embodiments, the outlet of the mixer is configured to be radially spaced to less than about 70° relative to the midpoint of the first inlet and second inlet. In certain embodiments, the outlet of the mixer is configured to be radially spaced to less than about 60° relative to the midpoint of the first inlet and second inlet. In certain embodiments, the outlet of the mixer is configured to be radially spaced to less than about 50° relative to the midpoint of the first inlet and second inlet. In certain embodiments, the outlet of the mixer is configured to be radially spaced to less than about 40° relative to the midpoint of the first inlet and second inlet. In certain embodiments, the outlet of the mixer is configured to be radially spaced to less than about 30° relative to the midpoint of the first inlet and second inlet. In certain embodiments, the outlet of the mixer is configured to be radially spaced to less than about 20° relative to the midpoint of the first inlet and second inlet. In certain embodiments, the outlet of the mixer is configured to be radially spaced to less than about 10° relative to the midpoint of the first inlet and second inlet.

[00113] In certain embodiments, the mixing portion of the mixer has a volume of less than

500 pL. In certain embodiments, the mixing portion of the mixer has a volume of less than

450 pL. In certain embodiments, the mixing portion of the mixer has a volume of less than

400 pL. In certain embodiments, the mixing portion of the mixer has a volume of less than

350 pL. In certain embodiments, the mixing portion of the mixer has a volume of less than

300 pL. In certain embodiments, the mixing portion of the mixer has a volume of less than

250 pL. In certain embodiments, the mixing portion of the mixer has a volume of less than

200 pL. In certain embodiments, the mixing portion of the mixer has a volume of less than

150 pL. In certain embodiments, the mixing portion of the mixer has a volume of less than

100 pL. In certain embodiments, the mixing portion of the mixer has a volume of less than

80 pL. In certain embodiments, the mixing portion of the mixer has a volume of less than 50 pL. In certain embodiments, the mixing portion of the mixer has a volume of less than 30 pL. In certain embodiments, the mixing portion of the mixer has a volume of less than 20 pL. In certain embodiments, the mixing portion of the mixer has a volume of less than 10 pL. In certain embodiments, the mixing portion of the mixer has a volume of less than 5 pL. In certain embodiments, the mixing portion of the mixer has a volume of between about 2 pL to about 450 pL, between about 2 pL to about 400 pL, between about 2 pL to about 350 pL, between about 2 pL to about 300 pL, between about 2 pL to about 250 pL, between about 2 pL to about 200 pL, between about 2 pL to about 150 pL, between about 2 pL to about 100 pL, between about 2 pL to about 80 pL, between about 2 pL to about 50 pL, between about 2 pL to about 30 pL, between about 2 pL to about 20 pL, between about 2 pL to about 10 pL, between about 2 pL to about 5 pL or about 4 pL.

[00114] The first and/or second reservoir of the present invention can be provided with at least one external pump or at least one integral pump. Alternatively, any other means can be provided to control the flow rate of the streams (A) and (B) of the reservoirs during operation. Any suitable pump can be used. In preferred embodiments, the pump is selected from the group consisting of a peristaltic pump, positive displacement pump, pneumatic pump, infusion pump, or a syringe pump.

[00115] In certain embodiments, the apparatus further comprises an equilibration conduit in fluid communication with the mixer. In certain embodiments, the equilibration conduit is in the form of a loop or coil. In certain embodiments, the equilibration conduit is configured to be at a temperature of less than about 40 °C, less than about 35 °C, preferably less than about 30 °C. In certain embodiments, the equilibration conduit is configured to be at a temperature between about 10 °C to about 40 °C. In certain embodiments, the equilibration conduit is configured to be at a temperature between about 10 °C to about 40 °C. In certain embodiments, the equilibration conduit is configured to be at a temperature between about 10 °C to about 35 °C. In certain embodiments, the equilibration conduit is configured to be at a temperature between about 15 °C to about 35 °C. In certain embodiments, the equilibration conduit is configured to be at a temperature between about 20 °C to about 30 °C. In certain embodiments, the equilibration conduit is configured to be at a temperature about 25 °C. In certain embodiments, the equilibration conduit is configured to be at ambient temperature.

[00116] As would be appreciated by a skilled addressee, the equilibration conduit can be any suitable volume. In some embodiments, the equilibration conduit has a volume less than about 20 mL, less than about 15 mL, less than about 12 mL, less than about 10 mL, less than about 8 mL, less than about 5 mL or less than about 3 mL. In some embodiments, the equilibration conduit has a volume between about 0.5 mL to about 20 mL, between about 0.5 mL to about 15 mL, between about 0.5 mL to about 12 mL, between about 0.5 mL to about 10 mL, between about 1 mL to about 10 mL, between about 1 mL to about 8 mL, between about 1 mL to about 5 mL, about 3 mL, or about 2.8 mL. In industrial settings, a significantly higher volume of the equilibration conduit can be used. In these embodiments, the volume of the equilibration conduit can be in the order of litres. In some embodiments, the equilibration conduit has a volume less than about 5 L, less than about 4 L, less than about 3 L, less than about 2 L, less than about 1 L, less than about 0.5 L or less than about 0.1 L.

Downstream processing of apparatus

[00117] As discussed above, the physical properties of the metastable polymersomes produced by the present invention can be manipulated downstream after initial formation including control of size, polydispersity or shape/morphology by using an external stimulus. The apparatus can be configured to provide the external stimulus using modular components in a plug-and-play fashion and integrate downstream processes without requiring discrete equipment.

[00118] In certain embodiments, the apparatus further comprises at least one of an equilibration conduit, an annealing conduit and a cooling conduit downstream of the mixer.

[00119] In certain embodiments, the apparatus further comprises an annealing conduit in fluid communication with the equilibration conduit or with the mixer. In certain embodiments, the annealing conduit is in the form of a loop or coil. In certain embodiments, the annealing conduit is configured to be at a temperature between about 20 °C to about 90 °C. In certain embodiments, the annealing conduit is configured to be at a temperature between about 15 °C to about 80 °C. In certain embodiments, the annealing conduit is configured to be at a temperature between about 20 °C to about 80 °C. In certain embodiments, the annealing conduit is configured to be at a temperature between about 20 °C to about 70 °C. In certain embodiments, the annealing conduit is configured to be at a temperature between about 30 °C to about 70 °C. In certain embodiments, the annealing conduit is configured to be at a temperature between about 40 °C to about 70 °C. In certain embodiments, the annealing conduit is configured to be at a temperature between about 50 °C to about 70 °C. In certain embodiments, the annealing conduit is configured to be at a temperature between about 60 °C to about 70 °C. In certain embodiments, the annealing conduit is configured to be at a temperature of about 70 °C.

[00120] As would be appreciated by a skilled addressee, any suitable volume of the annealing conduit can be used. In some embodiments, the annealing conduit has a volume less than about 20 mL, less than about 15 mL, less than about 12 mL, less than about 10 mL, less than about 8 mL, less than about 5 mL or less than about 3 mL. In some embodiments, the annealing conduit has a volume between about 0.5 mL to about 20 mL, between about 0.5 mL to about 15 mL, between about 0.5 mL to about 12 mL, between about 0.5 mL to about 10 mL, between about 1 mL to about 10 mL, between about 1 mL to about 8 mL, between about 1 mL to about 5 mL, between about 1 mL to about 3 mL, or about 2 mL. In industrial settings, a significantly higher volume of the annealing conduit can be used. In these embodiments, the volume of the annealing conduit can be in the order of litres. In some embodiments, the annealing conduit has a volume less than about 5 L, less than about 4 L, less than about 3 L, less than about 2 L, less than about 1 L, less than about 0.5 L or less than about 0.1 L.

[00121] In certain embodiments, the apparatus further comprises a cooling conduit in fluid communication with the annealing conduit. In certain embodiments, the annealing conduit is in the form of a loop or coil. In certain embodiments, the cooling conduit is configured to be at a temperature between about 20 °C to about 60 °C. In certain embodiments, the cooling conduit is configured to be at a temperature between about 30 °C to about 50 °C. In certain embodiments, the cooling conduit is configured to be at a temperature between about 35 °C to about 45 °C. In certain embodiments, the cooling conduit is configured to be at a temperature of about 40 °C.

[00122] As would be appreciated by a skilled addressee, any suitable volume of the cooling conduit can be used. In some embodiments, the cooling conduit has a volume less than about 100 mL, less than about 80 mL, less than about 70 mL, less than about 60 mL, less than about 50 mL, less than about 40 mL or less than about 35 mL. In some embodiments, the cooling conduit has a volume between about 1 mL to about 100 mL, between about 5 mL to about 80 mL, between about 5 mL to about 70 mL, between about 5 mL to about 60 mL, between about 5 mL to about 50 mL, between about 10 mL to about 50 mL, between about 20 mL to about 50 mL, between about 20 mL to about 40 mL, between about 25 mL to about 35 mL, about 32 mL or about 31.4 mL. In industrial settings, a significantly higher volume of the cooling conduit can be used. In these embodiments, the volume of the cooling conduit can be in the order of litres. In some embodiments, the cooling conduit has a volume less than about 5 L, less than about 4 L, less than about 3 L, less than about 2 L, less than about 1 L, less than about 0.5 L or less than about 0.1 L.

[00123] In some embodiments, apparatus further comprises a backpressure regulator prior to an apparatus outlet. Advantageously, the backpressure regulator can increase the boiling point of the organic phase of the mixed streams (A) and (B) to prevent solvent outgassing and/or evaporation during operation of the apparatus to form metastable polymersomes.

[00124] In some embodiments, the apparatus further comprises a downstream mixer having a first inlet in fluid communication with an outlet of the equilibration conduit, annealing conduit, cooling conduit or backpressure regulator and a second inlet in fluid communication with the outlet of a third reservoir for receiving a morphological change inducing additive, and an outlet to dispense downstream processed metastable polymersomes. Any suitable mixer can be used as the downstream mixer such as those as described herein.

[00125] In certain embodiments, the mixer comprises a third inlet in fluid communication with the outlet of a third reservoir for receiving a solution of an agent in either an aqueous or organic solvent.

[00126] In some embodiments, the apparatus further comprises a dialysis cell in fluid communication with an apparatus outlet. In certain embodiments, the dialysis cell can be adapted to receive a morphological change inducing additive such as an organic solvent, acid, base, salt and combinations thereof. Suitable acids, bases and salts have been described herein.

Metastable polymersomes

[00127] The present invention provides metastable polymersomes formed by the method and apparatus as described herein.

[00128] As would be appreciated by a skilled addressee, any suitable weight fraction of the hydrophilic block to the hydrophobic block of the amphiphilic block co-polymer can be used to form metastable polymersomes. The weight fraction of the hydrophilic block to the hydrophobic block can be estimated based on the total molecular weight of repeating units of the total hydrophophilic block relative to the total molecular weight of the amphiphilic block copolymers).

[00129] In certain embodiments, the ratio of the hydrophilic block to the hydrophobic block of an amphiphilic block co-polymer is between about 0.55 to 0.95, between about 0.6 to about 0.95, between about 0.7 to about 0.95, between about 0.8 to about 0.95 or preferably between 0.82 to 0.92.

[00130] Any suitable amphiphilic block co-polymer can be used in the present invention. In certain embodiments, the hydrophilic block of the amphiphilic block co-polymer comprises at least one monomer selected from the group consisting of polyacrylic acid (PAA), polymethacrylic acid (PMAA), poly((diisopropylamino)ethyl methacrylate) (PDPA), poly(A/, /- dimethylaminoethyl methacrylate) (PDMAEMA), poly(/V,/V-diethylaminoethyl methacrylate) (PDEAEMA), poly(/V-isopropylacrylamide) (PNIPAM), poly(2-methacryloyloxyethyl phosphorylcholine) (PM PC), polyethylene oxide (PEO), poly(2-vinylpyridine) (P2VP), poly(4- vinylpyridine) (P4VP), poly(2-methyl-2-oxazoline) (PMOXA), poly(oligo(ethylene glycol methyl ether acrylate)) (POEGMEA), poly(oligo(ethylene glycol methyl ether methacrylate)) (POEGMEMA), polyacrylamide, polyvinyl alcohol (PVA), and derivatives thereof.

[00131] In certain embodiments, the hydrophilic block of the amphiphilic block co-polymer is selected from the group consisting of at least one of polyacrylic acid (PAA), polymethacrylic acid (PMAA), poly((diisopropylamino)ethyl methacrylate) (PDPA), hydrophilic crosslinkable monomer (such as monomers comprising an acrylate group, a vinyl group, a hydroxyl group, a carboxyl group, an azide group, an alkyne, an epoxide group, an ether group, an acid anhydride group, an isocyanate group, an amine group and combinations thereof), hydrophilic drug monomer, poly(/V,/V-dimethylaminoethyl methacrylate) (PDMAEMA), poly(A/,A/- diethylaminoethyl methacrylate) (PDEAEMA), poly(/V-isopropylacrylamide) (PNIPAM), poly(2- methacryloyloxyethyl phosphorylcholine) (PMPC), polyethylene oxide (PEO), poly(2- vinylpyridine) (P2VP), poly(4-vinylpyridine) (P4VP), poly(2-methyl-2-oxazoline) (PMOXA), poly(oligo(ethylene glycol methyl ether acrylate)) (POEGMEA), poly(oligo(ethylene glycol methyl ether methacrylate)) (POEGMEMA), polyacrylamide, polyvinyl alcohol (PVA), and derivatives thereof. As would be appreciated by a skilled addressee, the hydrophilic block described herein include discrete blocks selected from two or more monomers and a statistically or randomly distributed combination of two or more monomers.

[00132] In certain embodiments, the hydrophobic block of the amphiphilic block co-polymer comprises at least one monomer selected from the group consisting of polycaprolactone (PCL), polylactide (PLA) such as poly(D,L-lactide), polyglycolic acid (PGA), polyvinyl acetate (PVAc), polyethyl ethylene (PEE), polybutadiene (PBD), polystyrene (PS), polydimethylsiloxane (PDMS), polypropylene oxide (PPG), poly(lactic acid-co-glycolic acid) (PLGA), poly(hydroxypropyl methacrylate) (HPMA), polybenzyl acrylate (PBzA), polymethyl methacrylate (PMMA), poly(tert-butyl acrylate) (PtBA), poly(n-butyl acrylate) (PBA), polypentafluorophenyl acrylate (PFPA), polypentafluorophenyl methacrylate (PFPMA), polytrimethylene carbonate (PTMC), poly(/V,/V-dimethylaminoethyl methacrylate) (PDMAEMA), poly(/V,/V-diethylaminoethyl methacrylate) (PDEAEMA), poly(/V- isopropylacrylamide) (PNIPAM), poly(2-vinylpyridine) (P2VP), poly(4-vinylpyridine) (P4VP) and derivatives thereof.

[00133] In certain embodiments, the hydrophobic block is selected from the group consisting of at least one of polycaprolactone (PCL), polylactide (PLA) such as poly(D,L-lactide), polyglycolic acid (PGA), polyvinyl acetate (PVAc), polyethyl ethylene (PEE), polybutadiene (PBD), polystyrene (PS), hydrophobic crosslinkable monomer (such as monomers comprising an acrylate group, a vinyl group, a hydroxyl group, a carboxyl group, an azide group, an alkyne, an epoxide group, an ether group, an acid anhydride group, an isocyanate group, an amine group and combinations thereof), hydrophobic drug monomer, polydimethylsiloxane (PDMS), polypropylene oxide (PPG), poly(lactic acid-co-glycolic acid) (PLGA), poly(hydroxypropyl methacrylate) (HPMA), polybenzyl acrylate (PBzA), polymethyl methacrylate (PMMA), poly(tert-butyl acrylate) (PtBA), poly(n-butyl acrylate) (PBA), polypentafluorophenyl acrylate (PFPA), polypentafluorophenyl methacrylate (PFPMA), polytrimethylene carbonate (PTMC), poly(/V,/V-dimethylaminoethyl methacrylate) (PDMAEMA), poly(/V,/V-diethylaminoethyl methacrylate) (PDEAEMA), poly(/V-isopropylacrylamide) (PNIPAM), poly(2-vinylpyridine) (P2VP), poly(4-vinylpyridine) (P4VP) and derivatives thereof. As would be appreciated by a skilled addressee, the hydrophobic block described herein include discrete blocks selected from two or more monomers and a statistically or randomly distributed combination of two or more monomers. [00134] As would be appreciated by a skilled addressee, poly((diisopropylamino)ethyl methacrylate) (PDPA), poly(/V,/V-dimethylaminoethyl methacrylate) (PDMAEMA), poly(A/,A/- diethylaminoethyl methacrylate) (PDEAEMA), poly(/V-isopropylacrylamide) (PNIPAM), poly(2- vinylpyridine) (P2VP), poly(4-vinylpyridine) (P4VP) and derivatives thereof have been disclosed to be able to be modified from a hydrophilic block to a hydrophobic block by treating the monomers or polymer comprising the monomers such as with temperature and/or pH.

[00135] The amphiphilic block co-polymer can be a diblock co-polymer or a triblock copolymer.

[00136] In some embodiments, the amphiphilic block co-polymer is selected from the group consisting of at least one of polyethylene oxide-b-polystyrene, polyacrylic acid-b-polystyrene, poly(4-vinylpyridine)-b-polystyrene, polyethylene oxide-b-poly(4-vinylpyridine)-b-polystyrene, polyethylene oxide-b-poly(D,L-lactide), polyethyl ethylene-b-polyethylene oxide, polybutadiene-b-polyethylene oxide, poly(2-methyl-2-oxazoline)-b-polydimethylsiloxane-b- poly(2-methyl-2-oxazoline), polyethylene oxide-b-polypropylene oxide-b-polyethylene oxide, polyethylene oxide-b-polycaprolactone-b-poly(2-methyl-2-oxazoline), poly(2- methacryloyloxyethyl phosphorylcholine)-b-poly((diisoproylamino)ethyl methacrylate), polyethylene oxide-b-polycaprolactone, polyethylene oxide-b-poly(hydroxypropyl methacrylate) and derivatives thereof.

[00137] In preferred embodiments, the amphiphilic block co-polymer is selected from the group consisting of at least one of PEO44-b-PSs6, PAA ₂₆ -b-PS ₈ i, P4VP ₂ rb-PSno, PEO44-b- P4VP ₂ i-b-PS ₃ oo, PEO ₄ 4-b-PDLLAi47, 1 :1 (w/w) PE0 ₄ 4-b-PS ₈ 6/P4VP ₂ i-b-PSiio, 1 :1 (w/w) PEO ₄ 4-b-PS86/PAA ₂₆ -b-PS8i and derivatives thereof.

[00138] As would be understood by a skilled addressee, the metastable state of the polymersomes have a certain lifetime as the metastable state transitions to an equilibrium state over time. In certain embodiments, the lifetime can be related to the temperature of the formed metastable polymersomes. For example, metastable polymersomes stored or formed in lower temperatures can have a longer lifetime while higher temperatures can have a shorter lifetime. In certain embodiments, the metastable state has a lifetime less than 14 days, less than 12 days, less than 10 days, less than 8 days, less than 7 days. In certain embodiments, the metastable state has a lifetime of about 7 days.

[00139] The polydispersity index (PDI) of the metastable polymersomes formed by the method and apparatus can be less than about 0.8, less than about 0.7, less than about 0.6, less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.18, less than about 0.15, less than about 0.1 or less than about 0.05. [00140] As would be appreciated by a skilled addressee, the average membrane thickness of the metastable polymersomes formed by the method and apparatus can be dependent on the conditions used to form the metastable polymersomes and the amphiphilic block copolymer chosen. In certain embodiments, the average membrane thickness of the metastable polymersomes is between about 18 to 26 nm, between about 18 to 24 nm, between about 20 to 24 nm or between about 20 to 23 nm.

[00141] The average membrane thickness of the metastable polymersomes formed by the method and apparatus of the present invention can increase until an equilibrium state is reached. In certain embodiments, the average membrane thickness of the metastable polymers increases between about 1 nm to 5 nm, between about 1 nm to 4 nm, between about 1 nm to 3 nm or between about 1 nm to 2 nm relative to the initial thickness. Without being bound by any one theory, the present inventors believe that the increase in membrane thickness is facilitated by a relaxation of membrane tension as the metastable polymersomes reach an equilibrium state.

[00142] As would also be appreciated by a skilled addressee, the average hydrodynamic diameter of the metastable polymersomes formed by the method and apparatus can be dependent on the conditions used to form the metastable polymersomes (including downstream processing if performed) and the amphiphilic block co-polymer chosen. In certain embodiments, the average hydrodynamic diameter of the metastable polymersomes is between about 50 nm to about 800 nm, between about 50 nm to about 700 nm, between about 50 nm to about 600 nm, between about 50 nm to about 500 nm, between about 50 nm to about 400 nm, between about 50 nm to about 300 nm, between about 50 nm to about 200 nm, between about 120 nm to about 300 nm, between about 150 nm to about 300 nm, between about 160 nm to about 300 nm, between about 180 nm to about 300 nm, between about 160 nm to about 280 nm, between about 160 nm to about 230 nm or between about 160 nm to about 200 nm.

[00143] The average hydrodynamic diameter of the metastable polymersomes formed by the method and apparatus of the present invention can also increase until an equilibrium state is reached. In certain embodiments, the average the metastable polymersomes increase in diameter between about 10% to about 65%, between about 20% to about 65%, between about 20% to about 65%, between about 25% to about 60%, between about 30% to about 65%, between about 40% to about 65%, between about 50% to about 65% or between about 55% to about 65%.

[00144] By controlling the conditions used to form the metastable polymersomes of the present invention, the formation of metastable polymersomes compared to other morphologies can be controlled. This can provide control over the purity of the formed metastable polymersomes of the present invention (i.e., the relative amount of metastable polymersomes formed compared to other morphologies such as micelles).

[00145] In certain embodiments, the purity of the formed metastable polymersomes is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99%.

EXAMPLES

[00146] The present invention will now be described with reference to the following examples.

Chemicals and materials

[00147] All chemicals (reagent grade) were purchased from Sigma-Aldrich (Australia) and used as received unless otherwise mentioned. Tetrahydrofuran (THF, HPLC grade) and 1 ,4- dioxane (analytical grade) were purchased from ChemSupply (Australia). Carbon-coated copper TEM grids (300 mesh) were purchased from ProSciTech (Australia). Styrene, 4- vinylpyridine (4VP) and tert-butyl acrylate (tBA) were deinhibited by passing through a basic alumina plug prior to use. Al BN was recrystallized from methanol prior to use. The polyethylene oxide-based macroRAFT agent (PEO44-DDMAT) was synthesized by reacting polyethylene oxide monomethyl ether (PEO44-OH) with 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT) in dichloromethane in the presence of 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) and 4-dimethylaminopyridine (DMAP), followed by purification by precipitation in hexane. Polytetrafluoroethylene (PTFE) syringe filters (0.45 pm, 33 mm) were purchased from Grace Davison Discovery Sciences (Australia). Polyethersulfone (PES) syringe filters (0.45 pm, 33 mm) were purchased from Merck Millipore Ltd (Ireland). Cellu Sep® regenerated cellulose dialysis tubing with 3.5 kDa molecular weight cut-off (MWCO) was purchased from Adelab Scientific (Australia). A list of continuous flow components used in a preferred embodiment(s) of the present invention is provided below:

Gel permeation chromatography (GPC)

[00148] Polymer molecular weight and dispersity were characterised on a Shimadzu modular system equipped with DGU-12A degasser, LC-10AT pump, SIL-10AD auto injector, CTO-10A column oven, RID-10A differential refractive index detector, and three Phenomenex 5.0 pm bead-size columns connected in series (10 ⁵ , 10 ⁴ and 10 ³ A). Dimethylformamide (DMF) containing 0.1% lithium bromide (LiBr) was used as the GPC eluent. The sample (~1 mg/mL) was filtered through a 0.45 pm PTFE syringe filter prior to injection. Number-average molecular weight (/W _n ,GPc) was estimated based on a narrow molecular weight (100 to 1 x 10 ⁶ g/mol) poly(methyl methacrylate) (PMMA) calibration standard. The /W _n ,GPC(DMF) values provided herein for all PS-containing block co-polymers are underestimates because PS chains are known to adopt a reduced chain conformation in DMF (Hildebrand solubility parameters: <5PS =16.6-20.2 MPa- ^1/2 and <5DMF = 24.7 MPa ^-1/2 ). These block co-polymers therefore exhibit increased apparent retention times in GPC and thus have smaller apparent /W _n ,GPC(DMF) values.

Transmission electron microscopy (TEM)

[00149] In a typical sample preparation, a 6 pL droplet of particle solution (<0.5 mg/mL) was placed onto a carbon-coated copper grid (300 mesh size) and allowed to sit for at least 10 min. A pre-cut filter paper was then used to blot the droplet, leaving behind a thin film. The sample was then left to evaporate under a benchtop fume exhaust arm. No staining was used. TEM analyses were performed either on an FEI Tecnai G2 20 TEM (120 kV) equipped with a BM Eagle 2K CCD Camera or a JEOL TEM-1400 (80 kV) equipped with an EMSIS Phurona CMOS Camera. Images were processed and analysed with Imaged.

Cryogenic transmission electron microscopy (Cryo-TEM)

[00150] In a typical sample preparation, 4 pL of polymersome solution was applied to a glow- discharged lacey formvar/carbon-coated copper grid (PELCO Ted Pella), blotted for 2.5 seconds at 15 °C with 90% humidity, and then plunged into liquid ethane at -176 °C using a Leica EM GP device (Leica Microsystems, Germany). Cryo-TEM data was collected on a FEI Talos Arctica transmission electron microscope operating at 200 kV acceleration voltage using TIA data collection software (ThermoFisher Scientific, United States). All datasets were collected using a Falcon 3EC detector.

Dynamic light scattering (DLS)

[00151] DLS measurements were conducted on a Zetasizer Nano ZSP instrument. Samples were measured at a backscatter angle of 173° at 25 °C. Samples in organic solvent/water mixtures were measured undiluted (concentrations between 0.1 -0.7 mg/mL) in a 600 pL quartz cuvette, while aqueous samples were measured at a concentration of 0.1 mg/mL in disposable 3 mL PMMA cuvettes.

Turbidity assay

[00152] Turbidity measurements were conducted on a Varian Cary® 50 UV-Vis spectrophotometer. Samples were measured in a 600 pL quartz cuvette with 0.2 cm pathlength (Thorlabs, Germany). For the turbidity (aging) experiment, a parent polymersome solution was prepared on day 0 and the following flow conditions: (i) Qorganic Qtotai = 0.7, (ii) Qtotai = 1 mL/min and (iii) c _poiy mer = 1 mg/mL. Aliquots from the parent polymersome solution were then removed for analysis on days 0, 2, 4, 7, 11 and 14. For the turbidity (annealing) experiment, an annealed polymersome solution was prepared and following flow conditions: (i) Qorganic/Qtotai = 0.7, (ii) Qtotai = 4 mL/min, (iii) c _poiy mer = 1 mg/mL and (iv) Tanneaiing = 70 °C. The sample was collected directly into a cuvette and immediately analysed at 6 s intervals for a total of 5 min.

RAFT polymerization of polyethylene oxide-b/ock-polystyrene (PEClu-b-PSse)

(Scheme I)

[00153] The polymerization was carried out in bulk using the following stoichiometry of [styrene]:[PEO44-DDMAT]:[AIBN] = [200]:[1]:[0.1], To a solution of styrene (4.40 g, 4.84 mL, 42.2 mmol) was added PEO44-DDMAT (0.50 g, 0.211 mmol) and AIBN (3.47 mg, 21.1 pmol). The reaction mixture was degassed for 15 min with nitrogen gas over an ice bath to prevent monomer evaporation. The polymerisation was initiated bysubmerging the reaction vessel into a pre-heated oil bath at 65 °C. After 36 h, the polymerisation was quenched by exposing the reaction mixture to air and submerging the reaction mixture in an ice bath. The crude product was diluted in THF and purified by precipitation into hexane. The purification process was repeated 3 times in total. The resulting precipitate was then dried overnight in a vacuum oven at 40 °C to yield PEO44-6-PS86 as a yellow powder. %Conversion = 43%; /WA.NMR = 11 ,260 g/mol; M _n .GPC(DMF) - 8,300 g/mol; B = 1.08.

RAFT polymerization of polyethylene oxide-b/ock-poly(4-vinylpyridine)-b/ock- polystyrene (PEO44-b-P4VP2i)

(Scheme II)

[00154] The polymerisation was carried out in methanol using the following stoichiometry of [4-vinylpyridine]:[PEO44-DDMAT]:[AIBN] = [25]:[1]:[0.1], To a solution of 4-vinylpyridine (1.00 g, 9.51 mmol) was added methanol (293 pL), PEO44-DDMAT (899 mg, 0.38 mmol) and AIBN (6.25 mg, 38.0 pmol). The reaction mixture was degassed for 15 min with nitrogen gas over an ice bath to prevent monomer evaporation. The polymerisation was initiated by submerging the reaction vessel into a pre-heated oil bath at 65 °C. After 22 h 20 min, the polymerisation was quenched by exposing the reaction mixture to air and submerging the reaction mixture in an ice bath. The crude product was diluted in DMF and purified by precipitation into 2:8 (v/v) diethyl ether/hexane. The purification process was repeated 3 times in total. The resulting precipitate was then dried overnight in a vacuum oven at 40 °C to yield PEO44-6-P4VP21 as a yellow powder. %Conversion = 84%; /WA.NMR = 4,570 g/mol; /W _n ,GPC(DMF) = 6,100 g/mol; £> = 1.10.

RAFT polymerization of polyethylene oxide-b/ock-poly(4-vinylpyridine)-b/ock- polystyrene (PE044-b-P4VP2i-b-PS3oo)

(Scheme III)

[00155] The polymerisation was carried out under dispersion conditions (25 wt% solids content) using the following stoichiometry of [styrene]:[PEC>44-P4VP2i]:[AIBN] = [300]:[1]:[0.1], To a solution of styrene (683 mg, 6.56 mmol) was added methanol (1.88 mL), water (470 pL), PEO44-P4VP21 (100 mg, 21.9 pmol) and AIBN (0.71 mg, 4.37 pmol). The reaction mixture was degassed for 20 min with nitrogen gas over an ice bath to prevent monomer evaporation. The polymerisation was initiated by submerging the reaction vessel into a pre-heated oil bath at 70 °C. After 22 h, the polymerisation was quenched by exposing the reaction mixture to air and submerging the reaction mixture in an ice bath. The crude product was purified by dialysis against methanol in a 3.5 kDa MWCO dialysis tubing. The resulting solution was evaporated using a rotary evaporator, and the product dried overnight in a vacuum oven at 40 °C to yield PE0 ₄ 4-6-P4VP ₂ i-b-PS3oo as a yellow powder. %Conversion = >99%; /W _n ,NMR = 35,820 g/mol; M _n ,GPC(DMF) - 19,200 g/mol; D = 1.18.

RAFT polymerization of poly(tert-butyl acrylate) (PtBAae)

[00156] The polymerisation was carried out in toluene using the following stoichiometry of [tBA]:[DDMAT]:[AIBN] = [50]:[1]:[0.1], To a solution of tBA (1.75 g, 2.00 mL, 0.014 mol) in toluene (0.277 mL) was added DDMAT (99.6 mg, 0.211 mmol) and AIBN (4.50 mg, 27.3 pmol). The reaction mixture was degassed for 20 min with nitrogen gas over an ice bath to prevent monomer evaporation. The polymerisation was initiated by submerging the reaction vessel into a pre-heated oil bath at 60 °C. After 1 h 50 min, the polymerisation was quenched by exposing the reaction mixture to air and submerging the reaction mixture in an ice bath. The solvent (toluene) and unreacted monomer were removed by reconstituting the crude polymer in toluene (10 mL) and evaporating to dryness for at least three times. The purified polymer was then dried overnight in a vacuum oven at 40 °C to yield PtBA ₂ 6 as a yellow viscous liquid. %Conversion = 52%; /W _n ,NMR = 3,700 g/mol; /W _n ,GPC(DMF) = 2,600 g/mol; D = 1.16.

RAFT polymerization of poly(tert-butyl acrylate)-b/ock-polystyrene (PtBA ₂ 6-b-PS8i)

(Scheme V) [00157] The polymerization was carried out in bulk using the following stoichiometry of [styrene]:[PtBA26-DDMAT]:[AIBN] = [300]:[1]:[0.1]. To a solution of styrene (3.38 g, 3.72 mL, 0.324 mmol) was added PtBA26-DDMAT (400 mg, 0.108 mmol) and Al BN (1.78 mg, 10.8 pmol). The reaction mixture was degassed for 25 min with nitrogen gas over an ice bath to prevent monomer evaporation. The polymerisation was initiated by submerging the reaction vessel into a pre-heated oil bath at 65 °C. After 24 h, the polymerisation was quenched by exposing the reaction mixture to air and submerging the reaction mixture in an ice bath. The crude product was diluted in THF and purified by precipitation into hexane. The purification process was repeated 3 times in total. The resulting precipitate was then dried overnight in a vacuum oven at 40 °C to yield PEO44-6-PS86 as a yellow powder. %Conversion = 27%; /W _n ,NMR= 12,140 g/mol; M _n ,GPC(DMF) - 7,700 g/mol; D = 1.08.

Deprotection of poly( tert-butyl acrylate)-b/ock-polystyrene (PtBA26-b-PSsi) to yield poly(acry//c ac/c/)-b/ock-polystyrene (PAA26-b-PSsi)

(Scheme VI)

[00158] The diblock co-polymer PtBA26-b-PS8i (1.00 g, 83.3 pmol) was dissolved in 3:1 (v/v) chloroform:THF (16 mL) and cooled to 0 °C in an ice bath. To the cooled solution was then added trifluoroacetic acid (TFA, 8 mL) dropwise under stirring. The reaction mixture was allowed to warm to room temperature and further stirred for 8 h. The solvent and TFA were removed by evaporation using a gentle stream of nitrogen, redissolved in THF and precipitated into 4:1 (v/v) hexane:diethyl ether. The product was then dried in a vacuum oven at 40 °C to yield PAA26-6-PS81 as a yellow solid. The degree of deprotection was estimated to be >99% by ¹ H NMR.

Continuous flow self-assembly procedure for PEO44-b-PS86

[00159] A 1 mg/mL stock solution of PEO44-6-PS86 in 1 :4 (v/v) dioxane/THF was prepared beforehand and stirred in an airtight Schott bottle at 100 rpm until required. The same stock solution was used in all experiments to minimize any concentration errors. Stock solutions with higher concentrations (up to 9 mg/mL) were prepared the same way as above. Whenever needed, the required volume of PEO44-6-PS86 stock solution was removed with a rubber gasket-free syringe and filtered through a 0.45 pm PTFE syringe filter to remove dust. To prevent or minimise clogging, 10 pm inline filters were placed before the micromixer in the continuous flow setup of the present invention. The inline filters can be replaced with Luer lock syringe filters. The filtered PEO44-6-PS86 solution was then loaded into a rubber gasket-free syringe and degassed at least 3 times before mounting the syringe onto a syringe pump. The water used in all experiments was obtained directly from a Milli-Q dispenser and filtered through a 0.45 pm PES syringe filter prior to use. The required volume of water was loaded into a syringe and similarly degassed for at least 5 times before mounting the syringe onto another syringe pump. Both syringes (one containing the organic phase and the other containing the aqueous phase) were then connected to the micromixer to complete the continuous flow setup. Next, the syringe pumps were set to dispense at any required combination of flow rates. Once dispensing has begun, the system is typically allowed to equilibrate for at least four times the volume of the equilibration loop (4 x 0.71 mL = 2.84 mL). After sufficient equilibration, the resulting mixture was collected directly into a glass vial and gently swirled to ensure homogeneity. The sample was then either analysed directly, allowed to age undisturbed, or quenched by extensive dialysis against water in a 3.5 kDa MWCO dialysis tubing.

Shape transformation of PEO44-b-PSs6 polymersomes

[00160] A fresh batch of polymersome solution was prepared by continuous flow selfassembly using the following flow conditions: Q _to tai = 1 mL/min, Q _or ganic = 0.7 mL/min, Q _wa ter = 0.3 mL/min and c _po iymer = 1 mg/mL. The solution was aged for 14 days before being subjected to shape transformation via osmotic pressure. Osmotic pressure was induced by adjusting the salinity of the aged polymersome solution to 50 mM NaCI using a stock solution of 5 M NaCI. The solution was then extensively dialyzed against 50 mM NaCI in a 3.5 kDa MWCO dialysis tubing to remove the organic solvents. The resulting solution was centrifuged at 12,000 rpm for 10 min to yield an off-white pellet of stomatocytes. The supernatant was carefully removed with a pipette and the pellet redispersed in water (n.b., the supernatant contains a small portion of <150 nm spherical polymersomes that were unaffected by the shape transformation procedure). The centrifugation/redispersion process was repeated three times in order to complete the solvent transfer process and to yield a pure stomatocyte phase for TEM analysis.

Continuous flow self-assembly procedure for PE044-b-P4VP2i-PS3oo

[00161] A 1 mg/mL stock solution of PEO44-6-P4VP21-PS300 in 1 :4 (v/v) dioxane/THF was prepared beforehand and stirred in an airtight Schott bottle at 100 rpm until required. The same stock solution was used in all experiments to minimize any concentration errors. Whenever needed, the required volume of PEO44-6-P4VP21-PS300 stock solution was removed with a rubber gasket-free syringe and filtered through a 0.45 pm PTFE syringe filter to remove dust. To prevent or minimise clogging, a 10 pm inline filter was placed before the micromixer in the continuous flow setup of the present invention. The inline filters can be replaced with Luer lock syringe filters. The filtered PEO44-6-P4VP21-PS300 solution was then loaded into a rubber gasket-free syringe and degassed at least 3 times before mounting the syringe onto a syringe pump. The water used was obtained directly from a Milli-Q dispenser and filtered through a 0.45 pm PES syringe filter prior to use. The required volume of water was loaded into a syringe and similarly degassed for at least 5 times before mounting the syringe onto another syringe pump. Both syringes (one containing the organic phase and the other containing the aqueous phase) were then connected to the micromixer to complete the continuous flow setup. Next, the syringe pumps were set to dispense at Qorganic = 0.8 mL/min and Qwater = 0.2 mL/min (Qtotai = 1 mL/min) to target the polymersome morphology. Once dispensing has begun, the system is typically allowed to equilibrate for at least four times the volume of the equilibration loop (4 x 0.7 mL = 2.8 mL). After sufficient equilibration, the resulting mixture was collected directly into a glass vial and gently swirled to ensure homogeneity. The sample was then allowed to age undisturbed or quenched by extensive dialysis against water in a 3.5 kDa MWCO dialysis tubing.

Continuous flow self-assembly procedure for PAA ₂₆ -b-PS8i

[00162] A 1 mg/mL stock solution of PAA26-£>-PSSI in THF was prepared beforehand and stirred in an airtight Schott bottle at 100 rpm until required. The same stock solution was used in all experiments to minimize any concentration errors. Whenever needed, the required volume of PAA ₂ 6-b-PS8i stock solution was removed with a rubber gasket-free syringe and filtered through a 0.45 pm PTFE syringe filter to remove dust. To prevent or minimise clogging, a 10 pm inline filter was placed before the micromixer in the continuous flow setup of the present invention. The inline filters can be replaced with Luer lock syringe filters. The filtered PAA ₂ 6-6-PS ₈ I solution was then loaded into a rubber gasket-free syringe and degassed at least 3 times before mounting the syringe onto a syringe pump. The aqueous phase used in this experiment is about 100-300 pM HCI solution, preferably, 100 pM HCI solution instead of water as the PAA chains must be partially deprotonated in order to target the polymersome morphology. The 100 pM HCI solution used was directly filtered through a 0.45 pm PES syringe filter prior to use. The required volume of 100 pM HCI solution was loaded into a syringe and similarly degassed for at least 5 times before mounting the syringe onto another syringe pump. Both syringes (one containing the organic phase and the other containing the aqueous phase) were then connected to the micromixer to complete the continuous flow setup. Next, the syringe pumps were set to dispense at Qorganic = 4.4 mL/min and Q o HCI = 3.6 mL/min (Qtotai = 8 mL/min) to target the polymersome morphology (i.e. , Qorgamc/Qtotai = 0.55). Once dispensing has begun, the system is typically allowed to equilibrate for at least four times the volume of the equilibration loop (4 x 0.71 mL = 2.84 mL). After sufficient equilibration, the resulting mixture was collected directly into a glass vial and gently swirled to ensure homogeneity. The sample was then allowed to age undisturbed or quenched by extensive dialysis against 100 pM HCI solution followed by water in a 3.5 kDa MWCO dialysis tubing. If necessary, prior to analysis by TEM and DLS, a drop of 0.1 M sodium hydroxide (NaOH) solution can be added to deprotonate the PAA chains on the polymersome surface, promoting chain repulsion and thus colloidal stability.

Continuous flow self-assembly and downstream annealing procedure for PEO44-b-PS86

[00163] A filtered PEC b-PSse solution was loaded into a 10 mL SGE gastight glass syringe and mounted onto a syringe pump. The degassing step is not required in this example because of the inclusion of a backpressure regulator (BPR) which prevents outgassing. Water was obtained directly from a Milli-Q dispenser and filtered through a 0.45 pm PES syringe filter prior to use. The required volume of water was loaded into another 10 mL SGE gastight glass syringe and mounted onto another syringe pump. Both syringes (one containing the organic phase (1 :4 (v/v) dioxane/THF) and the other containing the aqueous phase (water) were then connected to the micromixer as to complete the continuous flow setup. The 2 mL annealing loop was submerged (which gives a /residence, heating = 30 s at Qtotai = 4 mL/min) into a water bath pre-heated to the desired temperature (20-70 °C). After 10 min of thermal equilibration, the syringe pumps were set to dispense at flow rates of Q _org anic = 2.8 mL/min and Qwater = 1.2 mL/min (Qtotai = 4 mL/min) to target the polymersome morphology. Once dispensing has begun, the system is typically allowed to equilibrate for at least twice the volume of the sum of the equilibration loop and annealing loop (2 x (0.71 mL + 2 mL) = 5.42 mL). After sufficient equilibration, the resulting mixture was collected directly into a glass vial and then left to cool under ambient conditions (~10 min), before being quenched by extensive dialysis against water in a 3.5 kDa MWCO dialysis tubing.

Continuous flow self-assembly and downstream annealing procedure for PEO44-b-PSs6

[00164] A filtered PEO44-6-PS86 solution was loaded into a 10 mL SGE gastight glass syringe and mounted onto a syringe pump. The degassing step is not required in this example because of the inclusion of a backpressure regulator (BPR) which prevents outgassing. Water was obtained directly from a Milli-Q dispenser and filtered through a 0.45 pm PES syringe filter prior to use. The required volume of water was loaded into another 10 mL SGE gastight glass syringe and mounted onto another syringe pump. Both syringes (one containing the organic phase and the other containing the aqueous phase) were then connected to the micromixer as to complete the continuous flow setup. Note that a shut-off valve was placed between the syringe loaded with water and the micromixer to allow water to be replenished when necessary (n.b., the replenishment process can be performed as follows: (i) stop syringe pumps, (ii) set shut-off valve to “close” position, (iii) disconnect and replenish syringe with water, (iv) reconnect replenished syringe, (v) set shut-off valve to “open” position and (vi) start syringe pumps). The 2 mL annealing loop (which gives a ^residence, heating = 30 s at Qtotai = 4 mL/min) was submerged into a water bath pre-heated at 70 °C, while the 31.4 mL of cooling loop (which gives a ^residence, cooling = 7 min 51 s at Qtotai = 4 mL/min) was enclosed in an incubator pre-heated at 40 °C. Further down the line, a secondary micromixer was connected to the flow setup to introduce a stream of concentrated NaCI solution (CNaci = 5.05 M) via a syringe pump equipped with a plastic syringe (n.b., a gastight glass syringe is not required in this example because the system is no longer pressurized by the BPR at this point). After 10 min of thermal equilibration, the syringe pumps were set to dispense at flow rates of Qorgamc = 2.8 mL/min and water = 1.2 mL/min (Qtotai = 4 mL/min) to target the polymersome morphology, and QNaci = 0.04 mL/min to induce downstream shape transformation (CNaci, final = 50 mM). The first 8 mL of eluent is typically discarded to ensure the sample has been properly equilibrated prior to collection. In a typical experiment, 5 mL of sample would be collected and quenched by extensive dialysis against water in a 3.5 kDa MWCO dialysis tubing.

Example 1 - Metastable polymersomes

[00165] The present inventors have developed a continuous flow approach towards polymersome manufacturing (i.e., polymersome production and optionally downstream manipulation). The present inventors have found that self-assembly under continuous flow conditions can circumvent kinetic traps to form metastable polymersomes. Under ambient conditions (about 25 °C), the lifetime of the metastable state measures ca. 7 days, during which the polymersomes gradually grow in size as they transition into an equilibrium state. The metastable state of the formed polymersomes allows for the implementation of downstream processes. Significantly, the present inventors were able to demonstrate (i) unprecedented polymersome production rate of, but not limited to, >3 grams per hour — far surpassing the capabilities of common batch polymersome formation methods, which typically operate at rates of only several-to-tens of milligrams per hour, (ii) the general applicability of the continuous flow self-assembly process towards various block co-polymer types (e.g., diblock, triblocks, and different core and corona chemistries), and (iii) the modularity of the continuous flow apparatus to operate in a plug-and-play fashion.

[00166] The model polymer used in this Example is a diblock co-polymer, polyethylene oxide- b/oc -polystyrene (PEO44-6-PS86), synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization (/WA.NMR = 11 ,260 g/mol, £> = 1.08). The synthesis of the polymer is discussed above and the charaterisation data is shown in Figure 1. This diblock co-polymer was selected because it is a common building block for polymersome formation. The initial continuous flow apparatus used in this Example consists of two syringe pumps, a static mixing tee (micromixer), a 0.7-mL equilibration loop and a collection outlet. A photograph of the setup is shown in Figure 2.

[00167] To perform continuous flow self-assembly (i.e., the method of the present invention), the syringe pumps are equipped with two separate syringes; one containing the organic phase (1 mg/mL of PEO44-6-PS86 in 2:8 (v/v) 1 ,4-dioxane/tetrahydrofuran (THF)), and the other containing water. Both solutions were degassed thoroughly prior to the experiment to minimize outgassing or bubble formation. The degassing procedure is shown in Figure 3. It was surprisingly found that the syringe size, solution volume and length of the equilibration loop do not or have minimal influence the self-assembly process. These parameters can therefore be adjusted to suit the user’s needs (e.g., production scale) or mechanical limitations of their syringe pump.

Example 2 - Flow rate ratio

[00168] The effect of flow rate ratio was investigated on self-assembly of the amphiphilic block co-polymers. For this example, the ratio of the flow rate of the organic phase (Qorganic) and water (Qwater) was adjusted asymmetrically while maintaining a total flow rate of Qtotai = 1 mL/min. For simplicity, the data is discussed in terms of Qorganic/Qtotai. Any increment in Qorganic is thus accompanied by a concomitant decrease in Qwater.

[00169] The self-assembly process was performed at 7 different asymmetric flow rates ranging from Qorganic/Qtotai = 0.1-0.7 (in 0.1 increments) as shown in Figure 4A. In every case, the product was collected directly into a quartz cuvette and immediately analyzed by dynamic light scattering (DLS). The resulting particle size distributions are shown in Figure 1 B. Each sample’s intensity-averaged hydrodynamic diameter (Dh, intensity) and polydispersity index (PDI) are provided in Figure 1C. A summary of the DLS data is presented in Table 1.

[00170] Table 1 : Summary of DLS data in Example 2. [00171] ¹ Samples were analysed in their respective organic solvent/water mixtures.

[00172] All 7 asymmetric flow rates resulted in monomodal particle size distributions with relatively low PDIs of <0.16 as shown in Figure 4B-C and Table 1. At Qorganic/Qtotai 0.2, minimal changes in particle size were observed. Increments above this value, however, resulted in a linear increase in particle size (see Dh, intensity datapoints for Qorganic/Qtotai = 0.3-0.7 in Figure 4C). Asymmetric flow rates of Qorganic/Qtotai > 0.7 were also tested, but these flow conditions did not result in any particle formation because PEO44-6-PS86 remains molecularly dissolved when the organic solvent content exceeds 70 vol%.

[00173] Transmission electron microscopy (TEM) was then used to probe particle morphology. Shown in Figure 4D-F are three TEM images of particles produced at Qorganic/Qtotai = 0.2, 0.4 and 0.6, respectively. The gradual increase in Qorganic/Qtotai generated a morphological transition from micelles (Figure 4D) to a mixed phase of micelles/polymersomes (Figure 4E), and finally to polymersomes (Figure 4F). For clarity, the three accessible morphological phases are highlighted in Figure 4C using different shades of black.

[00174] Without being bound by any one theory, the present inventors believe that the selfassembly process of the present invention and Example 2 can be explained as follows. At low Qorganic/Qtotai (^ 0.2), the organic phase containing PEO44-6-PS86 is flowed at low flow rates (Qorganic 0.2 mL/min) and mixed with water, which is conversely flowed at much higher flow rates (Q _wa ter 0.8 mL/min). In other words, for all datapoints collected at Qorganic/Qtotai 0.2 (Figure 4B-C), the organic phase is asymmetrically mixed with a large volume excess (specifically, between 4- to 9-folds) of water. Since water is a non-solvent for the PS block, the applied flow conditions force PEO44-6-PS86 to undergo microphase separation immediately upon contact with water. Spherical micelles (Figure 4D) with a PS core and PEO corona are formed as a result as the morphology minimizes any unfavorable contact between the PS chains and water.

[00175] At 0.2 < Qorganic/Qtotai 0.6, near-symmetric flow rates is approached, which yields a mixture of micelles and polymersomes (the latter dominates in population as Qorganic/Qtotai approaches 0.6). Here, the onset of polymersome formation can be ascribed to the increase in Qorganic and concomitant decrease in Qwater. Under these flow conditions, there is an overall improvement in solvent quality, thus causing the PS chains to become more swollen and stretched. This leads to an increase in geometric packing parameter, which favors the formation of polymersomes over micelles. However, the inventors infer based on the coexistence of micelles and polymersomes (see pseudo-phase diagram Figure 4C) that not all PS chains adopt the same swollen/stretched chain conformation under the flow conditions of 0.2 S Qorganic/Qtotai — 0.6. [00176] Finally, flow conditions between 0.6 < Qorganic/Qtotai 0.7 lead to the formation of a pure phase of polymersomes. As mentioned above, polymersome formation is favored over micelle formation when PS chains adopt a swollen and stretched chain conformation. Under these flow conditions, polymersomes exclusively form because the bulk of PS chains in solution are well-solvated owing to the organic solvent content, which is as high 60-70 vol%.

[00177] To summarize the above, the present inventors propose a free energy diagram in Figure 5A to illustrate the two possible self-assembly pathways, namely “Pathway 1” (micelle formation) and “Pathway 2” (polymersome formation). Without being bound by any one theory, the present inventors believe that micelles formed via Pathway 1 at Qorganic Qtotai 0.2 are kinetically trapped structures, whose morphological transformation is hindered by an activation energy, EM » k&T. Polymersomes on the other hand, which are primarily formed via Pathway 2 at 0.6 < Qorganic/Qtotai 0.7, are more thermodynamically favorable structures occupying a lower free energy minimum. The present inventors note however, that this is simply a local minimum (EA2 “ k _e T) in the free energy landscape. Despite being thermodynamically favored, the polymersomes are in fact metastable (non-equilibrium) structures.

Example 3 - Metastable polymersome aging

[00178] To confirm whether (i) micelles are kinetically trapped structures and (ii) polymersomes are metastable structures, two fresh batches of micelles and polymersomes (at Qorganic/Qtotai = 0.1 and 0.7, respectively) were prepared. The samples were sealed and allowed to age at room temperature for 14 days, during which aliquots were periodically removed for turbidity analysis.

[00179] As shown in Figure 5B, no turbidity changes were observed in the micelle sample over the 14-day period. Since turbidity is a function of particle size, the data suggests that the micelles maintain a constant particle size throughout the experiment. The present inventors further verified this trend by repeating the same experiment, but this time aliquoting the samples for DLS analysis instead. As expected, no changes in micelle size were observed as shown in Figure 5I. The data is thus consistent with the hypothesis that the micelles are kinetically trapped structures. Here, thermal energy /c _B T at room temperature is insufficient to overcome the activation energy barrier E in the free energy landscape (referring back to Figure 5A). The system hence remains in a micellar state irrespective of aging period.

[00180] In contrast, the polymersome sample became increasingly turbid in the first 7 days, after which the turbidity plateaus. This suggests that the polymersomes are metastable with a finite lifetime of approximately 7 day. During this period, the polymersomes undergo a growth process, which is further substantiated by DLS analysis as shown in Figure 5I. This gradual increase in polymersome size occurs because thermal energy _B T at room temperature is high enough (EA2 “ keT) to push the system out of its metastable state into a more thermodynamically favorable state (Figure 5A). The process is also likely facilitated by the high chain mobility of PEO44-6-PS86 in the polymersome membrane (noting that the organic solvent content in this sample is as high as 70 vol% as it was produced using Qorganic/Qtotai = 0.7). The plateau occurs after about 7 days as the aged polymersomes eventually occupy a global free energy minimum and are thus considered to be in an equilibrium state.

[00181] The present inventors then explored the polymersomes’ metastability to generate polymersomes with controlled sizes in water. For this, the stability of the polymersomes into neat water were first tested. A fresh batch of metastable polymersomes was prepared (herein referred to as the parent solution). An aliquot was removed from the parent solution immediately after flow self-assembly and dialyzed against water to remove the organic solvents. The resulting sample was then probed by DLS (Figure 5C) and TEM (Figure 5D). The TEM data confirmed that the polymersome morphology is retained even in water. Although DLS showed a size decrease (-152 nm) upon solvent transfer from the initial organic solvent/water mixture to water, this is expected because polymersomes are known to swell in the presence of organic solvents. Note that the polymersomes no longer grow with age after being transferred into water because their membrane structure transitions into a glassy state in the absence of any plasticizing organic solvent. This so-called “quenched state” can be attributed to the high glass transition temperature of PS (Tg, _PS “ 100 °C).

[00182] To generate aqueous polymersomes with different sizes, the parent solution was allowed to age for different number of days and repeated the same aliquoting/dialysis procedure described above to quench the polymersome structures. The DLS data for six resulting aqueous polymersome samples quenched on days 0, 2, 4, 7, 11 and 14 are shown in Figure 5C.

[00183] The DLS is shown in Table 2.

[00184] Table 2. Summary of DLS data presented in Figure 5C.

[00185] Consistent with what was observed in Figure 5B, (i) a linear increase in size for polymersomes quenched between 0-7 days of aging, and (ii) no noticeable size change from day 7 onwards was observed. This demonstrates that size control is indeed possible in aqueous solution on polymersome metastability and its corresponding lifetime. Importantly, the ability to control polymersome size distribution with sub- 100 nm precision is a feat which is inconceivable using conventional polymersome formation methods.

[00186] Comparing two (cryo-)TEM images (Figure 5D-DE), the size difference between polymersomes is highlighted for polymersomes quenched on days 0 and 14, respectively. The difference in membrane thickness (dmembrane) between both samples were measured by performing statistical analyses on n = 115 polymersomes imaged by cryo-TEM (Figure 5E). From this, a dmembrane difference of ~2.1 nm was determined as shown in Figure 5F, which is statistically significant with P<0.0001 based on paired t-test. This indicates that the polymersome membrane structure undergoes some conformational changes during the aging process, possibly explaining the origins of the metastable state.

[00187] Without being bound by any one theory, the present inventors believe that this phenomenon is facilitated by a relaxation of membrane tension. Since polymersomes that are formed initially are relatively small in size (Dh, intensity = 121 ± 44 nm in the non-swollen state; see Figure 5D), they exhibit conversely high membrane curvatures (c = 1/r, where r= polymersome radius), which lead to the generation of an unfavorable amount of membrane tension (5). When left to age in their as-produced organic solvent/water mixture, the polymersomes grow in order to alleviate membrane tension until an equilibrium size is reached. As discussed above for Figure 5E, this growth process is also accompanied by an increase in membrane thickness (reflecting a looser packed membrane structure), which can further relieve the built-up membrane tension. [00188] Another advantage of aged polymersomes is their susceptibility to shape transformation. The polymersomes can be manipulated into non-spherical shapes using osmotic pressure as represented in Figure 5G. A requirement for this is a high degree of membrane flexibility, which the metastable polymersomes of the present invention possess, even after they have grown into an equilibrium size.

[00189] To confirm this, an unquenched 14-day aged polymersome solution was subjected to shape transformation by osmotic pressure as shown in Figure 5G-H. Specifically, the salinity of the polymersome solution was adjusted to 50 mw NaCI, followed by dialysis to quench the resulting structures. This salinity change generates an osmotic imbalance between the polymersomes’ inner compartment and their surrounding solution, thus leading to a net efflux of solvent out of the polymersomes. This drives a reduction in the polymersomes’ internal volume and causes the (initially spherical) polymersomes to deform into indented polymersomes known as stomatocytes as confirmed by TEM and cryo-TEM images in Figure 5H.

Example 4 - Polymer concentration and flow rate

[00190] The results discussed in Examples 2 and 3 are based on continuous flow selfassembly at a polymer concentration of c _poiy mer = 1 mg/mL and total flow rate of Qtotai = 1 mL/min. Although polymersome formation under such flow conditions are already more scalable than under batch conditions, the extent to which the method of the present invention can be scaled was assessed. To this end, the effect of polymer concentration (c _po iymer = 1-9 mg/mL) on polymersome formation was explored. The Qorgamc/Qtotai and Qtotai was maintained at 0.7 and 1 mL/min, respectively, to target the polymersome morphology as per the pseudo-phase diagram in Figure 4C. The polymersomes formed by the method represented in Figure 4A were quenched in water by dialysis, then analyzed by TEM and DLS (Figure 6A). Remarkably, the polymersome morphology was retained even at the highest polymer concentration tested (c _P oiymer = 9 mg/mL; rightmost TEM image inset, Figure 6A). DLS analysis revealed a linear increase in polymersome size with polymer concentration, possibly reflecting an increase in aggregation number (i.e., the number of block co-polymers that make up a single polymersome). The DLS data is shown in Table 3. [00191] Table 3. Summary of DLS data relative to polymer concentration

[00192] The effect of total flow rate (Qtotai = 1-8 mL/min) on polymersome formation was also assessed. The Qorganic/Qtotai and c _po iymer was maintained at 0.7 and 1 mg/mL, respectively, to once again target the polymersome morphology. The DLS data for five resulting quenched aqueous polymersome samples are shown in Figure 6B. As the data shows, polymersome size decreases slightly as a function of Qtotai, while PDI values (x datapoints in Figure 6B) decrease exponentially with Qtotai. For polymersomes prepared at the highest total flow rate (Qtotai = 8 mL/min), a notably low PDI of 0.045 ± 0.015 was measured, indicating near-monodisperse polymersomes (Figure 6C for TEM image). The decreasing size and PDI trends at higher Qtotai were attributed to an increase in flow turbulence during micromixing (Figure 7). The effect of Qtotai, however, diminishes beyond Qtotai s 8 mL/min as micromixing efficiency can no longer be reduced beyond the limitations imposed by the geometry of the micromixer. The DLS data is shown in Table 4.

[00193] Table 4. Summary of DLS data relative to flow rate

[00194] In the above, polymer concentration c _poiy mer and/or total flow rate Qtotai can be individually increased to improve the scalability of the method of the present invention. The combination of both polymer concentration and total flow rate was also explored. To this end, the self-assembly process was conducted using the following flow conditions: (i) Qorganic/Qtotai = 0.7 to target the polymersome morphology, (ii) c _poiy mer = 9 mg/mL, and (iii) Qtotai= 8 mL/min. The sample was quenched in water and then analyzed by TEM and DLS (Figure 6D). Even under these conditions, the formation of a pure phase of polymersomes (Dh, intensity = 121 ± 39 nm) with a relatively low PDI of 0.097 ± 0.015 was observed. Importantly, these continuous flow conditions equate to a production rate of >3 g of polymersomes/hour, far exceeding the capabilities of typical batch self-assembly processes. The production rate demonstrated herein can undoubtedly be improved by further increasing c _po iymer and Qtotai.

Example 5 - Alternative block co-polymers

[00195] The applicability of the method of the present invention to other block co-polymers was also investigated. Two polymers: (i) a triblock terpolymer, PEC>44-£>-P4VP2i-b-PS3oo (Chemical structure in Figure 6Ei and Figure 8 for characterisation data), which has an additional poly(4-vinylpyridine) (P4VP) middle block and a longer PS block to compensate the change in amphiphilicity incurred by the hydrophilicity of the P4VP block, and (ii) a diblock copolymer, PAA ₂ 6-6-PS ₈ I (Chemical structure in Figure 6Fi and Figure 9 for characterisation data), which has a hydrophilic polyacrylic acid (PAA) block to replace the corona-forming block, PEO were synthesised as discussed above (Scheme III and VI, respectively).

[00196] Indeed, for PEC>44-£>-P4VP2i-b-PS3oo and PAA26-£>-PSSI, the method of the present invention is applicable to the two polymers as shown in Figure 6E-F. In both cases, the polymersome morphology can be targeted with only minor alterations in self-assembly conditions, which were required to either (i) dissolve the longer PS block of PEO44-b-P4VP ₂ i- b-PSsoo or (ii) partially protonate the PAA chains of PAA ₂ 6-b-PS8i (as discussed above). Despite the changes introduced, aging studies (as shown in Figure 6E-F(ii-iv)) confirmed that both polymersome variants exhibited metastability, just as with PEO44-6-PS86 polymersomes.

[00197] The DLS data for PEC>44-£>-P4VP2i-b-PS3oo and PAA26-6-PS81 is shown in Tables 5 and 6, respectively.

[00198] Table 5. Summary of DLS data for PE044-£>-P4VP ₂ i-b-PS3oo [00199] Table 6. Summary of DLS data for PAA26-b-PS _8i

[00200] Exemplary amphiphilic block-co-polymers which have been used to form metastable polymersomes in the present invention include:

Example 6 - Downstream processing via continuous flow

[00201] Due to polymersome metastability, downstream processes can be incorporated in the method and apparatus of the present invention to control polymersome size and/or morphology and/or polydispersity in a similar manner to aging experiments as discussed in Example 3. The continuous flow apparatus was modified as shown in Figure 10A to include an annealing loop and a 100-psi back-pressure regulator down the line. A photograph of the setup is also shown in Figure 11. The annealing loop serves as a reactor to heat the polymersome solution and push the polymersomes out of their metastable state and into more stable, lower free energy states. The back-pressure regulator increases the boiling point of the organic phase and prevents solvent outgassing/evaporation.

[00202] The annealing loop used has a volume (Vanneaiing) of 2 mL and a total flow rate (Qtotai) of 4 mL/min to effectively yield an annealing residence time (tresidence/anneaiing = \/anneaiing/Qtotai) of 30 s. The other flow conditions used were Q _or ganic Qtotai = 0.7 and c _po iymer= 1 mg/mL to target the polymersome morphology. In a typical experiment, the following steps would be performed (i) produce and anneal the polymersomes underflow conditions at different temperatures ( T _an neaiing = 20-70 °C), (ii) collect and allow the polymersomes to cool to room temperature, (iii) dialyze them against water to remove the organic solvents, before finally (iv) analysing the collected product by DLS and TEM.

[00203] Indeed, as confirmed by DLS analysis (Figure 10B), polymersome size distribution was successfully manipulated/controlled by changing annealing temperature. The polymersome morphology was maintained across the entire range of annealing temperatures as confirmed by TEM images (Figure 10D-F). A minimum annealing temperature of 40 °C (Figure 10C) is preferred to induce an increase in polymersome size. Interestingly, the linear size increment observed when annealing temperature was increased from 40-70 °C. This strongly resembles the linear growth dynamics seen in Example 3 (see day 0-7 DLS datapoints in Figure 5C). The similarity in upper size limit of the aged (equilibrium) polymersomes and the polymersomes annealed at 70 °C (about 190-195 nm in both cases, see final entries of Table 2 and Table 7 for D _h . intensity comparison).

[00204] The DLS data of annealed metastable polymersomes is shown in Table 7.

[00205] Table 7. Summary of DLS data of annealed metastable polymersomes.

[00206] Accordingly, the time needed for the polymersomes to transition from their metastable state into a global equilibrium state (i.e. , from having to age the polymersomes for 7 days down to just 30 s by downstream annealing at 70 °C) can be significantly reduced by downstream processing via annealing.

[00207] To demonstrate the modularity of the present invention, an additional downstream process was implemented to manipulate/control polymersome shape/morphology, in addition to manipulating polymersome size. For this embodiment, the continuous flow apparatus was modified to include a cooling loop and a secondary micromixer connected to another syringe pump (Figure 12A and Figure 13).

[00208] The cooling loop allows the polymersomes to grow into its equilibrium size following annealing. To confirm this, Figure 12B shows a turbidity plot revealing how polymersomes that have been collected after annealing at 70 °C (using the setup in Figure 10A) require >2.5 min of cooling under ambient/batch conditions to achieve its maximum size. In order to translate the growth process under flow conditions, sufficient time should preferably be provided for the annealed polymersomes to grow prior to being subjected to shape transformation.

[00209] In a preferred embodiment, the cooling loop is preferably placed in an incubator at 40 °C. This can reduce the cooling rate sufficiently as to allow the annealed polymersomes to achieve their equilibrium size during the prescribed residence time.

[00210] The secondary micromixer, which is placed downstream of the cooling loop (Figure 12A), serves as a junction for the introduction of an additive (NaCI solution) needed to osmotically deform the annealed/grown polymersomes. In a typical experiment, a salinity change of 50 mM NaCI is generated by micromixing the annealed/grown polymersome solution with a concentrated NaCI solution (5.05 M) at a flow rate of 4 mL/min and 0.04 mL/min, respectively. A small amount of concentrated NaCI solution is typically preferred (as opposed to larger volumes of diluted NaCI solution) to minimise deviations in solvent quality after micromixing, thus preventing or minimising any morphological deviations beyond the intended shape transformation process.

[00211] To produce stomatocytes in a continuous flow fashion, the setup used was as shown in Figure 12A and the following flow conditions were applied: (i) c _po iymer = 1 mg/mL, (ii) Qorganic/Qtotal ⁼ 0.7, (Hi) Qtotal ⁼ 4 mL/min, (iv) Tannealing ⁼ 70 C With tresidence/annealing ⁼ 30 S, (v) Tcooling = 40 °C with tresidence/cooiing = 7.5 min, and (vi) c _N aci = 5.05 M at Q _Na ci = 0.04 mL/min. The as- produced solution was then dialysed to remove the organic solvents, and subsequently solvent exchanged with water to facilitate imaging. The stomatocyte morphology was confirmed using a combination of TEM (Figure 12C) and cryo-TEM (Figure 12D).

[00212] The metastable polymersomes of the continuous flow apparatus can also be downstream processed using a downstream purification process. In this embodiment, the downstream purification step can be performed using an in-line dialysis cell. An exemplary embodiment is shown in Figure 14. This embodiment can remove organic solvent from the polymersome samples under continuous flow conditions. The purification is based on crossflow dialysis, where a stream of water or another aqueous solution is flowed in the opposite direction relative to sample flow.

[00213] In certain embodiments, a pH stimulus can be used for downstream processing to control morphological transformation. This can be used in embodiments where the amphiphilic block co-polymer has a pH responsive group such as polyacrylic acid (PAA). An exemplary embodiment is shown in Figure 15 where the control of pH can induce morphological change from polymersomes (at equilibration loop 1) to micelles (equilibration loop 2) for a PAA ₂ 6-b- PSsi block co-polymer.

[00214] The use of a continuous flow apparatus as described herein can produce dynamic metastable polymersomes. At room temperature, the metastable state has a lifetime of about 7 days, after which the polymersomes reaches an equilibrium state. Despite being at equilibrium, the polymersome structures remain dynamic and can undergo shape transformation into stomatocytes. The method of the present invention is robust as it accommodates various flow conditions to, for instance, upscale polymersome production (>3 g/h) or yield polymer micelles. The self-assembly process is not polymer-specific and can be applied to block co-polymers with different block chemistries/lengths provided they are suitable to target the polymersome morphology. The continuous flow apparatus can be modified in a plug-and-play fashion. The present inventors have demonstrated that downstream processes such as thermal annealing and/or secondary micromixing can be performed in a continuous fashion to manipulate polymersome size (with sub- 100 nm precision) and/or polymersome shape/morphology. In other embodiments, the metastable polymersomes can be downstream processed in a batch wise manner via aging. The present invention as described herein is a significant contribution to the nanoparticle manufacturing sector, as it provides not only a fundamental basis for the production of near-monodisperse polymersomes at scale, but also showcases how polymersome properties can be tuned with precision on the nanoscale, in particular, under continuous flow conditions.

[00215] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is understood that the invention includes all such variations and modifications which fall within the spirit and scope of the present invention.