Copolymer for preparing polymersomes having improved electrostatic properties

Abstract

The present invention relates to polymersomes which have enhanced stability, and copolymer materials that are suitable for making such polymersomes. The polymersomes comprise a particular type of triblock copolymer which, under typical in vivo physiological conditions, is resistant to hydrolysis and which therefore allows the polymersomes to maintain a constant electrostatic potential (zeta potential) for a longer period of time. This allows for improved targeting of polymersomes to their site of action in vivo. Specifically, the present invention is directed to a triblock copolymer comprising a hydrophilic block, a linker moiety and a hydrophobic block, wherein the rate of hydrolysis of the linker moiety is lower under typical in vivo physiological conditions that the rate of hydrolysis of the hydrophobic block. The present invention is further directed to polymersomes comprising such a triblock copolymer, as well as pharmaceutical compositions comprising a plurality of such polymersomes and one or more pharmaceutically acceptable excipients or diluents. The present invention is further directed to uses of such polymersomes and pharmaceutical compositions in medicine, and to vaccines comprising such polymersomes.

Description

[0001] COPOLYMER FOR PREPARING POLYMERSOMES HAVING IMPROVED ELECTROSTATIC PROPERTIES

[0002] Field of the invention

[0003] The present invention relates to polymersomes which have enhanced stability, and copolymer materials that are suitable for making such polymersomes. The polymersomes comprise a particular type of triblock copolymer which, under typical in vivo physiological conditions, is resistant to hydrolysis and which therefore allows the polymersomes to maintain a constant electrostatic potential (zeta potential) for a longer period of time. This allows for improved targeting of polymersomes to their site of action in vivo.

[0004] Specifically, the present invention is directed to a triblock copolymer comprising a hydrophilic block, a linker moiety and a hydrophobic block, wherein the rate of hydrolysis of the linker moiety is lower under typical in vivo physiological conditions that the rate of hydrolysis of the hydrophobic block. The present invention is further directed to polymersomes comprising such a triblock copolymer, as well as pharmaceutical compositions comprising a plurality of such polymersomes and one or more pharmaceutically acceptable excipients or diluents. The present invention is further directed to uses of such polymersomes and pharmaceutical compositions in medicine, and to vaccines comprising such polymersomes.

[0005] Background to the invention

[0006] Polymersomes are self-assembled vesicles comprising synthetic polymers. The polymers that make up a polymersome are typically amphiphilic diblock copolymers, comprising a hydrophilic block which is exposed to the exterior surface of the polymersome vesicle, and a hydrophobic block which is exposed to the interior surface of the polymersome vesicle.

[0007] The use of polymersomes in drug delivery has been the subject of much research, because polymersomes possess key properties which makes them highly suited to this use. First, the formation of polymersomes into vesicles makes them attractive as carriers of encapsulated cargoes that are sensitive to degradation in e.g. blood plasma and / or the cell cytoplasm, such as nucleic acids (see, e.g., WO 2024 / 147020, the contents of which are herein incorporated by reference in their entirety). Second, the chemical properties of the copolymers which make up the polymersomes can be fine-tuned depending on the intended use, e.g. to promote disassembly of the polymersomes in a particular pH microenvironment, or escape premature lysosomal degradation (see, e.g., WO 2019 / 197834, the contents of which are herein incorporated by reference in their entirety). Third, polymersomes can be decorated with particular profiles of surface ligands which aid the selective targeting of the polymersomes to a particular cell or tissue of interest (see, e.g., WO 2020 / 144467, the contents of which are herein incorporated by reference in their entirety).

[0008] The potential for the polymeric components of polymersomes to be hydrolysed in systemic circulation however remains a challenge with developing polymersomes for therapeutic use. In particular, the hydrophobic block of the diblock copolymers employed in polymersomes is often a polyester or a polyamide. If the bond between the hydrophilic block and the hydrophobic block of such copolymers (which is typically an ester or an amide bond) is hydrolysed, the hydrophilic block dissociates from the bulk structure of the polymersome, leaving a free carboxylate group exposed to the external surface of the polymersome (see Fig. 1 for a schematic representation using PEG-PLA diblock copolymer as an example). These free carboxylate groups are negatively charged at physiological pH and therefore hydrolysis of the bond between the hydrophilic and hydrophobic blocks of the copolymer molecules that make up the polymersome results in an increase in the negative charge on the surface of the polymersome (i.e. a more negative zeta potential).

[0009] In turn, this has a negative impact on the properties of the polymersomes. For instance, some studies show that, compared to corresponding positively charged nanoparticles, negatively charged nanoparticles comprising a diblock copolymer of poly(ethylene glycol) and poly(lactic acid) (PEG-PLA) have lower cellular uptake and transport across epithelial cells (see Du et al., Biomater. Sci., 2018, 6:642) and lower tumour penetration than corresponding positively charged nanoparticles (see Wang etal., Nano Today, 2016, 11(2): 133-144). Another study demonstrated that charged micelles comprising a diblock copolymer of poly(ethylene glycol) and poly(s-caprolactone) (PEG-PCL) were degraded more rapidly by lipase than corresponding micelles with a neutral electrostatic potential (see Wan et al., Polym Chem, 2014, 5, 1720).

[0010] Thus, for optimum performance of polymersomes as drug delivery tools, it is important to be able to control the electrostatic potential on the polymersome surface. Changes in the electrostatic potential in vivo as a consequence of polymer hydrolysis is undesirable, and it is necessary to provide polymersomes that have a reliable and stable electrostatic potential. Previous approaches taken in the art to control the electrostatic potential of polymersomes include mixing the polymersomes with charged surfactants (as in Du et al. , Biomater. Sci. , 2018, 6:642; and Wang et al., Nano Today, 2016, 11(2): 133-144) or changing the termination groups of the hydrophilic block (as in Wan et al., Polym Chem, 2014, 5, 1720), rather than to try to prevent the degradation-induced formation of carboxylate.

[0011] There remains a need in the art to provide polymersomes comprising copolymers with reduced susceptibility to degradation by hydrolysis such that negative surface charge does not build up over time on the polymersomes.

[0012] Summary of the invention

[0013] The present inventors have surprisingly discovered that, by adding a particular type of linker moiety between the hydrophilic and hydrophobic blocks in a diblock copolymer, to create a triblock copolymer, the resulting polymersomes are less susceptible to degradation in such a way that exposes negatively charged carboxylate moieties on the polymersome surface. Thus, polymersomes can be provided which have more stable surface electrostatic potentials, which expands the utility of such polymersomes in vivo.

[0014] In particular, the present inventors have discovered that if a linker group is inserted between the hydrophilic and hydrophobic blocks such that the resulting bond between the linker and the hydrophilic group is less susceptible to hydrolysis than either (a) a direct bond between the hydrophilic and hydrophobic groups and (b) the bonds between the constituent monomer units of the hydrophobic group, then the polymersomes are surprisingly less susceptible to the development of increased negative charge on their external surface (more negative zeta potential). Without wishing to be bound by any particular theory, it is believed that in such polymersomes, hydrolysis preferentially occurs at the bonds between the constituent monomer units of the hydrophobic block, and such hydrolysis does not lead to the cleavage of the hydrophilic block; as a result, free carboxylate groups are not exposed on the external surface of the polymersome.

[0015] In the case where the hydrophobic block is a polyester, the inventors have discovered that to achieve the desired improvement in stability via insertion of the linker, the linker moiety may typically contain an ester monomer unit or an amide monomer unit. If the linker also contains an ester monomer unit (i.e. the linker is a sole ester monomer or is a polyester), then the relative length of the monomer units in (i) the linker moiety and (ii) the hydrophobic block is important. Thus, in this case, the linker moiety should comprise an ester monomer having more than 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy- substituted carbon, whilst the hydrophobic block should comprise an ester monomer having from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy- substituted carbon. In the case where the hydrophobic block is a polyester and the linker moiety comprises an amide monomer (i.e. the linker moiety is a sole amide monomer or is a polyamide), the relative lengths of the monomer units in the linker moiety and the hydrophobic block is a less critical parameter, as an amide bond is significantly less susceptible to hydrolysis than an ester bond.

[0016] Similarly, in the case where the hydrophobic block is a polyamide, the linker moiety should comprise an amide monomer having more carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon than the amide monomer which makes up the hydrophobic block. Preferably, the amide monomer in the hydrophobic block comprises 2 carbon atoms in a linear chain from the carbonyl carbon to the aminosubstituted carbon (i.e. is an a-amino acid) and the amide monomer in the linker moiety comprises more than 2 carbon atoms in a linear chain from the carbonyl carbon to the amino-subsituted carbon. Alternatively, the amide monomer in the hydrophobic block may comprise 3 carbon atoms in a linear chain from the carbonyl carbon to the aminosubstituted carbon (i.e. is a P-amino acid) and the amide monomer in the linker moiety comprises more than 3 carbon atoms in a linear chain from the carbonyl carbon to the amino-subsituted carbon.

[0017] Alternatively, in either case (i.e. where the hydrophobic block is either a polyester or a polyamide), the linker moiety may comprise an ether or a thioether monomer (which are less susceptible to hydrolysis than ester or amide bonds), particularly ether or thioether monomers that are sterically hindered, i.e. substituted with one, two, three or more bulky substituents.

[0018] The present inventors have also surprisingly discovered that the length of the linker moiety (i.e. the number of monomer units within the linker moiety) is important in achieving the desired stabilising effect. For small lengths of linker group, improved stability is observed as the number of monomer units is increased. However, there is a “sweet spot” in the length of the linker, and beyond a certain optimum length, the stabilising effect of the linker group decreases as further monomer units continue to be added. Without wishing to be bound by any particular theory, it is considered that if the linker moiety becomes too long, the conformation of the copolymer within the polymersome and / or the crystallinity of the polymersome may be affected in such a way that the bond between the linker and the hydrophilic group becomes more accessible to external water molecules, and thus more susceptible to hydrolysis.

[0019] In general terms, the present invention is directed to a triblock copolymer comprising a hydrophilic block, a linker moiety and a hydrophobic block, wherein:

[0020] - the hydrophilic block is covalently bonded to the hydrophobic block via the linker moiety;

[0021] - the hydrophobic block is a polyester or a polyamide; and the linker moiety consists of from 1 to 50 monomer units, wherein the bond between adjacent monomer units in the linker moiety and / or the bond between the linker moiety and the hydrophilic block is less susceptible to hydrolysis than the bond between adjacent monomer units in the hydrophobic block.

[0022] In one aspect, the present invention accordingly provides a triblock copolymer comprising a hydrophilic block, a linker moiety and a hydrophobic block, wherein the hydrophilic block is covalently bonded to the hydrophobic block via the linker moiety, and wherein:

[0023] (a) the hydrophobic block is a polyester, and the linker moiety consists of from 1 to 50 monomer units of:

[0024] (i) an ester monomer which comprises a longer linear chain of carbon atoms, from the carbonyl carbon to the hydroxy- substituted carbon within the monomer, than the ester monomer which makes up the polyester of the hydrophobic block;

[0025] (ii) an amide monomer;

[0026] (iii) an ether monomer; or

[0027] (iv) a thioether monomer; or

[0028] (b) the hydrophobic block is a polyamide, and the linker moiety consists of from 1 to 50 monomer units of: (i) an amide monomer which comprises a longer linear chain of carbon atoms, from the carbonyl carbon to the amino-substituted carbon within the monomer, than the amide monomer which makes up the polyamide of the hydrophobic block;

[0029] (ii) an ether monomer; or

[0030] (iii) a thioether monomer.

[0031] Preferably, when the hydrophobic block is a polyester, the linker moiety consists of from 1 to 50 monomer units of (i) an ester monomer which comprises a longer linear chain of carbon atoms, from the carbonyl carbon to the hydroxy-substituted carbon within the monomer, than the ester monomer which makes up the polyester of the hydrophobic block, or (ii) an amide monomer.

[0032] Preferably, when the hydrophobic block is a polyester and the linker moiety comprises an ester monomer, each ester monomer unit in the polyester comprises from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy-substituted carbon, and each ester monomer unit in the linker moiety comprises more than 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy- substituted carbon.

[0033] Preferably, when the hydrophobic block is a polyester and the linker moiety comprises an amide monomer, each ester monomer unit in the polyester comprises from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy-substituted carbon. More preferably, each amide monomer unit in the linker moiety comprises from 2 to 4 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon.

[0034] Preferably, when the hydrophobic block is a polyamide, each amide monomer unit in the polyamide comprises 2 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon, and the linker moiety consists of from 1 to 50 monomer units of an amide monomer comprising more than 2 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon.

[0035] Alternatively, when the hydrophobic block is a polyamide, each amide monomer unit in the polyamide comprises 3 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon, and the linker moiety consists of from 1 to 50 monomer units of an amide monomer comprising more than 3 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon.

[0036] The present invention also provides a polymersome comprising the triblock copolymer of the invention.

[0037] In a further aspect, the present invention provides a pharmaceutical composition comprising a plurality of the polymersomes according to the invention, and one or more pharmaceutically acceptable excipients or diluents.

[0038] In a further aspect, the present invention provides a polymersome or a pharmaceutical composition according to the invention for use in the treatment of a disease, optionally wherein the disease is cancer, an infectious disease, or a disorder of the nervous system.

[0039] In a further aspect, the present invention provides a method of treating cancer, an infectious disease or a disorder of the nervous system in a human patient, wherein said method comprises administration of a polymersome or a pharmaceutical composition according to the invention to a patient in need thereof.

[0040] In a further aspect, the present invention provides use of a polymersome or a pharmaceutical composition according to the invention for the manufacture of a medicament for the treatment of cancer, an infectious disease, or a disorder of a nervous system in a patient.

[0041] In a further aspect, the present invention provides a vaccine comprising a polymersome according to the invention, and one or more pharmaceutically acceptable excipients or diluents, preferably wherein the polymersome comprises an mRNA cargo encapsulated within the polymersome.

[0042] Brief description of the figures

[0043] Fig. l is a schematic diagram showing that under conditions which promote hydrolysis, the hydrophilic block of a block copolymer in a polymersome may dissociate from the bulk structure of the polymersome, leaving a free carboxylate group exposed to the external surface of the polymersome, which increases the magnitude of the negative zeta potential on the surface of the polymersome. Fig. 2 shows, for polymersomes formed from PEG(45)-OCO-PLA(150) comprising HOOC-PLA as impurity, (a) the particle size distribution by number as measured by dynamic light scattering, (b) the particle size distribution by intensity as measured by dynamic light scattering, (c) a correlogram of the DLS data, and (d) a transmission electron micrograph image of the polymersomes.

[0044] Fig. 3 shows, for polymersomes formed from PEG(45)-OCO-PLA(157) comprising trace HOOC-PLA, (a) the particle size distribution by number as measured by dynamic light scattering, (b) the particle size distribution by intensity as measured by dynamic light scattering, (c) a correlogram of the DLS data, and (d) a transmission electron micrograph image of the polymersomes.

[0045] Fig. 4 shows, for polymersomes formed from PEG(45)-NHCO-PCL(2)-PLA(150) comprising trace HOOC-PCL-PLA, (a) the particle size distribution by number as measured by dynamic light scattering, (b) the particle size distribution by intensity as measured by dynamic light scattering, (c) a correlogram of the DLS data, and (d) a transmission electron micrograph image of the polymersomes.

[0046] Fig. 5 shows the change in zeta potential (measured by ELS) over a 30-day time period for polymersomes stored in phosphate-buffered saline (PBS) at 5°C which are formed from different block copolymers: (1) PEG(45)-OCO-PLA(150) comprising HOOC-PLA as impurity; (2) PEG(45)-OCO-PLA(157) comprising trace HOOC-PLA; and (3) PEG(45)- NHCO-PCL(2)-PLA(150) comprising trace HOOC-PCL-PLA.

[0047] Fig. 6 shows, for polymersomes formed from PEG(45)-O-PCL(20)-PLA(149), (a) the particle size distribution by number as measured by dynamic light scattering, (b) the particle size distribution by intensity as measured by dynamic light scattering, (c) a correlogram of the DLS data, and (d) a transmission electron micrograph image of the polymersomes.

[0048] Fig. 7 shows, for polymersomes formed from PEG(45)-NH-PCL(2)-PLA(150), (a) the particle size distribution by number as measured by dynamic light scattering, (b) the particle size distribution by intensity as measured by dynamic light scattering, (c) a correlogram of the DLS data, and (d) a transmission electron micrograph image of the polymersomes. Fig. 8 shows, for polymersomes formed from PEG(45)-NH-PCL(7)-PLA(105), (a) the particle size distribution by number as measured by dynamic light scattering, (b) the particle size distribution by intensity as measured by dynamic light scattering, (c) a correlogram of the DLS data, and (d) a transmission electron micrograph image of the polymersomes.

[0049] Fig. 9 shows the change in zeta potential (measured by ELS) over a 30-day time period for polymersomes stored in phosphate-buffered saline (PBS) at 5°C which are formed from different block copolymers: (a) PEG(45)-O-PCL(20)-PLA(149); (b) PEG(45)-NH-PCL(2)- PLA(150); and (c) PEG(45)-NH-PCL(7)-PLA(105).

[0050] Detailed description

[0051] Definitions

[0052] As defined herein, the term “triblock copolymer” refers to a polymeric species comprising monomer units in which there is a linear arrangement of three blocks, a block being defined as a portion of a polymer molecule consisting of one or more monomeric units which have at least one constitutional feature absent from the adjacent portions. Thus, each block consists of monomer units derived from a specific, different species of monomer to the adjacent block(s). The triblock copolymers of the present invention have in particular a configuration A-B-C, where A, B and C represent blocks of three different monomer units (a, b and c respectively). Thus, the triblock copolymers of the present invention have a configuration [a]x-[b]y-[c]zwhere a, b and c represent different monomer units and x, y and z are whole number positive integers. Specifically, as will be discussed further below, in the copolymers of the present invention:

[0053] - A represents a hydrophilic block, and thus a represents a hydrophilic monomer unit, and x is a whole number positive integer that is at least 2;

[0054] - B represents a linker moiety, and thus b represents a linker monomer unit, and furthermore the number of monomer units in the linker moiety y is from 1 to 50; and

[0055] C represents a hydrophobic block, and thus c represents a hydrophobic monomer unit, and z is a whole number positive integer that is at least 2. For the avoidance of doubt, in the triblock copolymer of the present invention, one terminus of the linker moiety (B) is directly covalently bonded to the hydrophilic block (A) and the other terminus of the linker moiety is covalently bonded to the hydrophobic block (C). Thus, there are no further intermediate moieties located between the linker moiety and the hydrophilic block, or between the linker moiety and the hydrophobic block.

[0056] In some embodiments, the triblock copolymers of the present invention consist only of the components A-B-C (i.e. [a]x-[b]y-[c]z). In other embodiments, the hydrophilic block A and / or the hydrophobic block C may be “capped” at its / their terminal end with an organic moiety imparting particular functionality, e.g. a ligand (for e.g. binding to a receptor) or a reactive functional group. Thus, in some embodiments, the hydrophilic block A may be “capped” at its terminal end with an organic moiety imparting particular functionality, e.g. a ligand (for e.g. binding to a receptor) or a reactive functional group (e.g. the triblock copolymer may have the structure ligand-A-B-C or ligand-[a]x-[b]y-[c]z). In other embodiments, the hydrophobic block C may be “capped” at its terminal end with an organic moiety imparting particular functionality, e.g. a ligand (for e.g. binding to a receptor) or a reactive functional group (e.g. the triblock copolymer may have the structure A-B-C-ligand or [a]x-[b]y-[c]z-ligand). In other embodiments, the hydrophilic block A and the hydrophobic block C may both be “capped” at their respective terminal ends with an organic moiety imparting particular functionality, e.g. a ligand (for e.g. binding to a receptor) or a reactive functional group (e.g. the triblock copolymer may have the structure ligand-A-B-C-ligand or ligand-[a]x-[b]y-[c]z-ligand). Preferably, if a ligand is present, it is covalently bonded to the hydrophilic block A. However, further polymeric blocks may not be present. Thus, a triblock copolymer cannot comprise any blocks of repeating monomer units other than a single hydrophilic block A, a single linker moiety B and a single hydrophobic block C. For the avoidance of doubt, structures of the type A-B-C-D, where D is a block consisting of a monomeric repeat unit d, and structures of the type A-B-C-B- A, A-B-C-A-B-C or A-B-A-C, which contain further blocks of any of the monomeric repeat units a, b and / or c, are not triblock copolymers in accordance with the present invention. Likewise, structures of the type A-X-B-C or A-B-Y-C or A-X-B-Y-C where X and Y are additional groups that do constitute part of the hydrophilic block A, linker moiety B or hydrophobic block C, are not triblock copolymers in accordance with the present invention. Moreover, although structures of the type A-B-A are triblock copolymers within the most general meaning of the term, these are also not triblock copolymers according to the present invention, because copolymers of the present invention comprise blocks A, B and C, each derived from a different type of monomer unit.

[0057] As defined herein, the term “diblock copolymer” refers to a polymeric species comprising monomer units in which there is a linear arrangement of two blocks, a block being defined as a portion of a polymer molecule consisting of monomeric units which have at least one constitutional feature absent from the adjacent portions. Thus, each block consists of monomer units derived from a specific characteristic species of monomer. The diblock copolymers useful in the present invention have a configuration A-B, where A and B represent blocks of two different monomer units (a and b respectively). Thus, the diblock copolymers useful in the present invention have a configuration [a]x-[b]ywhere a and b represent different monomer units and x and y are whole number positive integers of 2 or greater. Typically, A represents a hydrophilic block (and thus a represents a hydrophilic monomer unit) and B represents a hydrophobic block (and thus b represents a hydrophobic monomer unit).

[0058] In some embodiments, a diblock copolymer consists only of the components A-B (i.e. [a]x- [b]y). In other embodiments, the hydrophilic block A and / or the hydrophobic block B may be “capped” at its / their terminal end with an organic moiety imparting particular functionality, e.g. a ligand (for e.g. binding to a receptor) or a reactive functional group. Thus, in some embodiments, the hydrophilic block A may be “capped” at its terminal end with an organic moiety imparting particular functionality, e.g. a ligand (for e.g. binding to a receptor) or a reactive functional group (e.g. the diblock copolymer may have the structure ligand-A-B or ligand-[a]x-[b]y). In other embodiments, the hydrophobic block B may be “capped” at its terminal end with an organic moiety imparting particular functionality, e.g. a ligand (for e.g. binding to a receptor) or a reactive functional group (e.g. the diblock copolymer may have the structure A-B-ligand or [a]x-[b]y-ligand). In other embodiments, both the hydrophilic block A and the hydrophobic block B may be “capped” at their respective terminal ends with an organic moiety imparting particular functionality, e.g. a ligand (for e.g. binding to a receptor) or a reactive functional group (e.g. the diblock copolymer may have the structure ligand-A-B -ligand or ligand-[a]x-[b]y- ligand). Preferably, if a ligand is present, it is covalently bonded to the hydrophilic block A. However, further polymeric blocks may not be present. Thus, a diblock copolymer cannot comprise any blocks of repeating monomer units other than a single (hydrophilic) block A and a single (hydrophobic) block B. For the avoidance of doubt, structures of the type A-B-C, where C is a block consisting of a monomeric repeat unit c, and structures of the type A-B-A, B-A-B or A-B-A-B, which contain further blocks of any of the monomeric repeat units a and / or b, are not diblock copolymers as defined herein.

[0059] As defined herein, the term “alkyl” refers to a linear or branched saturated monovalent hydrocarbon radical having the number of carbon atoms indicated in the prefix. Thus, the term “Ci-Ce alkyl” refers to a linear saturated monovalent hydrocarbon radical of one to six carbon atoms or a branched saturated monovalent hydrocarbon radical of three or to six carbon atoms, e.g. methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl and the like. Preferably an alkyl group is a Ci-Ce alkyl group, and more preferably a C1-C4 alkyl group.

[0060] As defined herein, the term “alkylene” refers to a linear saturated divalent hydrocarbon radical or a branched saturated divalent hydrocarbon radical having the number of carbon atoms indicated in the prefix, e.g. methylene, ethylene, propylene, 1 -methylpropylene, 2- methylpropylene, butylene, pentyl ene, and the like. Preferably, an alkylene group is a Ci- Ce alkylene group, and more preferably a C1-C4 alkylene group.

[0061] As defined herein, the term “alkenyl” refers to a linear or branched saturated monovalent hydrocarbon radical having the number of carbon atoms indicated in the prefix and containing at least one double carbon-carbon bond. Thus, the term “C2-C6 alkenyl” refers to a linear saturated monovalent hydrocarbon radical of two to six carbon atoms having at least one double bond, or a branched saturated monovalent hydrocarbon radical of three to six carbon atoms having at least one double carbon-carbon bond, e.g. ethenyl, propenyl, 1,3-butadienyl, (CH2)2CH=C(CH3)2, CH2CH=CHCH(CH3)2, and the like. Preferably, an alkenyl group is a C2-C6 alkenyl group, and more preferably a C2-C4 alkenyl group.

[0062] As defined herein, the term “alkynyl” refers to a linear or branched saturated monovalent hydrocarbon radical having the number of carbon atoms indicated in the prefix and containing at least one triple carbon-carbon bond. Thus, the term “C2-C6 alkynyl” refers to a linear saturated monovalent hydrocarbon radical of two to six carbon atoms having at least one triple carbon-carbon bond, or a branched saturated monovalent hydrocarbon radical of three to six carbon atoms having at least one triple carbon-carbon bond, e.g. ethynyl, propynyl, 1,3-butadiynyl, and the like. Preferably, an alkynyl group is a C2-C6 alkynyl group, and more preferably a C2-C4 alkynyl group.

[0063] As defined herein, the term “alkoxy” refers to an -OR radical where R is alkyl as defined above, e.g., methoxy, ethoxy, n-propoxy, iso-propoxy, n-butyl, iso-butyl, tert-butyl and the like. Preferably an alkoxy group is a Ci-Ce alkoxy group, and more preferably a C1-C4 alkoxy group.

[0064] As defined herein, the term “alkylamino” refers to an -NHR radical where R is alkyl as defined above, e.g. methylamino, ethylamino, n-propylamino, iso-propyl amino, and the like. Preferably an alkylamino group is a Ci-Ce alkylamino group, and more preferably a C1-C4 alkylamino group.

[0065] As defined herein, the term “aryl” refers to a monovalent monocyclic or bicyclic aromatic hydrocarbon radical of 6 to 10 ring atoms, e.g. phenyl or naphthyl, and the like.

[0066] As defined herein, the term “aralkyl” refers to an -(alkylene)-R radical where R is aryl as defined above.

[0067] As defined herein, the term “cycloalkyl” refers to a cyclic saturated monovalent hydrocarbon radical of three to ten carbon atoms wherein one or two carbon atoms may be replaced by an oxo group, e.g. cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, and the like. Preferably a cycloalkyl group is a C4-C6 cycloalkyl group.

[0068] As defined herein, the term “cycloalkylalkyl” refers to an -(alkylene)-R radical where R is cycloalkyl as defined above, e.g. cyclopropylmethyl, cyclobutylmethyl, cyclopentylethyl, or cyclohexylmethyl, and the like.

[0069] As defined herein, the term “halo” refers to fluoro, chloro, bromo, or iodo, preferably fluoro or chloro.

[0070] As defined herein, the term “haloalkyl” refers to an alkyl radical as defined above, which is substituted with one or more halogen atoms, preferably one to five halogen atoms, preferably fluorine or chlorine, including those substituted with different halogens, e.g. CH2CI, -CF3, -CHF2, -CH2CF3, -CF2CF3, -CF(CH3)2, and the like. Preferably a haloalkyl group is a Ci-Ce haloalkyl group, and more preferably a C1-C4 haloalkyl group. As defined herein, the term “haloalkoxy” refers to an -OR radical where R is haloalkyl as defined above, e.g. -OCF3, -OCHF2, and the like.

[0071] As defined herein, the term “heteroaryl” refers to a monovalent monocyclic or bicyclic aromatic radical of 5 to 10 ring atoms where one or more, preferably one, two, or three, ring atoms are heteroatom selected from N, O, or S, the remaining ring atoms being carbon. Representative examples include, but are not limited to, pyrrolyl, thienyl, thiazolyl, imidazolyl, furanyl, indolyl, isoindolyl, oxazolyl, isoxazolyl, benzothiazolyl, benzoxazolyl, quinolinyl, isoquinolinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl, tetrazolyl, and the like.

[0072] As defined herein, the term “heteroaralkyl” refers to an -(alkylene)-R radical where R is heteroaryl as defined above.

[0073] As defined herein, the term “heterocycyl” refers to a saturated or unsaturated monovalent monocyclic group of 4 to 8 ring atoms in which one or two ring atoms are heteroatoms selected from N, O, or S(O)Z, where z is an integer from 0 to 2, the remaining ring atoms being C. The heterocyclyl ring is optionally fused to a (one) aryl or heteroaryl ring as defined herein provided the aryl and heteroaryl rings are monocyclic. Additionally, one or two ring carbon atoms in the heterocyclyl ring can optionally be replaced by a -CO- group. More specifically the term heterocyclyl includes, but is not limited to, pyrrolidino, piperidino, homopiperidino, 2-oxopyrrolidinyl, 2-oxopiperidinyl, morpholino, piperazino, tetrahydropyranyl, thiomorpholino, and the like. When the heterocyclyl ring is unsaturated it can contain one or two ring double bonds, provided that the ring is not aromatic.

[0074] As defined herein, the term “heterocycloalkyl” refers to an -(alkyl ene)-R radical where R is heterocyclyl ring as defined above, e.g. tetraydrofuranylmethyl, piperazinylmethyl, morpholinylethyl, and the like.

[0075] As defined herein, the term “substituted” means that any given moiety is substituted with one or more (e.g. one, two, three, four, five, six or more) further groups selected from unsubstituted Ci-Ce alkyl, unsubstituted Ci-Ce alkoxy, unsubstituted Ci-Ce aminoalkyl, unsubstituted Ci-Ce haloalkyl, unsubstituted Ci-Ce haloalkoxy, unsubstituted C3-C10 cycloalkyl, unsubstituted C4-C16 cycloalkylalkyl, unsubstituted C2-C6 alkenyl, s unsubstituted C2-C6 alkynyl, unsubstituted Ce-Cio aryl, unsubstituted C7-C16 aralkyl, unsubstituted C4-C8 heterocycyl, unsubstituted C5-C14 heterocycloalkyl, unsubstituted C5- C10 heteroaryl, unsubstituted Ce-Ci6 heteroaralkyl, halo, hydroxy, amino, cyano or azido. Preferably, a “substituted” moiety comprises one, two, three or four such substituents. More preferably, a “substituted” moiety comprises one, two or three such substituents. Still more preferably, a “substituted” moiety comprises one or two such substituents. Most preferably, a “substituted” moiety comprises one such substituent.

[0076] As defined herein, a “microparticle” is any particle between 0.1 and 100 pm in size.

[0077] As defined herein, a “nanoparticle” is any particle between 1 and 100 nm in size.

[0078] As defined herein, the term “zeta potential” means the electrical potential at the slipping plane (i.e. the interface which separates mobile fluid from fluid that remains attached to the surface). It is a measure of electrokinetic potential in colloidal suspensions, and is caused by the net electrical charge contained within the region bounded by the slipping plane. The zeta potential is an important and readily measurable indicator of the stability of colloidal dispersions. Its magnitude indicates the degree of electrostatic repulsion between adjacent, similarly charged particles in a dispersion.

[0079] Typically, the zeta potential is measured using electrophoretic light scattering (ELS). This involves measuring the velocity of nanoparticles moving towards the electrode in the presence of an external electric field. In traditional electrophoretic light scattering (ELS), the electrophoretic mobility of particles suspended in a solution is measured via the Doppler shift of the scattered light. Similar to dynamic light scattering (DLS) experiments, a monochromatic coherent incident light beam illuminates suspended particles. Whereas in DLS the suspended particles are moving due to Brownian motion, in ELS the particles are also moving due to an applied electric field, if they have a net charge. Particles will move towards either the anode or the cathode, depending on the sign of their net charge. Because of this motion, the frequency and phase of the scattered light will be different from that of the incident light. This phenomenon is referred to as the Doppler effect. Typically, the zeta potential is measured using ELS using the method set out in ISO 13099.

[0080] As defined herein, the term “weight-average molecular weight”, or equivalently “Mw”, refers to the weighted arithmetic mean of a sample of polymer molecules based on their weight fraction. Thus, the weight-average molecular weight is calculated according to the following formula: wherein Mi is the molar mass of each polymer species i within the total sample and Ni is the number of each polymer species i within the total sample. Typically, the weightaverage molecular weight value of a polymer is measured using gel permeation chromatography (GPC). The detector type used at the end of the GPC column is not particularly limited, but typically the detector is a refractive index detector.

[0081] As defined herein, the term “polydispersity”, or equivalently “PDF’, is a measure of the broadness in the size of a polymersome particle. It can be measured using dynamic light scattering. Specifically, dynamic light scattering enables measurement of the “z-average size” (also known as the “z-average diameter” or the “cumulants mean”, which is an intensity weighted mean hydrodynamic size of the particles measured (i.e. a mean value calculated from the signal intensity) for the size of (e.g.) polymersome particles associated with a width parameter, or the polydispersity index. The cumulants analysis of dynamic light scattering data is the fit of a polynomial to the log of the G1 correlation function: ln[Gl(t)] = a + b / + c,t2+ ...

[0082] The value of b is known as the second order cumulant, or the z-average diffusion coefficient. This can be converted to the z-average size of the particles using the dispersity viscosity and some instrumental constants. The polydispersity is calculated as the coefficient of the squared term, c, when scaled as 2c / b2. Typically, the z-average size and the polydispersity are measured via dynamic light scattering using the method set out in ISO 22412:2017 . For further information on how to calculate the z-average and polydispersity values, see also https: / / www.malvernpanalytical.com / en / learn / knowledge- center / faqs / faqOO15averagediameter, the contents of which are incorporated herein by reference in their entirety.

[0083] As defined herein, the “polarity index” (P’) of a solvent is a well-known measure of how polar a solvent is. A higher polarity index figure indicates a more polar solvent. Polarity index is typically determined by measuring the ability of a solvent to interact with various test solutes. More typically, the polarity index (P’) of a solvent is as defined in Burdick and Jackson’s Solvent Guide (AlliedSignal, 1997; available online at https: / / macro.lsu. edu / howto / solvents / Polarity%20index.htm#:~:text=Polarity%20Index&te xt=Burdick%20%26%20Jackson%20solvents%20are%20arranged,with%20various%20po lar%20test%20solutes, the contents of which are incorporated herein by reference).

[0084] Burdick and Jackson rank solvents by reference to a numerical index that ranks solvents according to their different polarity. The Burdick and Jackson index is based on the structure of the solvents.

[0085] As defined herein, the term “DNA” refers to deoxyribonucleic acid and derivatives thereof, the molecule that carries most of the genetic instructions used in the development, functioning and reproduction of all known living organisms and many viruses. Most DNA molecules consist of two biopolymer strands coiled around each other to form a double helix. The two DNA strands are known as polynucleotides since they are composed of simpler units called nucleotides. Each nucleotide is composed of a nitrogen-containing nucleobase - cytosine (C), guanine (G), adenine (A), or thymine (T) - as well as a monosaccharide sugar called deoxyribose and a phosphate group. The nucleotides are joined to one another in a chain by covalent bonds between the sugar of one nucleotide and the phosphate of the next, resulting in an alternating sugar-phosphate backbone. According to base pairing rules (A with T, and C with G), hydrogen bonds bind the nitrogenous bases of the two separate polynucleotide strands to make double-stranded DNA.

[0086] As defined herein, the term “plasmid DNA” (pDNA) refers to a small, extrachromosomal DNA molecule that is physically separated from chromosomal DNA and can replicate independently. Typically, a plasmid is a small, circular, double-stranded helix of DNA. Typically, a plasmid encodes at least one (or more than one, e.g. two, three, four, five, six or more) gene. Plasmid DNA may be used in medical applications to induce translation and transcription of a gene within a target cell to which the plasmid is introduced. This can therefore result in an increase in the levels of a particular protein, for which the plasmid DNA codes, within the cell. For example, plasmids may be used for gene transfer as a potential treatment in gene therapy, so that the target cell may express a protein that is lacking in the cells. A plasmid may also be referred to as a “vector”. As defined herein, the term “RNA” refers to ribonucleic acid and derivatives thereof. RNA molecules are essential in various biological roles in coding, decoding, regulation and expression of genes. Unlike DNA, RNA is typically a single-stranded molecule folded onto itself, although in some cases RNA may be paired to form a double strand (e.g. in siRNA). Each RNA nucleotide is composed of a nitrogen-containing nucleobase - cytosine (C), guanine (G), adenine (A), or uracil (U) - as well as a monosaccharide sugar called deoxyribose and a phosphate group. In some cases, RNA nucleotides may contain a modified nucleobase, such as pseudouracil or N6-methyladenine. The nucleotides are joined to one another in a chain by covalent bonds between the sugar of one nucleotide and the phosphate of the next, resulting in an alternating sugar-phosphate backbone.

[0087] As defined herein, the term “mRNA” refers to messenger RNA, a family of RNA molecules that convey genetic information from DNA to the ribosome, where they specify the amino acid sequence of the protein products of gene expression. Following transcription of primary transcript mRNA (known as pre-mRNA) by RNA polymerase, processed, mature mRNA is translated into a polymer of amino acids: a protein. As in DNA, mRNA genetic information is in the sequence of nucleotides, which are arranged into codons consisting of three bases each. Each codon encodes for a specific amino acid, except the stop codons, which terminate protein synthesis. This process of translation of codons into amino acids requires two other types of RNA: transfer RNA (tRNA), that mediates recognition of the codon and provides the corresponding amino acid, and ribosomal RNA (rRNA), that is the central component of the ribosome's proteinmanufacturing machinery.

[0088] As defined herein, the term “small interfering RNA” (siRNA) refers to a class of doublestranded RNA molecules, which are typically 20-25 base pairs in length. siRNA plays many roles, but it is most notable in the RNA interference (RNAi) pathway, where it interferes with the expression of specific genes with complementary nucleotide sequences. siRNA functions by causing mRNA to be broken down after transcription, resulting in no translation. siRNA also acts in RNAi-related pathways, e.g. as an antiviral mechanism or in shaping the chromatin structure of a genome.

[0089] As defined herein, the term “small hairpin RNA” (shRNA) refers to an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors. shRNA is an advantageous mediator of RNAi in that it has a relatively low rate of degradation and turnover.

[0090] As defined herein, the term “micro RNA” (miRNA) refers to a small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals, and some viruses, which functions in RNA silencing and post-transcriptional regulation of gene expression.

[0091] As defined herein, the term “antisense oligonucleotide” (ASO) refers to a single strand of DNA or RNA that is complementary to a chosen sequence. Such oligonucleotides prevent translation of certain messenger RNA strands by hybridizing to them using standard Watson-Crick base pairing rules. Antisense oligonucleotides can be used to target a either a coding or non-coding RNA. This mechanism of action is therefore distinct from nucleic acids which act via RNA interference (e.g. siRNAs, miRNAs and shRNAs). Hybrids comprising ASOs can be degraded by the enzyme RNase H.

[0092] As defined herein, the term “PNA” refers to peptide nucleic acid, an artificially synthesized polymer similar to DNA or RNA invented by Peter E. Nielsen (Univ. Copenhagen), Michael Egholm (Univ. Copenhagen), Rolf H. Berg (Rise National Lab), and Ole Buchardt (Univ. Copenhagen) in 1991. PNA's backbone is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The various purine and pyrimidine bases are linked to the backbone by a methylene bridge (-CH2-) and a carbonyl group ( (C=O)-).

[0093] As defined herein, the term “antibody” includes whole antibodies and any antigen-binding fragment (ie., “antigen-binding portion”) or single chains thereof, as well as bispecific antibodies, and variants thereof. An antibody may also be referred to as an immunoglobulin (Ig). An antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. An antigen is any agent that causes the immune system of an animal body to produce an immune response, e.g. chemicals, bacteria, viruses or pollen. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0094] As defined herein, the term “antigen-binding portion” of an antibody refers to a fragment of an antibody that retains the ability to specifically bind to an antigen, such as a protein, polypeptide or peptide. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include a Fab fragment, a F(ab')2 fragment, a Fab’ fragment, an Fd fragment, an Fv fragment, a dAb fragment and an isolated complementarity determining region (CDR). Single chain antibodies such as scFv and heavy chain antibodies such as VHH and camel antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments may be obtained using conventional techniques known to those of skill in the art, and the fragments may be screened for utility in the same manner as intact antibodies.

[0095] As defined herein, antibody “fragments” may be made by truncation, e.g. by removal of one or more amino acids from its N and / or C-terminal ends. Up to 10, up to 20, up to 30, up to 40 or more amino acids may be removed from the N and / or C terminal in this way. Fragments may also be generated by one or more internal deletions. A fragment may comprise of at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 120, at least 150, at least 200, at least 250, at least 300 or at least 400 consecutive amino acids from an antibody or antibody variant sequence.

[0096] As defined herein, the term “amino acid” is a compound that comprises at least one amine functional group and at least one carboxylic acid functional group (i.e., a group of formula -CO2H). Usually the amino acid is an a-amino acid, although an amino acid can also, for example, be a P-amino acid or a y-amino acid or a 5-amino acid. The amino acid may be naturally occurring, synthetic, proteogenic, or nonproteogenic. As is well known, many amino acids have chiral centres. It is not important for the purposes of the present invention whether an amino acid is chiral or achiral, or whether it is present in a particular enantiomeric form. Examples of a-amino acids include the well-known 20 standard amino acids (specifically, Ala, Cys, Asp, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Vai, Trp and Tyr) as well as other proteinogenic amino acids (e.g. fMet, Sec, Pyl) and also non-proteinogenic amino acids. The amino acid is most often an L-amino acid but may also be a D-amino acid. An amino acid may comprise a chemical modification, including but not limited to a natural post-translation modification.

[0097] Triblock copolymers

[0098] In general terms, the present invention is directed to a triblock copolymer comprising a hydrophilic block, a linker moiety and a hydrophobic block, wherein:

[0099] - the hydrophilic block is covalently bonded to the hydrophobic block via the linker moiety;

[0100] - the hydrophobic block is a polyester or a polyamide; and the linker moiety consists of from 1 to 50 monomer units, wherein the bond between adjacent monomer units in the linker moiety and / or the bond between the linker moiety and the hydrophilic block is less susceptible to hydrolysis than the bond between adjacent monomer units in the hydrophobic block.

[0101] In general, the conditions under which the bond between adjacent monomer units in the linker moiety is “less susceptible to hydrolysis” than the bond between adjacent monomer units in the hydrophobic block are conditions of physiological temperature, pressure and pH, i.e. an aqueous solution at 37°C and atmospheric pressure at pH 7.5.

[0102] The present invention accordingly provides a triblock copolymer comprising a hydrophilic block, a linker moiety and a hydrophobic block, wherein the hydrophilic block is covalently bonded to the hydrophobic block via the linker moiety, and wherein:

[0103] (a) the hydrophobic block is a polyester, and the linker moiety consists of from 1 to 50 monomer units of:

[0104] (i) an ester monomer which comprises a longer linear chain of carbon atoms, from the carbonyl carbon to the hydroxy- substituted carbon within the monomer, than the ester monomer which makes up the polyester of the hydrophobic block;

[0105] (ii) an amide monomer; (iii) an ether monomer; or

[0106] (iv) a thioether monomer; or

[0107] (b) the hydrophobic block is a polyamide, and the linker moiety consists of from 1 to 50 monomer units of:

[0108] (i) an amide monomer which comprises a longer linear chain of carbon atoms, from the carbonyl carbon to the amino-substituted carbon within the monomer, than the amide monomer which makes up the polyamide of the hydrophobic block;

[0109] (ii) an ether monomer; or

[0110] (iii) a thioether monomer.

[0111] Typically, the bond between the linker moiety and the hydrophilic group is less susceptible to hydrolysis than either (a) a direct bond between the hydrophilic and hydrophobic groups or (b) a bond between two adjacent constituent monomer units of the hydrophobic group. Without wishing to be bound by any particular theory, it is thought that this contributes significantly to the improved stability to hydrolysis of the copolymer of the present invention when it is formulated into polymersomes.

[0112] Typically, when the hydrophobic block is a polyester, each ester monomer unit in the polyester comprises from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy-substituted carbon, and the linker moiety consists of from 1 to 50 monomer units of an ester monomer comprising more than 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy- substituted carbon.

[0113] Alternatively, however, when the hydrophobic block is a polyester, the linker moiety consists of from 1 to 50 monomer units of an amide monomer. Preferably in this embodiment, each ester monomer unit in the polyester comprises from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy- substituted carbon. Further preferably in this embodiment, the amide monomer in the linker moiety comprises from 2 to 4 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon. More preferably, each ester monomer unit in the polyester comprises from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy- substituted carbon and the amide monomer in the linker moiety comprises from 2 to 4 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon. Alternatively, when the hydrophobic block is a polyester, the linker moiety consists of from 1 to 50 monomer units of an ether monomer. Preferably in this embodiment, each ester monomer unit in the polyester comprises from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy- substituted carbon.

[0114] Alternatively, when the hydrophobic block is a polyester, the linker moiety consists of from 1 to 50 monomer units of a thioether monomer. Preferably in this embodiment, each ester monomer unit in the polyester comprises from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy- substituted carbon.

[0115] Preferably, when the hydrophobic block is a polyester, the linker moiety consists of from 1 to 50 monomer units of (i) an ester monomer which comprises a longer linear chain of carbon atoms, from the carbonyl carbon to the hydroxy-substituted carbon within the monomer, than the ester monomer which makes up the polyester of the hydrophobic block, or (ii) an amide monomer.

[0116] Typically, when the hydrophobic block is a polyamide, each amide monomer unit in the polyamide comprises 2 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon, and the linker moiety consists of from 1 to 50 monomer units of an amide monomer comprising more than 2 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon.

[0117] Alternatively, however, when the hydrophobic block is a polyamide, each amide monomer unit in the polyamide comprises 3 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon, and the linker moiety consists of from 1 to 50 monomer units of an amide monomer comprising more than 3 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon.

[0118] Alternatively, when the hydrophobic block is a polyamide, the linker moiety consists of from 1 to 50 monomer units of an ether monomer. Preferably in this embodiment, each amide monomer unit in the polyamide comprises 2 or 3 carbon atoms, and more preferably 2 carbon atoms, in a linear chain from the carbonyl carbon to the amino-substituted carbon.

[0119] Alternatively, when the hydrophobic block is a polyamide, the linker moiety consists of from 1 to 50 monomer units of a thioether monomer. Preferably in this embodiment, each amide monomer unit in the polyamide comprises 2 or 3 carbon atoms, and more preferably 2 carbon atoms, in a linear chain from the carbonyl carbon to the amino-substituted carbon.

[0120] Preferably, the linker moiety consists of from 2 to 40, more preferably from 3 to 30, and most preferably from 5 to 25, monomer units (i.e. monomer units of the ester monomer, the amide monomer, the ether monomer, or the thioether monomer).

[0121] Typically, the ratio of the number of monomer units in the linker moiety to the number of monomer units in the hydrophobic block is from 1 : 100 to 1 :2, preferably from 1 :60 to 1 :4, and more preferably from 1 :30 to 1 :6.

[0122] When the linker moiety consists of from 1 to 50 monomer units of an ester monomer comprising more than 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy-substituted carbon, the linker moiety preferably is of formula (A): wherein: m is an integer from 4 to 40; n is an integer from 1 to 50; each R1is independently selected from hydrogen, substituted or unsubstituted Ci- Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, substituted or unsubstituted Ci-Ce aminoalkyl, substituted or unsubstituted Ci-Ce haloalkyl, substituted or unsubstituted Ci-Ce haloalkoxy, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C4-C16 cycloalkylalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted Ce-Cio aryl, substituted or unsubstituted C7-C16 aralkyl, substituted or unsubstituted C4-C8 heterocycyl, substituted or unsubstituted C5-C14 heterocycloalkyl, substituted or unsubstituted C5-C10 heteroaryl, substituted or unsubstituted Ce-Ci6 heteroaralkyl, halo, hydroxy, amino, cyano or azido; each R2is independently selected from hydrogen, substituted or unsubstituted Ci- Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, substituted or unsubstituted Ci-Ce aminoalkyl, substituted or unsubstituted Ci-Ce haloalkyl, substituted or unsubstituted Ci-Ce haloalkoxy, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C4-C16 cycloalkylalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted Ce-Cio aryl, substituted or unsubstituted C7-C16 aralkyl, substituted or unsubstituted C4-C8 heterocyclyl, substituted or unsubstituted C5-C14 heterocycloalkyl, substituted or unsubstituted C5-C10 heteroaryl, substituted or unsubstituted Ce-Ci6 heteroaralkyl, halo, hydroxy, amino, cyano or azido;

[0123] * represents the point of attachment to the hydrophilic block; and A represents the point of attachment to the hydrophobic block.

[0124] Thus, in a preferred embodiment, the hydrophobic block is a polyester, each ester monomer unit in the polyester comprises from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy-substituted carbon, and the linker moiety is of formula (A).

[0125] In formula (A), the value of n is preferably from 2 to 40, more preferably from 3 to 30, and most preferably from 5 to 25.

[0126] In formula (A), the value of m is preferably from 4 to 30, more preferably from 4 to 20, and still more preferably from 5 to 10, and is most preferably 5.

[0127] In formula (A), preferably each R1is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl. Most preferably, each R1is hydrogen.

[0128] In formula (A), preferably each R2is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl. Most preferably, each R2is hydrogen.

[0129] Thus, in formula (A), preferably each R1is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl and each R2is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl. Most preferably, each R1is hydrogen and each R2is hydrogen.

[0130] Thus, in formula (A), preferably the value of m is from 4 to 30, each R1is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, and each R2is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl. More preferably, the value of m is from 5 to 10, each R1is hydrogen, and each R2is hydrogen. In a particular preferred embodiment, the value of m is 5, each R1is hydrogen and each R2is hydrogen, i.e. the monomer unit in the linker moiety is poly(caprolactone) (PCL). In an alternative embodiment, the value of m is 4, each R1is hydrogen and each R2is hydrogen, i.e. the monomer unit in the linker moiety is poly(valerolactone) (PVL).

[0131] Thus, in formula (A), preferably the value of m is from 4 to 30, the value of n is from 2 to 40, each R1is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, and each R2is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl. More preferably, the value of m is from 5 to 10, the value of n is from 3 to 30, each R1is hydrogen, and each R2is hydrogen. In a particular preferred embodiment, the value of m is 5, the value of n is from 5 to 25, each R1is hydrogen and each R2is hydrogen, i.e. the monomer unit in the linker moiety is poly(caprolactone) (PCL). In an alternative embodiment, the value of m is 4, the value of n is from 5 to 25, each R1is hydrogen and each R2is hydrogen, i.e. the monomer unit in the linker moiety is poly(valerolactone) (PVL).

[0132] In a particularly preferred embodiment, therefore, the hydrophobic block is a polyester, each ester monomer unit in the polyester comprises from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy-substituted carbon, the linker moiety is of formula (A), the value of m is from 4 to 30, the value of n is from 2 to 40, each R1is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, and each R2is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl.

[0133] Even more preferably, the hydrophobic block is a polyester, each ester monomer unit in the polyester comprises from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy- substituted carbon, the linker moiety is of formula (A), the value of m is from 5 to 10, the value of n is from 3 to 30, each R1is hydrogen, and each R2is hydrogen.

[0134] More preferably still, the hydrophobic block is a polyester, each ester monomer unit in the polyester comprises from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy-substituted carbon, the linker moiety is of formula (A), the value of m is 5, the value of n is from 5 to 25, each R1is hydrogen and each R2is hydrogen, i.e. the monomer unit in the linker moiety is poly(caprolactone) (PCL).

[0135] When the linker moiety consists of from 1 to 50 monomer units of an amide monomer, the linker moiety preferably is of formula (B): wherein: p is an integer from 1 to 40; q is an integer from 1 to 50; each R3is independently selected from hydrogen, substituted or unsubstituted Ci- Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, substituted or unsubstituted Ci-Ce aminoalkyl, substituted or unsubstituted Ci-Ce haloalkyl, substituted or unsubstituted Ci-Ce haloalkoxy, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C4-C16 cycloalkylalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted Ce-Cio aryl, substituted or unsubstituted C7-C16 aralkyl, substituted or unsubstituted C4-C8 heterocyclyl, substituted or unsubstituted C5-C14 heterocycloalkyl, substituted or unsubstituted C5-C10 heteroaryl, substituted or unsubstituted Ce-Ci6 heteroaralkyl, halo, hydroxy, amino, cyano or azido; each R4is independently selected from hydrogen, substituted or unsubstituted Ci- Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, substituted or unsubstituted Ci-Ce aminoalkyl, substituted or unsubstituted Ci-Ce haloalkyl, substituted or unsubstituted Ci-Ce haloalkoxy, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C4-C16 cycloalkylalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted Ce-Cio aryl, substituted or unsubstituted C7-C16 aralkyl, substituted or unsubstituted C4-C8 heterocyclyl, substituted or unsubstituted C5-C14 heterocycloalkyl, substituted or unsubstituted C5-C10 heteroaryl, substituted or unsubstituted Ce-Ci6 heteroaralkyl, halo, hydroxy, amino, cyano or azido; each R5is independently selected from hydrogen or unsubstituted Ci-Ce alkyl;

[0136] * represents the point of attachment to the hydrophilic block; and A represents the point of attachment to the hydrophobic block.

[0137] In a typical embodiment, the hydrophobic block is a polyamide, each amide monomer unit in the polyamide comprises 2 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon, and the linker moiety is of formula (B), further wherein p is from 2 to 40 (i.e. the amide monomer in the linker moiety comprises more than 2 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon).

[0138] Alternatively, the hydrophobic block is a polyamide, each amide monomer unit in the polyamide comprises 3 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon, and the linker moiety is of formula (B), further wherein p is from 3 to 40 (i.e. the amide monomer in the linker moiety comprises more than 3 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon).

[0139] Alternatively, the hydrophobic block is a polyester and the linker moiety is of formula (B). Preferably in this embodiment, each ester monomer unit in the polyester comprises from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy- substituted carbon,.

[0140] In formula (B), the value of q is preferably from 2 to 40, more preferably from 3 to 30, and most preferably from 5 to 25.

[0141] In formula (B), the value of p is preferably from 1 to 20, more preferably from 1 to 10, and still more preferably from 2 to 5.

[0142] In formula (B), preferably each R3is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl. Most preferably, each R1is hydrogen.

[0143] In formula (B), preferably each R4is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl. Most preferably, each R2is hydrogen.

[0144] In formula (B), preferably each R5is hydrogen. Thus, in formula (B), preferably each R3is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl and each R4is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl. Most preferably, each R3is hydrogen and each R4is hydrogen.

[0145] Thus, in formula (B), preferably each R3is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, each R4is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, and each R5is independently hydrogen. Most preferably, each R3is hydrogen, each R4is hydrogen and each R5is hydrogen.

[0146] Thus, in formula (B), preferably the value of p is from 1 to 20, each R3is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl and each R4is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl. Most preferably, the value of p is from 2 to 5, each R3is hydrogen and each R4is hydrogen.

[0147] Thus, in formula (B), preferably the value of p is from 1 to 20, each R3is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, each R4is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, and each R5is independently hydrogen. Most preferably, the value of p is from 2 to 5, each R3is hydrogen, each R4is hydrogen and each R5is hydrogen.

[0148] Thus, in formula (B), preferably the value of p is from 1 to 20, the value of q is from 2 to 40, each R3is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, each R4is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, and each R5is independently hydrogen. Most preferably, the value of p is from 2 to 5, the value of q is from 5 to 25, each R3is hydrogen, each R4is hydrogen and each R5is hydrogen.

[0149] In a particularly preferred embodiment, therefore, the hydrophobic block is a polyamide, each amide monomer unit in the polyamide comprises 2 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon, the linker moiety is of formula (B), the value of p is from 2 to 20, the value of q is from 2 to 40, each R3is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, each R4is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, and each R5is independently hydrogen.

[0150] Even more preferably, the hydrophobic block is a polyamide, each amide monomer unit in the polyamide comprises 2 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon, the linker moiety is of formula (B), the value of p is from 2 to 5, the value of q is from 5 to 25, each R3is hydrogen, each R4is hydrogen and each R5is hydrogen.

[0151] Alternatively, the hydrophobic block is a polyester, the linker moiety is of formula (B), the value of p is from 1 to 20, the value of q is from 2 to 40, each R3is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, each R4is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, and each R5is independently hydrogen.

[0152] Alternatively still, the hydrophobic block is a polyester, each ester monomer unit in the polyester comprises from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy-substituted carbon, the linker moiety is of formula (B), the value of p is from 1 to 5, the value of q is from 5 to 25, each R3is hydrogen, each R4is hydrogen and each R5is hydrogen.

[0153] When the linker moiety consists of from 1 to 50 monomer units of an ether or thioether monomer, the linker moiety preferably is of formula (C): wherein: t is an integer from 1 to 40 and u is an integer from 0 to 40, provided that (t+u) is an integer from 1 to 40; v is an integer from 1 to 50;

[0154] X is O or S; each R6is independently selected from hydrogen, substituted or unsubstituted Ci- Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, substituted or unsubstituted Ci-Ce aminoalkyl, substituted or unsubstituted Ci-Ce haloalkyl, substituted or unsubstituted Ci-Ce haloalkoxy, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C4-C16 cycloalkylalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted Ce-Cio aryl, substituted or unsubstituted C7-C16 aralkyl, substituted or unsubstituted C4-C8 heterocyclyl, substituted or unsubstituted C5-C14 heterocycloalkyl, substituted or unsubstituted C5-C10 heteroaryl, substituted or unsubstituted Ce-Ci6 heteroaralkyl, halo, hydroxy, amino, cyano or azido; each R7is independently selected from hydrogen, substituted or unsubstituted Ci- Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, substituted or unsubstituted Ci-Ce aminoalkyl, substituted or unsubstituted Ci-Ce haloalkyl, substituted or unsubstituted Ci-Ce haloalkoxy, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C4-C16 cycloalkylalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted Ce-Cio aryl, substituted or unsubstituted C7-C16 aralkyl, substituted or unsubstituted C4-C8 heterocyclyl, substituted or unsubstituted C5-C14 heterocycloalkyl, substituted or unsubstituted C5-C10 heteroaryl, substituted or unsubstituted Ce-Ci6 heteroaralkyl, halo, hydroxy, amino, cyano or azido; each R8is independently selected from hydrogen, substituted or unsubstituted Ci- Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, substituted or unsubstituted Ci-Ce aminoalkyl, substituted or unsubstituted Ci-Ce haloalkyl, substituted or unsubstituted Ci-Ce haloalkoxy, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C4-C16 cycloalkylalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted Ce-Cio aryl, substituted or unsubstituted C7-C16 aralkyl, substituted or unsubstituted C4-C8 heterocyclyl, substituted or unsubstituted C5-C14 heterocycloalkyl, substituted or unsubstituted C5-C10 heteroaryl, substituted or unsubstituted Ce-Ci6 heteroaralkyl, halo, hydroxy, amino, cyano or azido; each R9is independently selected from hydrogen, substituted or unsubstituted Ci- Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, substituted or unsubstituted Ci-Ce aminoalkyl, substituted or unsubstituted Ci-Ce haloalkyl, substituted or unsubstituted Ci-Ce haloalkoxy, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C4-C16 cycloalkylalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted Ce-Cio aryl, substituted or unsubstituted C7-C16 aralkyl, substituted or unsubstituted C4-C8 heterocyclyl, substituted or unsubstituted C5-C14 heterocycloalkyl, substituted or unsubstituted C5-C10 heteroaryl, substituted or unsubstituted Ce-Ci6 heteroaralkyl, halo, hydroxy, amino, cyano or azido;

[0155] * represents the point of attachment to the hydrophilic block; and A represents the point of attachment to the hydrophobic block.

[0156] In a typical embodiment, the hydrophobic block is a polyester and the linker moiety is of formula (C). Preferably in this embodiment, each ester monomer unit in the polyester comprises from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy-substituted carbon.

[0157] Alternatively, the hydrophobic block is a polyamide and the linker moiety is of formula (C). Preferably in this embodiment, each ester monomer unit in the polyester comprises 2 or 3 carbon atoms, more preferably 2 carbon atoms, in a linear chain from the carbonyl carbon to the hydroxy- substituted carbon.

[0158] In some embodiments, X is O. In other embodiments, X is S.

[0159] In formula (C), the value of v is preferably from 2 to 40, more preferably from 3 to 30, and most preferably from 5 to 25.

[0160] In formula (C), the value of (t+u) is preferably from 4 to 30, more preferably from 4 to 20, and still more preferably from 5 to 10. The value of t is preferably from 4 to 30, more preferably from 4 to 20, and still more preferably from 5 to 10. The value of u is preferably from 0 to 26, more preferably from 0 to 16, and still more preferably from 0 to 6.

[0161] In formula (C), preferably each R6is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl.

[0162] In formula (C), preferably each R7is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl. Most preferably, each R7is hydrogen.

[0163] In formula (C), preferably each R8is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl.

[0164] In formula (C), preferably each R9is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl. Most preferably, each R9is hydrogen. Thus, in formula (C), preferably each R6is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, each R7is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, each R8is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, and each R9is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl. Most preferably, each R7is hydrogen and each R9is hydrogen.

[0165] Thus, in formula (C), preferably the value of (t+u) is from 4 to 30, each R6is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, each R7is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, each R8is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, and each R9is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl. More preferably, the value of (t+u) is from 5 to 10, each R6is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, each R7is hydrogen, each R8is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, and each R9is hydrogen.

[0166] Thus, in formula (C), preferably the value of (t+u) is from 4 to 30, the value of v is from 2 to 40, each R6is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, each R7is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, each R8is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, and each R9is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl. More preferably, the value of (t+u) is from 5 to 10, the value of v is from 3 to 30, each R6is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, each R7is hydrogen, each R8is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, and each R9is hydrogen.

[0167] In a particularly preferred embodiment, therefore, the hydrophobic block is a polyester, the linker moiety is of formula (C), the value of (t+u) is from 4 to 30, the value of v is from 2 to 40, each R6is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, each R7is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, each R8is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, and each R9is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl.

[0168] Even more preferably, the hydrophobic block is a polyester, each ester monomer unit in the polyester comprises from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy- substituted carbon, the linker moiety is of formula (C), the value of (t+u) is from 5 to 10, the value of v is from 3 to 30, each R6is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, each R7is hydrogen, each R8is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, and each R9is hydrogen.

[0169] Alternatively, the hydrophobic block is a polyamide, the linker moiety is of formula (C), the value of (t+u) is from 4 to 30, the value of v is from 2 to 40, each R6is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, each R7is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, each R8is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, and each R9is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl.

[0170] Alternatively still, the hydrophobic block is a polyamide, each amide monomer unit in the polyamide comprises 2 or 3 carbon atoms, and preferably 2 carbon atoms, in a linear chain from the carbonyl carbon to the amino-substituted carbon, the linker moiety is of formula (C), the value of (t+u) is from 5 to 10, the value of v is from 3 to 30, each R6is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, each R7is hydrogen, each R8is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, and each R9is hydrogen.

[0171] The nature of the polyester or the polyamide which makes up the hydrophobic block of the triblock copolymer is not particularly limited in structure, other than in the embodiments described herein in which it must contain from 1 to 4 atoms in a linear chain from the carbonyl carbon to the hydroxy- substituted carbon (in the case of a polyamide) or 2 atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon (in the case of a polyamide). Thus, to take one example, poly(lactic acid) (PLA) is a polyester which has 2 carbons in a linear chain: the carbonyl carbon, and the alpha-carbon, which is in turn covalently bound to both a hydroxy group and a methyl group:

[0172] PLA

[0173] PLA contains a stereogenic centre; that is to say that it may exist in both L and D enantiomeric forms. It is not important for the purposes of the present invention whether PLA (or any other monomer unit within a hydrophobic block or hydrophilic block that may be chiral) is present in a particular enantiomeric form or as a racemate. Herein, therefore, any reference to “PLA” encompasses both enantiomeric forms in any proportion, i.e. the single L-enantiomeric form, the single D-enantiomeric form, a racemate, or a mixture of the two enantiomeric forms in unequal proportions. In the art, poly(L-lactic acid) is commonly abbreviated to PLLA, and poly(D,L-lactic acid) (i.e. racemic PLA) is commonly abbreviated to PDLLA. For the avoidance of doubt, any reference to “PLA” herein also embraces references to PLLA and PDLLA.

[0174] Typically, the hydrophobic block is a polyester. Representative polyesters include poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(3 -hydroxybutyrate) (P3HB), poly(3-hydroxyval erate) (PHV), and poly(4- hydroxybutyrate) (P4HB). Thus, in some embodiments, the triblock copolymer comprises a hydrophobic block which is poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(3 -hydroxybutyrate) (P3HB), poly(3- hy dr oxy valerate) (PHV), or poly(4-hydroxybutyrate) (P4HB). Preferably, the hydrophobic block is poly(lactic acid) (PLA).

[0175] Alternatively, the hydrophobic block is a polyamide. Representative polyamides include polypeptides (i.e. a polymer block consisting of amino acid residues) or polypeptoids (i.e. a polymer block consisting of TV- substituted glycine residues). Non-limiting examples of polypeptides are those consisting of a-amino acids (2 linear carbon atoms between the carbonyl and amino-bearing carbons), P-amino acids (3 linear carbon atoms between the carbonyl and amino-bearing carbons) or y-amino acids (4 linear carbon atoms between the carbonyl and amino-bearing carbons). Non-limiting examples of polypeptides are those consisting of amino acid residues selected from methionine, histidine, lysine, glutamic acid, phenylalanine and derivates thereof. Derivatives of these amino acid residues are not limited, but include, for instance, derivatives of lysine and glutamic acid that incorporate an imidazolyl substituent, and derivatives of glutamic acid in which the carboxylic acid side group is protected (e.g. esterified). In general, TV-substituted glycine residues that can be present in the polypeptoid may have the general formula -C(=0)-CH2-NR- wherein R is an organic side group (substituent). As with the amino acid residues discussed above, there is no particularly limitation on the chemical structure of the group R. Those skilled in the art would readily appreciate that the invention can routinely be carried out with a wide range of polypeptoids and could readily implement the principles of the invention to prepare amphiphilic copolymers having polypeptoid polymer blocks composed of any N- substituted glycine residues.

[0176] The size of the hydrophobic block is not particularly limited. However, in preferred embodiments, typically the weight-average molecular weight (Mw) of the hydrophobic block is from 5,000 to 20,000 Da, and preferably from 7,500 Da to 14,000 Da (e.g. from 10,000 Da to 12,000 Da, or about 11,000 Da). In preferred embodiments, the number of monomer units in the hydrophobic block is from 50 to 300, more preferably from 100 to 200 (e.g. from 110 to 180, or about 150).

[0177] The hydrophilic polymer block is not particularly limited and can, in general, be constituted from any hydrophilic polymerizable monomer such as those well-known and widely used in the art for producing amphiphilic polymers, e.g. for use in constructing micelles and / or polymersomes. As discussed above, the increased stability is thought to be largely due to the difference in the potential for bonds in the linker group to be hydrolysed compared to direct bonds between the hydrophilic and hydrophobic blocks, or bonds between adjacent monomer units within the hydrophobic block. This prevents exposure of free carboxylate groups on the surface of a polymersome comprising such triblock copolymers, which results in a more favourable zeta potential. The skilled person will therefore appreciate that the structure of the hydrophilic block, and in particular the type of bond between adjacent monomer units within the hydrophilic block, is unlikely to affect the electrostatic properties, and thus stability, of these polymersomes.

[0178] A wide variety of suitable such hydrophilic polymers blocks are described in documents such as US 2010 / 0003336 Al, WO 2017 / 144849, WO 2017 / 158382, WO 2017 / 199023, WO 2017 / 191444, WO 2019 / 197834, WO 2020 / 144467 and WO 2020 / 225538, the contents of each of which are herein incorporated by reference in their entirety. Nonlimiting, representative examples of such polymers include a polyether, poly(oligo(ethylene glycol methyl ether methacrylate) (POEGMA), poly(2- methacryloyloxyethyl phosphorylcholine) (PMPC), poly(vinyl pyrrolidine) (PVP), or polysarcosine. Thus, typically, the triblock copolymer comprises a hydrophilic block which is a polyether, poly(oligo(ethylene glycol methyl ether methacrylate) (POEGMA), poly(2 -methacryloyl oxy ethyl phosphorylcholine) (PMPC), poly(vinyl pyrrolidine) (PVP), or polysarcosine. Preferably, the hydrophilic block is a polyether. More preferably, the hydrophilic block is a polyether selected from poly(ethylene glycol), amine-terminated poly(ethylene glycol), polypropylene glycol), or amine-terminated polypropylene glycol). Even more preferably, the hydrophilic block is a polyether selected from polypthylene glycol) or amine-terminated polypthylene glycol), collectively referred to as PEG.

[0179] For the avoidance of doubt, the structure of the monomer repeat unit in PEG is as follows:

[0180] PEG

[0181] A PEG block may terminate in a hydroxy group (-OH), that is to say a PEG block which comprises PEG monomers as depicted above and which terminates at each end with the terminal unit -(CH2)a0H, wherein a is an integer from 1 to 6, and is preferably 2 or 3. Alternatively, a PEG block may terminate in an amino group (-NH2) i.e. is an amine- terminated polypropylene glycol), that is to say a PEG block which comprises PEG monomers as depicted above and which terminates at each end with the terminal unit - (CH2)bNH2, wherein b is an integer from 1 to 6, and is preferably 2 or 3. All embodiments referencing a PEG block throughout this specification relate equally to a PEG block which terminates in a hydroxy group in an amine-terminated PEG block which terminates in an amino group. Thus, in some embodiments, the PEG block terminates in a hydroxy group. In other embodiments, the PEG block terminates in an amino group.

[0182] In some embodiments, therefore, the hydrophilic block is a polyether (e.g. PEG) which is covalently bound to the linker moiety through a terminal -OH group. In other embodiments, the hydrophilic block is a polyether (e.g. PEG) which is covalently bound to the linker moiety through a terminal -NH2 group. The size of the hydrophilic block is not particularly limited. However, in preferred embodiments, typically the weight-average molecular weight (Mw) of the hydrophilic block is from 44 to 5,000 Da, and preferably from 1,500 Da to 2,500 Da (e.g. from 1,800 Da to 2,200 Da). In preferred embodiments, the number of monomer units in the hydrophobic block is from 2 to 100, more preferably from 30 to 60 (e.g. from 40 to 45, or about 45). In a preferred embodiment, the hydrophilic block is polyethylene glycol), amine- terminated poly(ethylene glycol), polypropylene glycol) or amine-terminated polypropylene glycol) and the hydrophobic block is poly(lactic acid) or poly(glycolic acid). More preferably, the hydrophilic block is poly(ethylene glycol) or amine-terminated polyethylene glycol) and the hydrophobic block is poly(lactic acid).

[0183] In such embodiments, typically, the hydrophilic block of the copolymer is PEG having a weight-average molecular weight (Mw) of from 1,500 to 2,500 Da. Preferably, the hydrophilic block is PEG having an Mwof from 1,600 to 2,400 Da. More preferably, the hydrophilic block is PEG having an Mwof from 1,700 to 2,300 Da. Still more preferably, the hydrophilic block is PEG having an Mwof from 1,800 to 2,200 Da, e.g. from 1,900 to 2,100 Da, or about 2,000 Da.

[0184] In such embodiments, typically, the hydrophilic block of the copolymer consists of from 35 to 56 repeat units of PEG (i.e. PEG35-PEG56). Preferably, the hydrophilic block of the copolymer consists of from 37 to 54 repeat units of PEG (i.e. PEG37-PEG54). More preferably, the hydrophilic block of the copolymer consists of from 39 to 52 repeat units of PEG (i.e. PEG39-PEG52). Still more preferably, the hydrophilic block of the copolymer consists of from 41 to 50 repeat units of PEG (i.e. PEG41-PEG50), e.g. from 43 to 48 repeat units of PEG (i.e. PEG43-PEG48) or about 45 repeat units of PEG (i.e. PEG45).

[0185] In such embodiments, typically, the hydrophobic block of the copolymer is PLA having a weight-average molecular weight (Mw) of from 7,500 to 14,000 Da. Preferably, the hydrophobic block is PLA having an Mwof from 8,000 to 14,000 Da. More preferably, the hydrophobic block is PLA having an Mwof from 9,000 to 13,000 Da. Even more preferably, the hydrophobic block is PLA having an Mwof from 10,000 to 12,000 Da. Still more preferably, the hydrophobic block is PLA having an Mwof about 11,000 Da, i.e. from 10,500 to 11,500 Da.

[0186] In such embodiments, typically, the hydrophobic block of the copolymer consists of from 110 to 194 repeat units of PLA (i.e. PLA110-PLA194). Preferably, the hydrophobic block of the copolymer consists of from 125 to 181 repeat units of PLA (i.e. PLA125-PLA181). More preferably, the hydrophobic block of the copolymer consists of from 139 to 166 repeat units of PLA (i.e. PLA139-PLA166). Still more preferably, the hydrophobic block of the copolymer consists of about 150 repeat units of PLA (i.e. PLA150). Thus, in such embodiments, typically the hydrophilic block is PEG having an Mwof from 1,500 to 2,500 Da and the hydrophobic block is PLA having an Mwof from 8,000 to 14,000 Da. Preferably, the hydrophilic block is PEG having an Mwof from 1,600 to 2,400 Da and the hydrophobic block is PLA having an Mwof from 9,000 to 13,000 Da. More preferably, the hydrophilic block is PEG having an Mwof from 1,700 to 2,300 Da and the hydrophobic block is PLA having an Mwof from 10,000 to 12,000 Da. Still more preferably, the hydrophilic block is PEG having an Mwof from 1,800 to 2,200 Da and the hydrophobic block is PLA having an Mwof about 11,000 Da.

[0187] Thus, in such embodiments, typically, the hydrophilic block consists of from 35 to 56 repeat units of PEG and the hydrophobic block consists of from 110 to 194 repeat units of PLA. Preferably, the hydrophilic block consists of from 37 to 54 repeat units of PEG and the hydrophobic block consists of from 125 to 181 repeat units of PLA. More preferably, the hydrophilic block consists of from 39 to 52 repeat units of PEG and the hydrophobic block consists of from 139 to 166 repeat units of PLA. Still more preferably, the hydrophilic block consists of from 41 to 50 repeat units of PEG and the hydrophobic block consists of about 150 repeat units of PLA.

[0188] The ratio of the Mwof the hydrophilic block to the Mwof the hydrophobic block in the copolymer is not particularly limited. However, typically this ratio is from 1 :9 to 1:3. Preferably, this ratio is from 1 :8 to 1:3. More preferably, this ratio is from 1 :7 to 1 :4. Still more preferably, this ratio is from 1 :6 to 1 :4.

[0189] The ratio of the Mwof the linker moiety to the Mwof the hydrophobic block in the copolymer is not particularly limited, other than by the number of monomer units that may be present in the linker moiety. However, typically this ratio is from 1 : 150 to 1:1. Preferably, this ratio is from 1 : 100 to 1 :2. More preferably, this ratio is from 1 :50 to 1:3. Still more preferably, this ratio is from 1:20 to 1:4.

[0190] In a particularly preferred embodiment, the triblock copolymer comprises a hydrophilic block, a linker moiety and a hydrophobic block, wherein:

[0191] (a) the hydrophilic block is PEG and is covalently bonded to the hydrophobic block via a linker moiety;

[0192] (b) the hydrophobic block is PLA; and

[0193] (c) the linker moiety consists of from 1 to 50 monomer units of PCL. In such embodiments, typically the hydrophilic block is PEG having an Mwof from 1,500 to 2,500 Da, the hydrophobic block is PLA having an Mwof from 8,000 to 14,000 Da, and the linker moiety is PCL having an Mwof from 114 to 5,700 Da. Preferably, the hydrophilic block is PEG having an Mwof from 1,600 to 2,400 Da, the hydrophobic block is PLA having an Mwof from 9,000 to 13,000 Da, and the linker moiety is PCL having an Mwof from 228 to 4,560 Da. More preferably, the hydrophilic block is PEG having an Mwof from 1,700 to 2,300 Da, the hydrophobic block is PLA having an Mwof from 10,000 to 12,000 Da, and the linker moiety is PCL having an Mwof from 342 to 3,420 Da. Still more preferably, the hydrophilic block is PEG having an Mwof from 1,800 to 2,200 Da, the hydrophobic block is PLA having an Mwof about 11,000 Da, and the linker moiety is PCL having an Mwof from 570 to 2,850 Da.

[0194] Thus, in such embodiments, typically, the hydrophilic block consists of from 35 to 56 repeat units of PEG, the hydrophobic block consists of from 110 to 194 repeat units of PLA, and the linker moiety consists of from 1 to 50 repeat units of PCL. Preferably, the hydrophilic block consists of from 37 to 54 repeat units of PEG, the hydrophobic block consists of from 125 to 181 repeat units of PLA and the linker moiety consists of from 2 to 40 repeat units of PCL. More preferably, the hydrophilic block consists of from 39 to 52 repeat units of PEG, the hydrophobic block consists of from 139 to 166 repeat units of PLA and the linker moiety consists of from 3 to 30 repeat units of PCL. Still more preferably, the hydrophilic block consists of from 41 to 50 repeat units of PEG, the hydrophobic block consists of about 150 repeat units of PLA and the linker moiety consists of from 5 to 25 repeat units of PCL.

[0195] Polymersomes

[0196] Polymersomes are synthetic vesicles formed from amphiphilic block copolymers, i.e. a block copolymer that comprises a hydrophilic block and a hydrophobic block. They are typically self-assembled structures. Examples of polymersomes are described in US 2010 / 0003336 Al, WO 2017 / 144849, WO 2017 / 158382, WO 2017 / 199023, WO 2017 / 191444, WO 2019 / 197834, WO 2020 / 144467, WO 2020 / 225538, WO 2023 / 094810 and WO 2024 / 147020, the contents of each of which are herein incorporated by reference in their entirety. Over the last twenty years they have attracted significant research attention as versatile carriers because of their colloidal stability, tuneable membrane properties and ability in encapsulating or integrating other molecules (for one representative review article, see Lee and Feijen; J Control Release, 2012, 161(2), 473-483, the contents of which are herein incorporated by reference in their entirety).

[0197] The present invention provides polymersomes comprising the triblock copolymer as defined herein. Typically, a polymersome of the present invention is formed from one type of copolymer, i.e. the triblock copolymer described above. Alternatively, polymersomes of the present invention may be formed from more than one type of copolymer, e.g. two, three, four, five or six different types of copolymer. In these embodiments, the polymersome comprises the triblock copolymer as defined herein and one or more further types of copolymer. There is no particular restriction on the types of other copolymer that may be present. The skilled person is well aware of suitable (amphiphilic) copolymers that are useful in polymersome preparation.

[0198] In one particular embodiment, a polymersome according to the present invention comprises:

[0199] (a) a triblock copolymer as described herein; and

[0200] (b) a diblock copolymer comprising a hydrophilic block that is directly covalently bonded to a hydrophobic block, wherein the hydrophobic block is (i) a polyester, preferably wherein each ester monomer unit in the polyester comprises from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy-substituted carbon, or (ii) a polyamide, preferably wherein each amide monomer unit in the polyamide comprises 2 or 3 carbon atoms, more preferably 2 carbon atoms, in a linear chain from the carbonyl carbon to the amino-substituted carbon.

[0201] In this embodiment, the ratio of copolymer (a) to diblock copolymer (b) in the polymersome is preferably from 1 : 1 to 100: 1, e.g. from 2: 1 to 50: 1, or from 5: 1 to 20: 1.

[0202] In this embodiment, the nature of the polyester or the polyamide which makes up the hydrophobic block of diblock copolymer (b) is not particularly limited in structure, other than in preferred embodiments in which it contains from 1 to 4 atoms in a linear chain from the carbonyl carbon to the hydroxy- substituted carbon (in the case of a polyamide) or 2 atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon (in the case of a polyamide). Typically, the hydrophobic block is a polyester. Representative polyesters include poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co- glycolic acid) (PLGA), poly (3 -hydroxybutyrate) (P3HB), poly(3-hydroxyval erate) (PHV), and poly(4-hydroxybutyrate) (P4HB). Alternatively, the hydrophobic block is a polyamide. Representative polyamides include polypeptides (i.e. a polymer block consisting of amino acid residues) or polypeptoids (i.e. a polymer block consisting of N- substituted glycine residues), in particular a-amino acids. The size of the hydrophobic block is not particularly limited. However, in preferred embodiments, typically the weight-average molecular weight (Mw) of the hydrophobic block is from 5,000 to 20,000 Da, and preferably from 7,500 Da to 14,000 Da (e.g. from 10,000 Da to 12,000 Da, or about 11,000 Da). In preferred embodiments, the number of monomer units in the hydrophobic block is from 50 to 300, more preferably from 100 to 200 (e.g. from 110 to 180, or about 150).

[0203] In this embodiment, the nature of the hydrophilic block of diblock copolymer (b) is not particularly limited in structure and can, in general, be constituted from any hydrophilic polymerizable monomer such as those well-known and widely used in the art for producing amphiphilic polymers, e.g. for use in constructing micelles and / or polymersomes. Non-limiting, representative examples of such polymers include a polyether, poly(oligo(ethylene glycol methyl ether methacrylate) (POEGMA), poly(2- methacryloyloxyethyl phosphorylcholine) (PMPC), poly(vinyl pyrrolidine) (PVP), or polysarcosine. Preferably, however, the hydrophilic block is a polyether. More preferably, the hydrophilic block is a polyether selected from poly(ethylene glycol), amine-terminated poly(ethylene glycol), polypropylene glycol), or amine-terminated polypropylene glycol). Even more preferably, the hydrophilic block is a polyether selected from poly(ethylene glycol) or amine-terminated poly(ethylene glycol), collectively referred to as PEG. The size of the hydrophilic block is not particularly limited. However, in preferred embodiments, typically the weight-average molecular weight (Mw) of the hydrophilic block is from 44 to 5,000 Da, and preferably from 1,500 Da to 2,500 Da (e.g. from 1,800 Da to 2,200 Da). In preferred embodiments, the number of monomer units in the hydrophobic block is from 2 to 100, more preferably from 30 to 60 (e.g. from 40 to 45, or about 45).

[0204] In this embodiment, preferably the hydrophobic block of copolymer (a) is the same as the hydrophobic block of diblock copolymer (b), and / or the hydrophilic block of copolymer (a) is the same as the hydrophilic block of diblock copolymer (b). Thus, typically, the hydrophobic block of copolymer (a) is the same as the hydrophobic block of diblock copolymer (b). Thus, typically, the hydrophilic block of copolymer (a) is the same as the hydrophilic block of diblock copolymer (b). Thus, typically, the hydrophobic block of copolymer (a) is the same as the hydrophobic block of diblock copolymer (b), and the hydrophilic block of copolymer (a) is the same as the hydrophilic block of diblock copolymer (b).

[0205] Polymersomes are typically nanoparticles or microparticles. The polymersomes of the present invention may be of any feasible geometry, e.g. substantially spherical, ellipsoidal, cylindrical or bilayer form, but typically they are substantially spherical. A typical (largest) diameter of a polymersome of the present invention is in the range 50 to 5000 nm. More typically, the z-average diameter, as measured using dynamic light scattering, is in the range 50 to 1000 nm. Typically, the polymersomes of the present invention have a z- average diameter of less than 300 nm, preferably less than 250 nm, most preferably less than 200 nm or 150 nm. Preferably, the polymersomes of the present invention have a z- average diameter measured by dynamic light scattering of from 50 to 150 nm, more preferably from 80 to 120 nm, e.g. approximately 100 nm. In one aspect, the polymersome of the present invention is a nanoparticle. Alternatively, the polymersome of the present invention is a microparticle. Typically, particle size is measured using dynamic light scattering (DLS). Alternatively, particle size is measure using transmission electron microscopy (TEM). Typically, particle size distribution is measured using DLS.

[0206] The polymersomes of the present invention typically have a polydispersity index of less than 0.2 as measured by dynamic light scattering. Preferably, the polymersomes of the present invention have a polydispersity index of less than 0.15 as measured by dynamic light scattering. More preferably, the polymersomes of the present invention have a polydispersity index of less than 0.12 as measured by dynamic light scattering. Still more preferably, the polymersomes of the present invention have a polydispersity index of less than 0.1 as measured by dynamic light scattering.

[0207] Typically, the polymersomes of the present invention have a polydispersity index of at least 0.01 as measured by dynamic light scattering. Preferably, the polymersomes of the present invention have a polydispersity index of at least 0.02 as measured by dynamic light scattering. More preferably, the polymersomes of the present invention have a polydispersity index of at least 0.04 as measured by dynamic light scattering. Still more preferably, the polymersomes of the present invention have a polydispersity index of at least 0.06 as measured by dynamic light scattering.

[0208] Typically, therefore, the polymersomes of the present invention have a polydispersity index of from at least 0.01 to less than 0.2 as measured by dynamic light scattering. Preferably, the polymersomes of the present invention have a polydispersity index of from at least 0.02 to less than 0.15 as measured by dynamic light scattering. More preferably, the polymersomes of the present invention have a polydispersity index of from at least 0.04 to less than 0.12 as measured by dynamic light scattering. Still more preferably, the polymersomes of the present invention have a polydispersity index of from at least 0.06 to less than 0.1 as measured by dynamic light scattering.

[0209] Typically, a polymersome of the present invention does not comprise any, or substantially any, lipid component. Thus, typically, a polymersome of the present invention does not comprise any, or substantially any, phospholipid, cholesterol, triglyceride or free fatty acid. In particular, a polymersome of the present invention does not comprise any, or substantially any, phospholipid. By “phospholipid” is meant an amphiphilic molecule with a hydrophilic “head” group comprising a phosphate moiety and two hydrophobic “tails” derived from fatty acids, joined by an alcohol residue (typically glycerol).

[0210] Thus, a polymersome of the present invention will typically comprise between from 0% to no more than 25% lipid, more preferably the polymersome will comprise from 0% to no more than 15% lipid, even more preferably, the polymersome will comprise from 0% to no more than 10% lipid, still more preferably no more than 5% lipid, yet more preferably no more than 2% lipid, and even more preferably still, will comprise between 0% and no more than 1% lipid, and most preferably no more than 0.2% lipid. Preferably, the polymersome does not comprise any lipid. Thus, a polymersome of the present invention will typically comprise no more than 25% phospholipid, more preferably no more than 15% phospholipid, even more preferably no more than 10% phospholipid, still more preferably no more than 5% phospholipid, yet more preferably no more than 2% phospholipid, even more preferably still no more than 1% phospholipid, and most preferably no more than 0.2% phospholipid. Preferably, the polymersome does not comprise any phospholipid. Polymersomes are often substantially spherical. Polymersomes typically comprise an amphiphilic membrane. The membrane is generally formed from two monolayers of amphiphilic molecules, which align and entangle to form an enclosed core with hydrophilic head groups facing the core and the exterior of the vesicle, and hydrophilic tail groups forming the interior of the membrane.

[0211] The thickness of the bilayer is generally between 2 and 100 nm, more typically between 2 and 50 nm (for instance between 5 and 20 nm). These dimensions can routinely be measured, for example by using Transmission Electron Microscopy (TEM) and / or and Small Angle X-ray Scattering (SAXS) (see, for example, Battaglia et al.,' J. Am. Chem. Soc., 2005, 127, 8757-8764, the contents of which are herein incorporated by reference in their entirety).

[0212] Polymersomes of the present invention may be formed from one type of copolymer, i.e. the triblock copolymer described above. Alternatively, polymersomes of the present invention may be formed from more than one type of copolymer, e.g. two, three, four, five or six different types of copolymer. When a polymersome is formed from more than one different type of copolymer, different regions of the polymersome typically have different bilayer thicknesses. For example, if a polymersome is formed from two different types of copolymer, preferably the thickness of the polymersome bilayer of a first region is from 1 to 10 nm, more preferably from 2 to 5 nm. Preferably the thickness of the polymersome bilayer of a second region is from 5 to 50 nm, for instance from 10 to 40 nm. More preferably the thickness of the polymersome bilayer of the second region is from 5 to 20 nm. Preferably the thickness of the polymersome bilayer of the first region is less than the thickness of the polymersome bilayer of the second region. Alternatively, the copolymers can have same thickness but different chemical compositions, which in turn create two different permeabilities with one copolymer forming a bilayer which is less permeable than the other.

[0213] It is known that when two different polymersome-forming copolymers are mixed to form a hybrid vesicle they phase-separate and thus give rise to polymersomes that contain discrete regions corresponding to the discrete copolymers. For example, this phenomenon is described in detail in LoPresti et al., ACS NANO, 2011, 5(3), 1775-1784, the contents of which is herein incorporated by reference in their entirety. Polymersomes can be readily manufactured by applying these known synthetic principles. In aqueous solution, normally an equilibrium exists between different types of structures, for instance between polymersomes and micelles. It is preferred that at least 80%, more preferably at least 90% or 95% by weight and most preferably all of the structures in solution are present as polymersomes. Thus, in other words, it is preferred that at least 80%, more preferably at least 90% or 95% by weight and most preferably all of the structures in solution are present in the form of vesicles. This can be achieved using the methods outlined herein.

[0214] Methods of preparation

[0215] Methods of preparing the PEG-PCL-PLA triblock copolymers of the present invention are not particularly limited. Exemplary methods that can be used for polymerising the monomers are atom-transfer radical polymerisation (ATRP) (see, e.g., an exemplary method described by Du el al. in Journal of the American Chemical Society, 2005, 127(51), 17982-17983), living radical polymerisation process, functional NCA (N- carb oxy anhydride) polymerisation with efficient postpolymerization modification and ring opening polymerisation (ROP). Living radical polymerisation has been found to provide polymers of monomers having a polydispersity (of molecular weight) of less than 1.5, as judged by gel permeation chromatography. Polydispersities in the range of from 1.0 to 1.4 for each block are preferred.

[0216] Ring-opening polymerization is particularly preferred when the linker moiety comprises an ester monomer unit and / or the hydrophobic block comprises an ester monomer unit. In such embodiments, the copolymers can be synthesized using a hydrophilic polymer (e.g. hydroxyl-terminated PEG) as the initiator, in combination with a lactone (e.g. caprolactone) to first produce a diblock copolymer of the hydrophilic block and the linker moiety. After isolation of this diblock copolymer a further ROP reaction can be carried out in the presence of a different lactone to produce the desired triblock copolymer. The ROP reaction can be catalyzed by an organic base (e.g. l,8-diazabicyclo(5.4.0)undec-7- ene; DBU) or an inorganic complex (e.g. tin(II) 2-ethylhexanoate. Typically the polymerization reactions are carried out under anhydrous conditions. The diblock copolymer intermediate and / or the triblock copolymer product may be filtered through an amino-funcitionalised column (e.g. a silica column) in order to remove impurities with free carboxylate groups which are the byproduct of hydrolysis of the diblock / triblock copolymers. Optionally, one or more ligands may be covalently attached to the terminus of the triblock copolymer. Preferably, any ligand is covalently attached to the terminus of the hydrophilic block of the triblock copolymer (i.e. via the end of the hydrophilic block that is not covalently bound to the linker moiety). Ligands are discussed further below. Ligands may be attached to triblock copolymers via any standard reaction technique(s) known in the art.

[0217] Optionally, the terminal exposed group of the hydrophilic block (i.e. the end of the hydrophilic block that is not covalently bound to the linker moiety) can be transformed into an alternative reactive functional group by any standard reaction technique(s) known in the art. Examples of alternative reactive functional groups include amino (-NH2), azido (-N3), thio (-SH) or halo (-F, -Cl, -Br or -I).

[0218] The method of preparing polymersomes using the triblock copolymer is not particularly limited. However, this method is typically a method of self-assembly. Such methods are known to a skilled person, and include the following:

[0219] - Film rehydration: also known as an emulsion evaporation or solvent evaporation method, this involves the evaporation of organic solvent under vacuum from a solution comprising the copolymer building blocks of polymersomes (and any material to be encapsulated in the polymersomes). As the solvent evaporates, a layer of polymer is formed on the wall of the flask. Subsequently, the thin film of polymersomes is hydrated in aqueous media (optionally comprising a soluble material to be encapsulated by the polymersomes), where it swells to produce polymersomes. This rehydration step may optionally occur under reduced pressure and / or at elevated temperature.

[0220] - Solvent switch: Copolymer building blocks (and any material to be encapsulated) are dissolved in a water-miscible organic solvent. Aqueous solvent is added to enable a solvent switch, and polymersomes form on the inside of the reaction vessel. Optionally the polymersomes are electroporated to allow encapsulation of water-soluble cargo.

[0221] - Solvent displacement: Copolymer building blocks (and any material to be encapsulated) are dissolved in an organic solvent. A different organic solvent is added to enable a solvent displacement, and polymersomes form on the inside of the reaction vessel. Optionally the polymersomes are electroporated to allow encapsulation of water-soluble cargo. - Solvent injection: Copolymer building blocks (and any material to be encapsulated) are dissolved in an organic solvent. This solution is then injected into aqueous medium at elevated temperature and / or reduced pressure in order to form polymersomes. Optionally the polymersomes are electroporated to allow encapsulation of water-soluble cargo.

[0222] In an embodiment, a method of forming polymersomes of the present invention involves the following steps: (i) dispersing the triblock copolymer in an aqueous medium; (ii) acidifying the composition formed in step (i); (iii) optionally, adding any cargo to be encapsulated to the acidified composition; and (iv) raising the pH to around neutral to encapsulate the cargo. This method preferably comprises a preliminary step wherein the amphiphilic copolymer is dispersed in an organic solvent in a reaction vessel and the solvent is then evaporated to form a film on the inside of the reaction vessel.

[0223] In another embodiment, a method of forming polymersomes of the present invention involves the following steps: (i) dispersing the triblock copolymer, and optionally any cargo to be encapsulated, in an organic solvent (e.g. a 2: 1 chlorofornrmethanol mixture) in a reaction vessel; (ii) evaporating the solvent to form a film on the inside of the reaction vessel; and (iii) re-hydrating the film with an aqueous solution, optionally comprising a solubilised cargo to be encapsulated.

[0224] In another embodiment, a method of forming polymersomes of the present invention involves the following steps: (i) dispersing the triblock copolymer, and optionally any cargo to be encapsulated, in an organic solvent in a reaction vessel; (ii) adding an aqueous solvent to enable solvent switch and the formation of polymersomes on the inside of the reaction vessel; and (iii) optionally electroporating the obtained polymersomes to allow encapsulation of water-soluble cargo.

[0225] In another embodiment, a method of forming polymersomes of the present invention involves the following steps: (i) dispersing the triblock copolymer, and optionally any cargo to be encapsulated, in an organic solvent in a reaction vessel; (ii) adding a different organic solvent to enable solvent displacement and the formation of polymersomes on the inside of the reaction vessel; and (iii) optionally electroporating the obtained polymersomes to allow encapsulation of water-soluble cargo. In another embodiment, a method of forming polymersomes of the present invention involves the following steps: (i) dispersing the triblock copolymer, and optionally any cargo to be encapsulated, in an organic solvent in a reaction vessel; (ii) adding this solution to an aqueous solvent (i.e. via solvent injection) which causes formation of polymersomes on the inside of the reaction vessel; and (iii) optionally electroporating the obtained polymersomes to allow encapsulation of water-soluble cargo.

[0226] Solvent injection methods are considered to be particularly preferable methods of forming polymersomes according to the present invention. Without wishing to be bound by any particular theory, it has been surprisingly found that use of a solvent injection method provides polymersomes having particularly low (i.e. desirable) polydispersity. Moreover, it is also considered that use of an organic solvent in such methods which has a high polarity index may also contribute to low polydispersity of polymersome products.

[0227] Thus, in a particularly preferred embodiment, a method of preparing the polymersomes of the present invention comprises:

[0228] (i) dissolving the triblock copolymer in an organic solvent having a polarity index of greater than 3.9; and

[0229] (ii) adding the resulting organic polymer solution to an aqueous solution.

[0230] Preferably, the organic solvent has a polarity index of from greater than 5.0 to 7.5, preferably from 5.5 to 7.0, and more preferably from 6.0 to 6.5. In some embodiments, the organic solvent is selected from acetone, methanol, ethanol, pyridine, 2-methoxyethanol, acetonitrile, propylene carbonate, N,N- dimethylformamide (DMF), dimethyl acetamide, Mmethylpyrrolidine or dimethyl sulfoxide (DMSO). Preferably, the organic solvent is DMF.

[0231] The concentration of copolymer in the organic solution is not particularly limited, but is typically from 5 to 40 mg / mL, more preferably 5 to 30 mg / mL.

[0232] Typically, the organic solution (i.e. organic solvent containing dispersed triblock copolymer) is added rapidly to the aqueous solution. Thus, typically, all the organic solvent is injected (e.g. using a syringe) into the aqueous solution as a single batch. The injection rate is therefore typically 10 to 250 pL / min, and more preferably from 20 to 150 pL / min. The organic solvent is not typically added to the aqueous solution dropwise, portionwise or with a low injection rate.

[0233] The aqueous solution may be water. Alternatively, the aqueous solution may be a solution comprising water and one or more organic or inorganic salts (i.e. a buffer solution). Typical inorganic salts include, but are not limited to, the halides, oxides, hydroxides, sulfates, carbonates, phosphates, nitrates, acetates and oxalates of the alkali metals (e.g. lithium, sodium and potassium), alkaline earth metals (e.g. magnesium and calcium), aluminium, zinc and ammonium. Typical organic salts include, but are not limited to, the acetates, citrates, lactates, oxamates, tartrates, stearates, benzoates and formates of the alkali metals (e.g. lithium, sodium and potassium), alkaline earth metals (e.g. magnesium and calcium), aluminium, zinc and ammonium, as well as zwitterionic salts including CAPS (3 -(Cyclohexylamino)- 1 -propanesulfonic acid), HEPES (4-(2 -hydroxyethyl)- 1- piperazineethanesulfonic acid), CHES (A-cyclohexyl-2-aminoethanesulfonic acid), MES (2-(A-morpholino)ethanesulfonic acid), MOPS (3-(A-morpholino)propanesulfonic acid), HEPBS (N-(2 -Hydroxy ethyl)piperazine-N'-(4-butanesulfonic acid)) and

[0234] PIPES (piperazine-A,A-bis(2-ethanesulfonic acid)). Preferably, the aqueous solution is water or phosphate-buffered saline (PBS).

[0235] The ratio of organic solution that is added to aqueous solution is not particularly limited, but typically the ratio of organic:aqueous solution is from 1 :6 to 4: 1 (v / v), and is more preferably about 4:6 (v / v).

[0236] Optionally, the triblock polymer is freeze dried prior to being dissolved in the organic solvent.

[0237] Typically, after the organic polymer solution has been added to the aqueous solution, the solvent is subsequently removed to leave behind the self-assembled polymer structures. The solvent may be removed by any method known in the art, e.g. by dialysis or under vacuum. Preferably, the solvent is removed by dialysis.

[0238] In embodiments wherein the polymersomes comprise an encapsulated cargo, the cargo may be dissolved or dispersed in the organic solvent alongside the copolymer. In such embodiments, the cargo is typically encapsulated in the polymersomes during the selfassembly process after addition of the organic solution to the aqueous solution. For example, from 0.01% to 10% (w / w) of material to be encapsulated is mixed with copolymer in the organic solvent.

[0239] Alternatively, in embodiments wherein the polymersomes comprise an encapsulated cargo, the cargo may be loaded into the polymersomes after the polymersomes have selfassembled. The polymersomes may be loaded, for example, using a pH change system, electroporation or film hydration.

[0240] In a pH change system process, polymer is dispersed in aqueous liquid in ionised form, in which it solubilises at relatively high concentrations without forming polymersomes. Subsequently the pH is changed such that some or all of the ionised groups become deprotonated so that they are in non-ionic form. At the second pH, the hydrophobicity of the block increases and polymersomes are formed spontaneously.

[0241] An alternative method for forming polymersomes with an encapsulated material may involve simple electroporation of the material and polymer vesicles in water. For instance, the cargo may be contacted in solid form with an aqueous dispersion of polymer vesicles and an electric field applied to allow the formation of pores on the polymersomes membrane. The solubilised material molecules may then enter the polymersome vesicles though the pores. This is followed by membrane self-healing process with the consecutive entrapment of the material molecules inside the polymersomes.

[0242] Alternatively, material dissolved in organic solvent may be emulsified into an aqueous dispersion of polymer vesicles, whereby solvent and the material become incorporated into the core of the vesicles, followed by evaporation of solvent from the system.

[0243] UV spectroscopy and HPLC chromatography may be used to calculate the encapsulation efficiency of a polymersome, using techniques well known in the art.

[0244] As discussed above, the polymersomes of the present invention may be formed from two or more different block copolymers. In such embodiments, in the method of forming polymersomes, a mixture of the two or more block copolymers is used.

[0245] Encapsulated cargoes

[0246] The polymersomes of the present invention may comprise an encapsulated cargo. Alternatively, the polymersomes of the present invention may not comprise any encapsulated cargo. For the avoidance of doubt, it is also possible to encapsulate a plurality of different cargoes within a single polymersome, or to provide a plurality of polymersome each containing a particular encapsulated cargo.

[0247] The encapsulated cargo may typically be a small molecule drug (preferably having a molecular weight of less than 5,000 Da), a nucleic acid or an antibody. As will be readily understood, the encapsulated cargo is selected in accordance with the disorder to be treated. Non-limiting examples of such disorders are described elsewhere in this disclosure.

[0248] Non-limiting examples of small molecule drugs include: a drug that is effective for the treatment or prevention of a brain disorder; a drug that is effective for the treatment or prevention of the immune and / or inflammatory disorder; and a drug that is effective for the treatment or prevention of a cancer. There is no particular limitation on the identity of the drug and so drugs can be selected from those known in the art for treatment or prevention of the disorder of interest in any given embodiment.

[0249] Non-limiting examples of drugs include neuroprotectants, immunomodulatory drugs (“immunomodulators”), non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, disease-modifying antirheumatic drugs (DMARDs,) immunosuppressants, TNF-alpha inhibitors and anti -cancer drugs.

[0250] Illustrative and non-limiting examples of specific drugs that may be encapsulated include fumarate and fumarate esters, glutamate antagonists (e.g., Estrogen, Ginsenoside Rd, Progesterone, Simvastatin, Memantine), antioxidants (e.g., Acetylcysteine, Crocin, Fish oil, Minocycline, Pyrroloquinoline quinone (PQQ), Resveratrol, Vinpocetine, Vitamin E), Stimulants (e.g., Selegiline, Nicotine, Caffeine), Caspase inhibitors, Trophic factors (e.g., CNTF, IGF-1, VEGF, and BDNF), Anti protein aggregation agents (e.g. sodium 4- phenylbutyrate, trehalose, and polyQ-binding peptide), Erythropoietin, Lithium, carnosine, asiatic acid, flavonoids (e.g. xanthohumol, naringenin, galangin, fisetin and baicalin), cannabinoids (e.g., WIN55, 212-2, JWH-133 and TAK-937), citicoline, minocycline, cerebrolysin, ginsenosoid-Rd, granulocyte-colony stimulating factor, Tat-NR2B9c, magnesium, albumin, paracetamol, aspirin, choline and magnesium salicylates, celecoxib, diclofenac (e.g. diclofenac potassium, diclofenac sodium), diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate, mefenamic acid, meloxicam, nabumetone, naproxen (including naproxen sodium), oxaprozin, piroxicam, rofecoxib, salsalate, sodium salicylate, sulindac, tolmetin, valdecoxib, corticosteroids, alemtuzumab, interferon beta-lb, fingolimod, glatiramer acetate, natalizumab, plegridy, peginterferon beta la, teriflunomide, methotrexate, sulfasalazine, leflunomide, adalimumab, etanercept, golimumab, ustekinumab, azathioprine, cyclosporine, infliximab, golimumab, certolizumab, hydroxychloroquine, methotrexate, azathioprine, my cophenol ate, acitretin, hydrea, isotretinoin, mycophenolate mofetil, sulfasalazine, 6- thioguanine, calcipotriol, calcitriol, tacalcitol, tacrolimus, pimecrolimus, dithranol, endamustine, bendamustine, carmustine, chlorambucil, cyclophosphamide, dacarbazine, ifosfamide, melphalan, procarbazine, streptozocin, temozolomide, capecitabine, 5-Fluoro Uracil, Fludarabine, Gemcitabin, Methotrexate, Pemetrexed, Raltitrexed, Actinomycin D, Bleomycin, Doxorubicin, Epirubicin, Mitomycin, Mitoxantrone, Etoposide, Docetaxel, Irinotecan, Paclitaxel, Topotecan, Vinblastine, Vincristine, Vinorelbine, Eribulin, Carboplatin, Cisplatin, Oxaliplatin, Afatinib, Aflibercept, BCG, Bevacizumab, Brentuximab, Cetuximab, Crizotinib, Denosumab, Erlotinib, Gefitinib, Imatinib, Interferon, Ipilimumab, Lapatinib, Panitumumab, Pertuzumab, Rituximab, Sunitinib, Sorafenib, Trastuzumab emtansine, Temsirolimus, Trastuzumab, Vemurafenib, Clodronate, Ibandronic acid, Pamidronate, Zolendronic acid, Anastrozole, Abiraterone, Bexarotene, Bicalutamide, Buserelin, Cyproterone, Degarelix, Exemestane, Flutamide, Folinic acid, Fulvestrant, Goserelin, Lanreotide, Lenalidomide, Letrozole, Leuprorelin, Medroxyprogesterone, Megestrol, Mesna, Octreotide, Stilboestrol, Tamoxifen and Thalidomide.

[0251] Non-limiting examples of nucleic acid cargos include a ribonucleic acid (an RNA), a deoxyribonucleic acid (a DNA), and a peptide nucleic acid (a PNA). Preferably, the nucleic acid is selected from an RNA and a DNA. RNAs may be single-stranded or double-stranded RNAs and include messenger RNA (mRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), transfer RNA (tRNA) and RNA-based antisense oligonucleotides (ASOs). DNAs may be single-stranded or doublestranded DNAs and include plasmid DNA (pDNA) and DNA-based antisense oligonucleotides (ASOs). Plasmid DNA cargo typically comprises from 2,000 to 400,000 base pairs, more preferably from 2,000 to 12,000 base pairs, and most preferably from 2,000 to 6,000 base pairs. Double-stranded DNA cargoes typically comprise from 100 to 5000 base pairs.

[0252] Typically, single-stranded messenger RNA cargo comprises from 300 to 3000 nucleotides, depending on the identity of the delivered gene. A single strand of other nucleic acid cargoes typically comprises from 3 to 300 nucleotides, preferably from 5 to 100 nucleotides, more preferably from 10 to 50 nucleotides, still more preferably from 15 to 30 nucleotides, and most preferably from 20 to 25 nucleotides.

[0253] Typically, when the encapsulated cargo comprises a nucleic acid, the encapsulated cargo also comprises a nucleic acid-binding protein. This nucleic acid-binding protein is selected as a suitable binding partner for the nucleic acid cargo. Proteins that are specialised to bind to each different type of nucleic acid cargo are well-known in the art and an appropriate binding protein for a specific cargo can be readily selected by the skilled person.

[0254] For instance, if the nucleic acid is a DNA, then the nucleic acid-binding protein is preferably a histone protein. Typically, in this embodiment, the nucleic acid-binding protein is a histone selected from an Hl, H2A, H2B, H3 or H4 histone. Hl histones are sometimes referred to as H5 histones. Histones H2A, H2B, H3 and H4 are sometimes known as core histones, and H1 / H5 histones are known as linker histones. Typically, therefore, if the nucleic acid is a DNA, the nucleic acid-binding protein may be a linker histone. Alternatively, the nucleic acid-binding protein may be a core histone. Preferably, the histone is selected from Hl and H3. Most preferably, the histone is Hl. In a particularly preferred embodiment, the nucleic acid cargo is a plasmid DNA (pDNA) and the nucleic acid-binding protein is a histone, preferably Hl or H3, and more preferably Hl. Alternatively, the nucleic acid cargo is a DNA-based antisense oligonucleotide and the nucleic acid-binding protein is a histone, preferably Hl or H3, and more preferably Hl. Alternatively, the nucleic acid cargo is a single-stranded DNA (ssDNA) and the nucleic acid-binding protein is a histone, preferably Hl or H3, and more preferably Hl.

[0255] If the nucleic acid is an RNA, then the nucleic acid-binding protein is preferably a eukaryotic translation initiation factor 4E (EIF4E) or a polyA binding protein. In one embodiment, the nucleic acid cargo is an mRNA, and the nucleic acid-binding protein is EIF4E or a polyA binding protein.

[0256] An antibody cargo may be a monoclonal antibody or a polyclonal antibody. Monoclonal antibodies (mAbs) are immunoglobulin molecules that are identical to each other and have a single binding specificity and affinity for a particular epitope. Monoclonal bispecific antibodies (BsmAbs) are mAbs that can bind simultaneously to two different types of antigen. Polyclonal antibodies are antibodies that are derived from different B cell lines. A polyclonal antibody may comprise a mixture of different immunoglobulin molecules that are directed against a specific antigen. A polyclonal antibody may comprise a mixture of different immunoglobulin molecules that bind to one or more different epitopes within an antigen molecule. Polyclonal antibodies may be produced by routine methods such as immunisation with the antigen of interest. For example a mouse or sheep capable of expressing antibodies may be immunised with an immunogenic conjugate. The animals may optionally be capable of expressing human antibody sequences. Blood may be subsequently removed and the Ig fraction purified to extract the polyclonal antibodies.

[0257] A polymersome is preferably capable of dissociating and releasing any encapsulated cargo once it has reached the tissue of interest (i.e. the target tissue). Non-limiting, exemplary tissues of interest are discussed in more detail later and include cancer cells, immune cells and cells (e.g. CNS cells) beyond the blood-brain barrier. Preferably the polymersome is capable of dissociating and releasing the encapsulated cargo after it has been internalised, via endocytosis, within a target cell (e.g. a cancer cell, an immune cell, or a CNS cell).

[0258] Dissociation may be promoted by a variety of mechanisms, such as pH sensitivity of the block copolymer, thermal sensitivity of the block copolymer, hydrolysis (i.e. water sensitivity of the block copolymer) and / or redox sensitivity of the block copolymer.

[0259] Targeting ligands

[0260] In certain embodiments, the polymersome of the present invention is for binding to the surface of a cell and comprises (a) a polymer brush and (b) at least a first ligand type on its external surface, wherein said first ligand type is capable of binding to a first receptor type on said cell surface. This aids the binding of the polymersomes to a target cell, thus improving binding to, and internalisation within, the target cell. A “ligand” may also be referred to herein as a “targeting moiety”. By “on its external surface” is meant that each ligand is located such that it is able to interact with its target (as opposed to being located at an inaccessible position that precludes interaction with the target, for example by being encapsulated within the nanoparticle or microparticle).

[0261] In further embodiments, the polymersome of the present invention may additionally comprise at least a second ligand type on its external surface, wherein said second ligand type is capable of binding to a second receptor type on said cell surface. Without wishing to be bound by any particular theory, it is believed that the multiplexing of ligands on the surface of a nanoparticle or microparticle in this fashion confers the property of “superselectivity” for the target cells. In other words, such “multiplexed” polymersomes provide the advantage that polymersome-cell binding via multiple different ligand types results in enhanced selectivity in delivery of the encapsulated cargo carried within the polymersome, reducing undesired off-target binding. A further advantage of this embodiment is that by using a multiplexed polymersome, the cell surface receptors on a target cell (e.g. a cancer cell or an immune cell) are less readily able to mutate in such a fashion that would prevent effective binding of the polymersome over time. The concept of “super-selectivity” of polymersomes is discussed in detail in WO 2020 / 144467, the contents of which are incorporated herein by reference in their entirety.

[0262] In some embodiments, therefore, the polymersome of the present invention is for binding to the surface of a cell, and comprises at least a first ligand type on its external surface and at least a second ligand type on its external surface, wherein said first ligand type is capable of binding to a first receptor type on said cell surface, and said second ligand type is capable of binding to a second receptor type on said cell surface. The polymersome of the present invention may, for example, comprise from two to seven different ligand types on its external surface (e.g. from two to six different ligand types, or from three to five different ligand types, or two ligand types), each of which is capable of binding to a complementary receptor type on the cell surface.

[0263] For super-selective interactions to be observed in the context of polymersomes, it is also advantageous that each ligand individually has a very low binding affinity for its target receptor. In practice, selective ligands with such a low binding energy to a target receptor are not readily available. This problem may, however, be overcome by also providing on the surface of the polymersomes a moiety which creates an interference steric potential with the surface of the target cell, such as a polymer brush. Typically a polymer brush may comprise a naturally occurring polymer, such as a polypeptide or polysaccharide, or a synthetic polymer, such as any of the amphiphilic block copolymers described above. Components on the external surface of the target cell, such as glycans, glycoproteins and glycolipids (collectively referred to as the “glycocalyx”), are also believed to contribute to this repulsive steric potential. Preferred polymeric components of the polymer brush include polyethylene glycol) (PEG), poly(vinyl pyrrolidone) (PVP), poly(2- methacryloyloxyethyl phosphorylcholine) (PMPC), poly(glycerol)s, poly(sulfobetaine), poly(carboxybetaine), poly(amino acid)s, polysarcosine, poly(2-oxazoline)s, poly(N-(2- hydroxypropyl)methacrylamide), polyglycols, heparin, dextran, poly(ethylene glycol)- poly(2-(diisopropylamino)ethyl methacrylate) and / or poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA).

[0264] Preferably in such embodiments, the polymer brush has a degree of polymerisation of at least 5, more preferably at least 10. Preferably, the degree of polymerisation of the polymer brush is no more than 500, e.g. no more than 300, or no more than 200. Preferably, the polymer brush has a length of from 1.5 to 350 nm, and more preferably from 3 to 210 nm.

[0265] The number of each type of ligand on the external surface of the polymersome may be optimised once certain physical parameters of a ligand-receptor system (which can readily be determined by a person of skill in the art) are known. Specifically, in certain embodiments, the optimum number of ligands of the ithligand type (A) on the external surface of the polymersome may be defined according to the following equation (1): wherein: a is the nanoparticle or microparticle activity and is calculated as a = [P]VPNA, wherein [P] is the molar concentration of nanoparticles or microparticles in bulk solution, NA is the Avogadro constant, and Vp is the geometrical volume; kB is the Boltzmann constant;

[0266] T is the absolute temperature;

[0267] EB(I) is the total energy of binding of the ithligand-receptor pair, given by the sum of (a) the ligand / receptor binding affinity kBTlnKo(i), wherein, Ko(i) is the dissociation constant for the ith receptor / ligand couple, and (b) the steric interference, Es, between the polymersome and the cell surface; and n is the density of receptors of type i on the cell surface.

[0268] In the case of a spherical (or substantially spherical) particle, the parameter a can be calculated as a = [P]NA( 3)[3(R+d)3-2R3)], wherein R is the radius of the nanoparticle or microparticle, and d is the ligand tether length.

[0269] The number (and density) of each type of ligand on the external surface of a polymersome can typically be controlled during synthesis of the polymersome by varying the ratio of ligand-bound copolymer and “pristine” copolymer (i.e. diblock copolymer that does not have a ligand attached). For any given system, the number of ligands per polymersome is then given by the copolymer self-assembly parameter (related to the polymer molecular weight and the packing factor) and the polymersome size. The number of each type of ligand on the external surface of a polymersome (and hence the density of receptors) can typically be verified using mass spectrometry.

[0270] Typically in such embodiments, the ligand is attached to a polymer component on the external surface of the polymersome, i.e. the hydrophilic block of the amphiphilic diblock copolymer. Thus, the ligand tether length d is given by the molecular mass of the hydrophilic block. Typically, d = 0.3N nm, where N is the polymerisation degree of the hydrophilic block.

[0271] The overall steric potential Esis the sum of the steric potential arising from the glycocalyx brush on the cell surface and the steric potential arising from the polymer brush that coats the polymersome. The magnitude of both depends on how accessible the ligands and receptor are. This in turn depends on: (i) the relative height of the receptor with respect to the glycan / glycoprotein / glycolipid etc. chains on the cell surface (Soho where ho is the glycan / glycoprotein / glycolipid length and 5G is between 0 and 1 and is a measure of how buried the receptor is in the glycocalyx), and (ii) the tether length of the ligands relative to the length of the polymer chains of the brush on the external surface of the nanoparticle (Sphp where hp is the polymer chain length and 5p is between 0 and 1 and is a measure of how buried the ligand is in the polymer brush). These parameters can readily be obtained for any given system from structural biology databases known in the art.

[0272] Typically in such embodiments, the polymersome comprises from 2 to 1000 ligands of the first ligand type. Preferably, the polymersome comprises from 5 to 1000 ligands of the first ligand type, more preferably from 10 to 500 ligands of the first ligand type, even more preferably from 20 to 200 ligands of the first ligand type, and most preferably from 50 to 100 ligands of the first ligand type.

[0273] Typically in such embodiments, the polymersome comprises from 2 to 1000 ligands of the second ligand type. Preferably, the polymersome comprises from 5 to 1000 ligands of the second ligand type, more preferably from 10 to 500 ligands of the second ligand type, even more preferably from 20 to 200 ligands of the second ligand type, and most preferably from 50 to 100 ligands of the second ligand type.

[0274] Typically in such embodiments, the polymersome comprises from 2 to 1000 ligands of a subsequent (i.e. third or greater) ligand type. Preferably, the polymersome comprises from 5 to 1000 ligands of the subsequent ligand type, more preferably from 10 to 500 ligands of the subsequent ligand type, even more preferably from 20 to 200 ligands of the subsequent ligand type, and most preferably from 50 to 100 ligands of the subsequent ligand type.

[0275] Typically in such embodiments, the combination of ligands on the surface of the nanoparticle or microparticle leads to a total binding energy of from 81<BT to 301<BT, where kB is Boltzmann’s constant and T is the temperature. This leads to on-off association profiles of the polymersomes wherein the receptors are saturated only above a given onset receptor density, whilst the polymersomes do not bind at all at lower receptor densities.

[0276] Typically in such embodiments, each ligand type is adapted to enable the polymersomes to bind to a target. Typically the ligand binds selectively to the target. The target is a chemical substance that is located on or in the vicinity of the tissue of interest (and thus enables the polymersome to accumulate specifically at the tissue of interest in preference to other sites). The target is preferably a receptor, e.g. a receptor that is present in particularly high quantity at the target tissue of interest. Most preferably, the target is a receptor on or within a cell surface membrane.

[0277] Typically in such embodiments, each ligand type can be any ligand that binds specifically to the target. As is well known in the art, for example from the well-developed field of bioconjugates, a wide range of substances can be used as ligands, e.g. to target receptors.

[0278] In one such embodiment, each ligand is a moiety that is attached to the external surface of the polymersomes. Examples of suitable ligands include antibodies, antibody fragments, aptamers, oligonucleotides, small molecules, peptides and carbohydrates. Peptide, protein, antibody and antibody fragment ligands are particularly preferred. However, any such moiety can be used as a ligand in the present invention. The suitability of any given moiety to target any given receptor can be determined using routine assay methods, involving testing for the ability of the moiety to bind specifically to the receptor.

[0279] One example of a ligand is a ligand that is adapted to enable the nanoparticle or microparticle to cross the blood-brain barrier (BBB). This property of the ligand arises through the ability of the ligand to bind to a target (e.g. a receptor) at the blood-brain barrier, wherein the target (e.g. receptor) mediates transcytosis across the blood-brain barrier. Examples of receptors for receptor-mediated transcytosis that are highly expressed on the endothelial cells that form the blood-brain barrier include low-density lipoprotein receptor-related protein 1 (LRP-1), scavenger receptor class B, member 1 (SCARB1), insulin receptor (IR) and transferrin receptor 1 (TFRC), all of which are suitable targets for the targeting moiety.

[0280] Thus, in one embodiment, at least one of the ligand types, and preferably one ligand type, targets the LRP-1 receptor. LRP-1 is a member of the LDL receptor family that plays diverse roles in various biological processes including lipoprotein metabolism, degradation of proteases, activation of lysosomal enzymes and cellular entry of bacterial toxins and viruses. Deletion of the LRP-1 gene leads to lethality in mice, revealing a critical, but as of yet, undefined role in development. Tissue-specific gene deletion studies reveal an important contribution of LRP-1 in the vasculature, central nervous system, in macrophages and in adipocytes. Three important properties of LRP-1 dictate its diverse role in physiology: first, its ability to recognise more than thirty distinct ligands; second, its ability to bind a large number of cytoplasmic adaptor proteins via determinants located on its cytoplasmic domain in a phosphorylation-specific manner; and third, its ability to associate with and modulate the activity of other transmembrane receptors such as integrins and receptor tyrosine kinases.

[0281] Polymersomes that feature a ligand that targets the LRP-1 receptor may cross the BBB and deliver efficiently the encapsulated cargo into both the CNS parenchyma and CNS cells. In particular, it has been found that the endothelial transcytosis mechanism does not involve acidification of the polymersome in membrane-trafficking organelles, which is important to avoid premature distintegration of the polymersome and concomitant release of the encapsulated cargo. Still further, the LRP-1 receptor is associated with traditional endocytosis in CNS cells, which, subsequent to navigation across the BBB, aids the delivery of the drug within their cytosol (via disintegration of the nanoparticle or microparticle).

[0282] Peptides that bind to the receptor LRP-1 are known in the art. For example, Angiochem (Montreal, Canada) have developed peptides that the leverage the LRP-1 mediated pathway to cross the blood-brain barrier when conjugated to drug cargos. One specific example of a peptide that is suitable for use in the present invention is Angiopep-2, which is a peptide having the sequence TFFYGGSRGKRNNFKTEEY. Further examples of suitable targeting moieties are disclosed in WO 2013 / 078562, the contents of which are herein incorporated by reference in their entirety (and, specifically, the ligand peptides disclosed in which are herein incorporated by reference).

[0283] In one embodiment, at least one of the ligand types, and preferably one ligand type, targets the SCARB1 receptor. The protein encoded by this gene is a plasma membrane receptor for high density lipoprotein cholesterol (HDL). The encoded protein mediates cholesterol transfer to and from HDL. In addition, this protein is a receptor for hepatitis C virus glycoprotein E2.

[0284] Malignant tumours display remarkable heterogeneity to the extent that even at the same tissue site different types of cells with varying genetic background may be found. In contrast, a relatively consistent marker the scavenger receptor type Bl (SR-B1) has been found to be consistently overexpressed by most tumour cells. Scavenger Receptor Class B Type I (SR-BI) is a high-density lipoprotein (HDL) receptor that facilitates the uptake of cholesterol esters from circulating lipoproteins. Additional findings suggest a critical role for SR-BI in cholesterol metabolism, signalling, motility, and proliferation of cancer cells and thus a potential major impact in carcinogenesis and metastasis. Recent findings indicate that the level of SR-BI expression correlate with aggressiveness and poor survival in breast and prostate cancer. Moreover, genomic data show that depending on the type of cancer, high or low SR-BI expression may promote poor survival. SR-BI is considered a diagnostic as well as prognostic indicator of cancer to help elucidate the contributions of this protein to cancer development, progression, and survival.

[0285] Ligands that bind to SCARB1 are known in the art. One such ligand is poly(2- (methacryloyloxy)ethyl phosphorylcholine) (PMPC).

[0286] In certain embodiments, one ligand type on the external surface of the polymersome targets LRP-1 and another ligand type on the external surface of the polymersome targets SCARB1.

[0287] In another embodiment, at least one of the ligand types, preferably one ligand type, is a ligand that is adapted to enable the polymersome to bind to a cancer cell. Cancer cells typically have a high density of membrane receptors. Illustrative and non-limiting examples of such targeting moieties include proteins (mainly antibodies and their fragments), peptides, nucleic acids (aptamers), small molecules, vitamins and carbohydrates.

[0288] Examples of receptors for receptor-mediated transcytosis that are highly expressed on tumour cells include LRP-1, SCARB1, TFRC, folate receptor 1 (F0LR1) and epidermal growth factor receptor (EGFR). For example, SCARB1 is highly expressed in HeLa cells (cervical cancer) and FaDu cells (squamous cell carcinoma of the hypopharynx).

[0289] In one embodiment, at least one of the ligand types, and preferably one ligand type, targets LRP-1. In another embodiment, at least one of the ligand types, and preferably one ligand type, targets SCARB1. In another embodiment, at least one of the ligand types, and preferably one ligand type, targets TFRC. In another embodiment, at least one of the ligand types, and preferably one ligand type, targets FOLR1. In another embodiment, at least one of the ligand types, and preferably one ligand type, targets EGFR.

[0290] In one embodiment, at least one of the ligand types, and preferably one ligand type, targets the TFRC receptor. This gene encodes a cell surface receptor necessary for cellular iron uptake by the process of receptor-mediated endocytosis. This receptor is required for erythropoiesis and neurologic development.

[0291] Iron as an important element plays crucial roles in various physiological and pathological processes. Iron metabolism behaves in systemic and cellular two levels that usually are in balance conditions. The disorders of the iron metabolism balances relate with many kinds of diseases including Alzheimer’s disease, osteoporosis and various cancers. In systemic iron metabolism that is regulated by hepcidin-ferroportin axis, plasma iron is bound with transferrin (TF) which has two high-affinity binding sites for ferric iron. The generic cellular iron metabolism consists of iron intake, utilization and efflux. During the iron intake process in generic cells, transferrin receptors (TFRs) act as the most important receptor mediated controls. TFR1 and TFR2 are two subtypes of TFRs those bind with iron-transferrin complex to facilitate iron into cells. TFR1 is ubiquitously expressed on the surfaces of generic cells, whereas TFR2 is specially expressed in liver cells. TFR1 has attracted more attention than TFR2 by having diverse functions in both invertebrates and vertebrates. Recently reports showed that TFR1 involved in many kinds of diseases including anemia, neurodegenerative diseases and cancers. Most importantly, TFR1 has been verified to be abnormally expressed in various cancers. Thus, TFR1 is postulated as a potential molecular target for diagnosis and treatment for cancer therapy.

[0292] In one embodiment, at least one of the ligand types, and preferably one ligand type, targets folate receptor 1 (F0LR1). The protein encoded by this gene is a member of the folate receptor family. Members of this gene family bind folic acid and its reduced derivatives, and transport 5-methyltetrahydrofolate into cells. This gene product is a secreted protein that either anchors to membranes via a glycosyl-phosphatidylinositol linkage or exists in a soluble form. Mutations in this gene have been associated with neurodegeneration due to cerebral folate transport deficiency.

[0293] The folate cycle sustains key metabolic reactions and is essential for rapidly growing cells. Under physiologic conditions, exogenous reduced folates (water-soluble B vitamins) are predominantly transported into cells via the low-affinity, high-capacity, ubiquitously expressed reduced folate carrier (RFC; bidirectional anion-exchange mechanism). Once in the cell, folates play an essential role in the biosynthesis of purines and thymidine, which in turn are required for DNA synthesis, methylation, and repair. Folates are also transported by high-affinity FRs. In humans, there are four isoforms of the FR (FRa, FRP, FRy, and FR5). FRa, FRp, and FR5 are attached to the cell surface by a glycosylphosphatidylinositol anchor, while FRy is a secreted protein. Because FRa is expressed on the cell surface in a tumour-specific manner, it provides the potential to allow not only tumour localization, but also selected delivery of therapeutic agents to the malignant tissue, minimizing collateral toxic side-effects.

[0294] There are a number of unique advantages to exploiting FR as a diagnostic and therapeutic target. First, FRa is located on the luminal surface of epithelial cells in most proliferating nontumor tissues and is inaccessible to circulation. In contrast, FRa is expressed all over the cell in malignant tissue and is accessible via circulation. Second, FR has the ability to bind to folic acid, a relatively innocuous, small molecule that can rapidly penetrate solid tumours and is amenable to chemical conjugation with other molecules. Once a folate conjugate is bound to FR, it is internalized into the cell and the FRa is rapidly recycled to the cell surface via the FR-mediated endocytic pathway. These factors all emphasize the potential role of FRa in the diagnosis and treatment of specific tumour types.

[0295] In one embodiment, at least one of the ligand types, and preferably one ligand type, targets epidermal growth factor receptor (EGFR). The protein encoded by this gene is a transmembrane glycoprotein that is a member of the protein kinase superfamily. This protein is a receptor for members of the epidermal growth factor family. EGFR is a cell surface protein that binds to epidermal growth factor. Binding of the protein to a ligand induces receptor dimerization and tyrosine autophosphorylation and leads to cell proliferation.

[0296] Epidermal growth factor receptors (EGFRs) are a large family of receptor tyrosine kinases (TK) expressed in several types of cancer, including breast, lung, esophageal, and head and neck. EGFR and its family members are the major contributors of a complex signaling cascade that modulates growth, signaling, differentiation, adhesion, migration and survival of cancer cells. EGFR binds to its cognate ligand EGF, which further induces tyrosine phosphorylation and receptor dimerization with other family members leading to enhanced uncontrolled proliferation. Due to their multi-dimensional role in the progression of cancer, EGFR and its family members have emerged as attractive candidates for anticancer therapy. Specifically, the aberrant activity of EGFR has shown to play a key role in the development and growth of tumor cells, where it is involved in numerous cellular responses including proliferation and apoptosis. The epidermal growth factor receptor (EGFR) signalling pathway is also a strong contender for both initiating and determining clinical outcomes in many respiratory diseases. Deregulation of the EGFR pathway causing aberrant EGFR signalling is associated with the early stage pathogenesis of lung fibrosis, cancer and numerous airway hypersecretory diseases, including COPD, asthma and cystic fibrosis.

[0297] Ligands for binding to each of these receptor are well known in the art. Example ligands for LRP-1 and SCARB1 are discussed above. Example ligands for TFRCs, e.g. TFR1, are transferrin and transferrin mimic peptide. An example ligand for F0LR1 is folic acid. An example ligand for EGFR is the peptide YHWYGYTPQNVI peptide.

[0298] A further example of a ligand is a ligand that is adapted to enable the polymersome to bind to an immune cell. Illustrative and non-limiting examples of such ligands include phosphoryl choline (as discussed in more detail below), peptidoglycan, lipoproteins, glycolipids, lipopolysaccharide, lipopeptides, synthetic compounds such as loxoribine and bropirimine, peptidoglycans, acetylated / malelylated proteins, modified low-density lipoproteins, polyanionic ligands, sulfated sugars, mannose- modified polysaccharides, fucose- modified polysaccharides, galactose-modified polysaccharides, proteins and P- glucan. In immune system cells targeting, the specific and precise targeting requires a particularly high level of discrimination / precision, which can be afforded by the polymersomes of the present invention.

[0299] Targeting of immune cells is believed to be important in treating immune-related diseases, such as autoimmune diseases and graft rejection, and for improving preventive / therapeutic vaccines. The cell membrane provides a remarkable example of spatiotemporal control of complex biological interactions thanks to hundreds of different ligands-receptors interactions selected trough evolution with the right amount of affinity and multi- combinatorial binding. Targeting of immune cells may also be important in embodiments where the nucleic acid cargo is an mRNA, and the polymersome forms part of an mRNA vaccine which is devised to induce production of an antigen in vivo by the subject to whom the polymersome is administered.

[0300] Therapies that target components of the defence system such as neutrophils and neutrophil-associated effectors are promising for adjunct host-directed therapies to improve antibiotic efficacy, i.e. in tuberculosis treatment, and reduce both treatment time and long-term pathological sequelae. Neutrophils have however proven very difficult cells to manipulate, and to the knowledge of the inventors, no commercially available vector exists that enables the efficient intracellular delivery of cargo within their short life span without compromising their viability and activation state. Expression of high levels of immune cells including neutrophils has been associated with detrimental outcome in several solid tumours and new strategies to decrease their presence and activity are currently under clinical development. Accordingly, neutrophils are desirable targets for the polymersomes of the present invention.

[0301] A ligand can be attached to the external surface of the polymersome using routine techniques, for example by adapting well known methods for attaching ligands to polymers, drugs, nucleic acids, antibodies and other substances. The attachment may be non-covalent (e.g. electrostatic) or covalent, though it is preferably covalent. For example, the targeting moiety can be attached by reacting a suitable functional group on the targeting moiety (including but not limited to a carboxyl group) with a corresponding functional group on at least one of the copolymers that form, or will form, the polymersome. The attachment can be effected either before the polymersome structure is formed from the copolymers, or after the polymersomes have been formed. In particular, the targeting moiety can be attached via a covalent bond to the terminal hydroxy (-OH) group on the PEG block of the PEG-PLA copolymers or the terminal amino (-NH2) group on an amine-terminated PEG block of the PEG-PLA copolymers. Typical bonds between a targeting moiety and the PEG-PLA copolymers include, but are not limited to, ester and amide bonds.

[0302] It is also possible to provide for attachment of the ligand to the copolymers by first chemically activating either or both of the ligand and the copolymers. For example, a peptide ligand may be activated by adding a reactive species to one of its termini, such as a cysteine moiety (whose thiol group is well known to react readily with functional groups such as the widely used maleimide moiety). Similarly, a copolymer (e.g. a PEG-PLA copolymer) can be activated by functionalising it with a reactive species (e.g. a maleimide moiety when the targeting moiety carries a thiol group). The copolymer may be provided with such a reactive species either by functionalisation of the copolymer itself, or by providing suitable monomers prior to the polymerisation that forms the copolymer, or by providing a suitable initiator for the polymerisation. A ligand may be attached directly to the external surface of the polymersome, or alternatively it may be attached via a chemical spacer. Suitable chemical spacers, or linkers, may comprise e.g. a triazole, thiosuccinamide, ester, amide, isourea, urethane, dihydropyrazine or disulfide moiety.

[0303] Pharmaceutical compositions

[0304] The polymersomes of the present invention can be formulated as a pharmaceutical composition using routine techniques known in the art.

[0305] The pharmaceutical composition comprises a plurality of the polymersomes of the present invention. It also comprises one or more pharmaceutically acceptable excipients or diluents. The one or more pharmaceutically acceptable excipients or diluents may be any suitable excipients or diluents. The pharmaceutical composition is typically aqueous, i.e. it contains water (in particular sterile water).

[0306] A typical pH of the aqueous pharmaceutical composition is 7.0 to 7.6, preferably 7.2 to 7.4. Pharmaceutically acceptable buffers may be used to achieve the required pH. The pharmaceutical composition may be in the form of a sterile, aqueous, isotonic saline solutions.

[0307] Typically the pharmaceutical composition is an injectable composition, e.g. it is suitable for intravenous delivery, for example it is suitable for infusion.

[0308] Medical uses of the polymersomes

[0309] The polymersomes and pharmaceutical formulations of the present invention are able to target tissues including, but not limited to cells (e.g. CNS cells) beyond the blood-brain barrier, immune cells and cancer cells and to release the nucleic acid / nucleic acid-binding protein (and any further encapsulated drugs) once localised at the target. As discussed above, the polymersomes may comprise ligands on their external surface (e.g. as part of the polymers themselves or as distinct moieties attached thereto) which results in a high targeting efficiency for the target cell type.

[0310] As will be readily understood, any encapsulated cargo, and any ligand present on the external surface of the polymersome, are selected in accordance with the disease to be treated. The polymersomes of the present invention may be useful in the treatment of cancer. Examples of cancers include: cancers of the skin, such as melanoma; lymph node; breast; cervix; uterus; gastrointestinal tract; lung; ovary; prostate; colon; rectum; mouth; brain; head and neck; throat; testes; thyroid; kidney; pancreas; bone; spleen; liver; bladder; larynx; nasal passages; AIDS-related cancers; cancers of the blood and bone marrow, such as multiple myeloma and acute and chronic leukemias, for example, lymphoblastic, myelogenous, lymphocytic, and myelocytic leukemias; advanced malignancy, amyloidosis, neuroblastoma, meningioma, hemangiopericytoma, multiple brain metastase, glioblastoma multiforms, glioblastoma, brain stem glioma, poor prognosis malignant brain tumor, malignant glioma, recurrent malignant glioma, anaplastic astrocytoma, anaplastic oligodendroglioma, neuroendocrine tumor, rectal adenocarcinoma, Dukes C & D colorectal cancer, unresectable colorectal carcinoma, metastatic hepatocellular carcinoma, Kaposi's sarcoma, karotype acute myeloblastic leukemia, chronic lymphocytic leukemia (CLL), Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous T-Cell lymphoma, cutaneous B-Cell lymphoma, diffuse large B-Cell lymphoma, low grade follicular lymphoma, metastatic melanoma (localized melanoma, including, but not limited to, ocular melanoma), malignant mesothelioma, malignant pleural effusion mesothelioma syndrome, peritoneal carcinoma, papillary serous carcinoma, gynecologic sarcoma, soft tissue sarcoma, scleroderma, cutaneous vasculitis, Langerhans cell histiocytosis, eiomyosarcoma, fibrodysplasia ossificans progressive, hormone refractory prostate cancer, resected high-risk soft tissue sarcoma, unrescectable hepatocellular carcinoma, Waldenstrom's macroglobulinemia, smoldering myeloma, indolent myeloma, fallopian tube cancer, androgen independent prostate cancer, androgen dependent stage IV non- metastatic prostate cancer, hormone-insensitive prostate cancer, chemotherapy-insensitive prostate cancer, papillary thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma, and leiomyoma.

[0311] The polymersomes of the present invention may also be useful in the treatment of infectious disease. Examples of infectious diseases include: Acinetobacter infections, Actinomycosis, Adenovirus infection, African sleeping sickness (African trypanosomiasis), AIDS (acquired immunodeficiency syndrome), Amoebiasis, Anaplasmosis, Angiostrongyliasis, Anisakiasis, Anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, Ascariasis, Aspergillosis, Astrovirus infection, Babesiosis, Bacillus cereus infection, Bacterial meningitis, Bacterial pneumonia, Bacterial vaginosis, Bacteroides infection, Balantidiasis, Bartonellosis, Baylisascaris infection, BK virus infection, Black piedra, Blastocystosis, Blastomycosis, Bolivian hemorrhagic fever, Botulism (and Infant botulism), Brazilian hemorrhagic fever, Brucellosis, Bubonic plague, Burkholderia infection, Buruli ulcer, Calicivirus infection (Norovirus and Sapovirus), Campylobacteriosis, Candidiasis (Moniliasis; Thrush), Capillariasis, Dental caries, Carrion's disease, Cat-scratch disease, Cellulitis, Chagas disease (American trypanosomiasis), Chancroid, Chickenpox, Chikungunya, Chlamydia, Chlamydophila pneumoniae infection (Taiwan acute respiratory agent or TWAR), Cholera, Chromoblastomycosis, Chytridiomycosis, Clonorchiasis, Clostridium difficile colitis, Coccidioidomycosis, Colorado tick fever (CTF), Common cold (Acute viral rhinopharyngitis; Acute coryza), Coronavirus disease 2019 (COVID-19), Coxsackie B virus infection, Creutzfeldt-Jakob disease (CJD), Crimean-Congo hemorrhagic fever (CCHF), Cryptococcosis, Cryptosporidiosis, Cutaneous larva migrans (CLM), Cyclosporiasis, Cysticercosis, Cytomegalovirus infection, Dengue fever, Desmodesmus infection, Dientamoebiasis, Diphtheria, Diphyllobothriasis, Dracunculiasis, Eastern equine encephalitis (EEE), Ebola hemorrhagic fever, Echinococcosis, Ehrlichiosis, Enterobiasis (Pinworm infection), Enterococcus infection, Enterovirus infection, Epidemic typhus, Erythema infectiosum (Fifth disease), Exanthem subitum (Sixth disease), Fasciolasis, Fasciolopsiasis, Fatal familial insomnia (FFI), Filariasis, Food poisoning by Clostridium perfringens, Free-living amebic infection, Fusobacterium infection, Gas gangrene (Clostridial myonecrosis), Geotrichosis, Gerstmann-Straussler-Scheinker syndrome (GSS), Giardiasis, Glanders, Gnathostomiasis, Gonorrhea, Granuloma inguinale (Donovanosis), Group A streptococcal infection, Group B streptococcal infection, Haemophilus influenzae infection, Hand, foot and mouth disease (HFMD), Hantavirus Pulmonary Syndrome (HPS), Heartland virus disease, Helicobacter pylori infection, Hemolytic-uremic syndrome (HUS), Hemorrhagic fever with renal syndrome (HFRS), Hendra virus infection, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Herpes simplex, Histoplasmosis, Hookworm infection, Human bocavirus infection, Human ewingii ehrlichiosis, Human granulocytic anaplasmosis (HGA), Human metapneumovirus infection, Human monocytic ehrlichiosis, Human papillomavirus (HPV) infection, Human parainfluenza virus infection, Human T-lymphotropic virus 1 infection, Hymenolepiasis, Epstein-Barr virus infectious mononucleosis (Mono), Influenza (flu), Isosporiasis, Japanese encephalitis, Kawasaki disease, Keratitis, Kingella kingae infection, Kuru, Lassa fever, Legionellosis (Legionnaires' disease), Pontiac fever, Leishmaniasis, Leprosy, Leptospirosis, Listeriosis, Lyme disease (Lyme borreliosis), Lymphatic filariasis (Elephantiasis), Lymphocytic choriomeningitis, Malaria, Marburg hemorrhagic fever (MHF), Measles, Middle East respiratory syndrome (MERS), Melioidosis (Whitmore's disease), Meningitis, Meningococcal disease, Metagonimiasis, Microsporidiosis, Molluscum contagiosum (MC), Monkeypox, Mumps, Murine typhus (Endemic typhus), Mycoplasma pneumonia, Mycoplasma genitalium infection, Mycetoma, Myiasis, Neonatal conjunctivitis (Ophthalmia neonatorum), Nipah virus infection, Norovirus, (New) Variant Creutzfeldt- Jakob disease (vCJD, nvCJD), Nocardiosis, Onchocerciasis (River blindness), Opisthorchiasis, Paracoccidioidomycosis (South American blastomycosis), Paragonimiasis, Pasteurellosis, Pediculosis capitis (Head lice), Pediculosis corporis (Body lice), Pediculosis pubis (pubic lice, crab lice), Pelvic inflammatory disease (PID), Pertussis (whooping cough), Plague, Pneumococcal infection, Pneumocystis pneumonia (PCP), Pneumonia, Poliomyelitis, Prevotella infection, Primary amoebic meningoencephalitis (PAM), Progressive multifocal leukoencephalopathy, Psittacosis, Q fever, Rabies, Relapsing fever, Respiratory syncytial virus infection, Rhinosporidiosis, Rhinovirus infection, Rickettsial infection, Rickettsialpox, Rift Valley fever (RVF), Rocky Mountain spotted fever (RMSF), Rotavirus infection, Rubella, Salmonellosis, Severe acute respiratory syndrome (SARS), Scabies, Scarlet fever, Schistosomiasis, Sepsis, Shigellosis (bacillary dysentery), Shingles (Herpes zoster), Smallpox (variola), Sporotrichosis, Staphylococcal food poisoning, Staphylococcal infection, Strongyloidiasis, Subacute sclerosing panencephalitis, Bejel, Syphilis, and Yaws, Taeniasis, Tetanus (lockjaw), Tick- borne encephalitis, Tinea barbae (barber's itch), Tinea capitis (ringworm of the scalp), Tinea corporis (ringworm of the body), Tinea cruris (Jock itch), Tinea manum (ringworm of the hand), Tinea nigra, Tinea pedis (athlete’s foot), Tinea unguium (onychomycosis), Tinea versicolor (Pityriasis versicolor), Toxic shock syndrome (TSS), Toxocariasis (ocular larva migrans (OLM)), Toxocariasis (visceral larva migrans (VLM)), Toxoplasmosis, Trachoma, Trichinosis, Trichomoniasis, Trichuriasis (whipworm infection), Tuberculosis, Tularemia, Typhoid fever, Typhus fever, Ureaplasma urealyticum infection, Valley fever, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, Vibrio vulnificus infection, Vibrio parahaemolyticus enteritis, Viral pneumonia, West Nile fever, White piedra (tinea blanca), Yersinia pseudotuberculosis infection, Yersiniosis, Yellow fever, Zeaspora, Zika fever and Zygomycosis. Further disorders that may be susceptible to treatment or prevention with the polymersomes of the invention include brain disorders, inflammatory or autoimmune diseases, atherosclerosis, ischemic heart disease, liver disorders, kidney disorders, diseases associated with ageing, and genetic diseases.

[0312] In one embodiment, the disorder that may be susceptible to treatment or prevention with the polymersomes of the invention is a genetic disease. The genetic disease may be selected from lp36 deletion syndrome, lq21.1 deletion syndrome, 2q37 deletion syndrome, 5q deletion syndrome, 5, 10-methenyl tetrahydrofolate synthetase deficiency, 17ql2 microdeletion syndrome, 17ql2 microduplication syndrome, 18p deletion syndrome, 21 -hydroxylase deficiency, Alpha 1 -antitrypsin deficiency, AAA syndrome (achalasia-addisonianism-alacrima syndrome), Aarskog-Scott syndrome, ABCD syndrome, Absence deformity of leg-cataract syndrome, Aceruloplasminemia, Acheiropodia, Achondrogenesis type II, achondroplasia, Acute intermittent porphyria, Adenylosuccinate lyase deficiency, Adrenoleukodystrophy, Alagille syndrome, ADULT syndrome, Aicardi-Goutieres syndrome, Albinism, Alexander disease, Alfi's syndrome, alkaptonuria, Alport syndrome, Alternating hemiplegia of childhood, Aortic arch anomaly - peculiar facies - intellectual disability, Amish lethal microcephaly, Amyotrophic lateral sclerosis - Frontotemporal dementia, Angel-shaped phalango-epiphyseal dysplasia, Alstrbm syndrome, Alzheimer's disease, Amelogenesis imperfecta, Aminolevulinic acid dehydratase deficiency porphyria, Androgen insensitivity syndrome, Angelman syndrome, Aphalangy-syndactyly-microcephaly syndrome, Apert syndrome, Arthrogryposis-renal dysfunction-cholestasis syndrome, Ataxia telangiectasia, Axenfeld syndrome, Bainbridge- Ropers syndrome, Beare-Stevenson cutis gyrata syndrome, Beckwith-Wiedemann syndrome, Benjamin syndrome, biotinidase deficiency, Bjbrnstad syndrome, Blepharophimosis intellectual disability syndromes, Bloom syndrome, Birt-Hogg-Dube syndrome, Brody myopathy, Brunner syndrome, CADASIL syndrome, Cat eye syndrome, CRASIL syndrome, Chronic granulomatous disorder, Campomelic dysplasia, Camptodactyly-taurinuria syndrome, Canavan disease, Carpenter Syndrome, CDKL5 deficiency disorder, Cerebral dysgenesis-neuropathy-ichthyosis-keratoderma syndrome (CEDNIK), Cleft palate short stature vertebral anomalies syndrome, Combined malonic and methylmalonic aciduria (CMAMMA), Combined malonic and methylmalonic aciduria (CMAMMA), Congenital muscular dystrophy-infantile cataract-hypogonadism syndrome, Cystic fibrosis, Charcot-Mari e-Tooth disease, CHARGE syndrome, Chediak-Higashi syndrome, Chondrodysplasia, Grebe type, Cleidocranial dysostosis, Cockayne syndrome, Coffin-Lowry syndrome, Cohen syndrome, collagenopathy, types II and XI, Congenital insensitivity to pain with anhidrosis (CIPA), Congenital Muscular Dystrophy, Corneal dystrophy-perceptive deafness syndrome, Cornelia de Lange syndrome (CDLS), Cowden syndrome, CPO deficiency (coproporphyria), Cranio-lenticulo-sutural dysplasia, Cri du chat, Crohn's disease, Crouzon syndrome, Crouzonodermoskeletal syndrome (Crouzon syndrome with acanthosis nigricans), Currarino syndrome, Darier's disease, Dent's disease (Genetic hypercalciuria), Denys-Drash syndrome, De Grouchy syndrome, Dolichonychia, Down Syndrome, DiGeorge syndrome, Distal hereditary motor neuropathies, multiple types, Distal muscular dystrophy, Duchenne muscular dystrophy, Dravet syndrome, Ectrodactyly-polydactyly syndrome, Edwards Syndrome, Ehlers-Danlos syndrome, Emanuel syndrome, Emery-Dreifuss syndrome, Epidermolysis bullosa, Erythropoietic protoporphyria, Fanconi anemia (FA), Fabry disease, Factor V Leiden thrombophilia, Fatal familial insomnia, Familial adenomatous polyposis, Familial dysautonomia, Familial Creutzfeld-Jakob Disease, Familial episodic pain syndrome, Familial thoracic aortic aneurysm and aortic dissection, Feingold syndrome, FG syndrome, FBXW7 neurodevelopmental syndrome, Fibular aplasia-ectrodactyly syndrome, Fine-Lubinsky syndrome, Fragile X syndrome, Friedreich's ataxia, G6PD deficiency, Galactosemia, Gaucher disease, Gerstmann-Straussler-Scheinker syndrome, Gillespie syndrome, Glutaric aciduria, type I and type 2, GRACILE syndrome, GRIN2B-related neurodevelopmental disorder, Griscelli syndrome, Gustavson syndrome, Hailey-Hailey disease, Harlequin type ichthyosis, Hemochromatosis type 1, Hemochromatosis type 2A, Hemochromatosis type 2B, Haemochromatosis type 3, Hemochromatosis type 4, Hemochromatosis type 5, Hemophilia, Hepatoerythropoietic porphyria, Hereditary coproporphyria, Hereditary hemorrhagic telangiectasia (Osler-Weber-Rendu syndrome), Hereditary inclusion body myopathy, Hereditary multiple exostoses, Hereditary spastic paraplegia (infantile-onset ascending hereditary spastic paralysis), Herman sky-Pudlak syndrome, Hereditary neuropathy with liability to pressure palsies (HNPP), Heterotaxy, Homocystinuria, Huntington's disease, Hunter syndrome, Hurler syndrome, Hutchinson- Gilford progeria syndrome, Hyperlysinemia, Hyperoxaluria, primary, Hyperphenylalaninemia, Hypoalphalipoproteinemia (Tangier disease), Hypochondrogenesis, Hypochondroplasia, Immunodeficiency-centromeric instabilityfacial anomalies syndrome (ICF syndrome), Incontinentia pigmenti, Infantile cerebral and cerebellar atrophy with postnatal progressive microcephaly, Ischiopatellar dysplasia, Isodicentric 15, PRICKLEI -related progressive myoclonus epilepsy with ataxia, Jackson- Weiss syndrome, Jacobsen syndrome, Joubert syndrome, Juvenile-onset dystonia, Juvenile primary lateral sclerosis (JPLS), Keloid disorder, KIF1 A- Associated Neurological Disorder, Kleefstra syndrome, Kniest dysplasia, Kosaki overgrowth syndrome, Krabbe disease, Kufor-Rakeb syndrome, LCAT deficiency, Lesch-Nyhan syndrome, Li-Fraumeni syndrome, Limb-Girdle Muscular Dystrophy, Lynch syndrome, lipoprotein lipase deficiency, Malignant hyperthermia, Maple syrup urine disease, Marfan syndrome, Maroteaux-Lamy syndrome, McCune- Albright syndrome, McLeod syndrome, MEDNIK syndrome, Mediterranean fever, familial, Menkes disease, Methemoglobinemia, Methylmalonic acidemia, Micro syndrome, Microcephaly, Miller-Dieker syndrome, Morquio syndrome, Mowat-Wilson syndrome, Muenke syndrome, Multiple endocrine neoplasia type 1 (Weimer's syndrome), Multiple endocrine neoplasia type 2, Muscular dystrophy, Muscular dystrophy, Duchenne and Becker type, Myostatin-related muscle hypertrophy, myotonic dystrophy, Natowicz syndrome, NEDAMSS (neurodevelopmental disorder with regression, abnormal movements, loss of speech and seizures), Neurofibromatosis type I, Neurofibromatosis type II, Niemann-Pick disease, Nonketotic hyperglycinemia, Nonsyndromic deafness, Noonan syndrome, Norman-Roberts syndrome, Ogden syndrome, Omenn syndrome, Osteogenesis imperfecta, Ostravik- Lindemann-Solberg syndrome, Pantothenate kinase-associated neurodegeneration, Patau syndrome (Trisomy 13), PCC deficiency (propionic acidemia), Porphyria cutanea tarda (PCT), Pendred syndrome, Peutz-Jeghers syndrome, Pfeiffer syndrome, Phelan- McDermid syndrome, Phenylketonuria, Pipecolic acidemia, Pitt-Hopkins syndrome, Polycystic kidney disease, Polycystic ovary syndrome (PCOS), Porphyria, Prader-Willi syndrome, Primary ciliary dyskinesia (PCD), Primary pulmonary hypertension, Protein C deficiency, Protein S deficiency, Proximal 18q deletion syndrome, Pseudo-Gaucher disease, Pseudoxanthoma elasticum, Retinitis pigmentosa, Rett syndrome, Roberts syndrome, Rubinstein-Taybi syndrome (RSTS), Sandhoff disease, Sanfilippo syndrome, Scheuermann's disease, Schwartz-Jampel syndrome, Sjogren-Larsson syndrome, Skin fragility-woolly hair-palmoplantar keratoderma syndrome, Spondyloepiphyseal dysplasia congenita (SED), Shprintzen-Goldberg syndrome, Sickle cell anemia, Siderius X-linked mental retardation syndrome, Sideroblastic anemia, Sly syndrome, Smith-Lemli-Opitz syndrome, Smith-Magenis syndrome, Snyder-Robinson syndrome, Spinal muscular atrophy, Spinocerebellar ataxia (types 1-29), Split hand split foot-nystagmus syndrome, SSB syndrome (SADDAN), Stargardt disease (macular degeneration), Stickler syndrome (multiple forms), Strudwick syndrome (spondyloepimetaphyseal dysplasia, Strudwick type), Tay-Sachs disease, Tetrahydrobiopterin deficiency, Thanatophoric dysplasia, Thickened earlobes-conductive deafness syndrome, Treacher Collins syndrome, Tuberous sclerosis complex (TSC), Turner syndrome, Usher syndrome, Variegate porphyria, Viljoen-Kallis-Voges syndrome, von Hippel-Lindau disease, von Willebrand disease, Waardenburg syndrome, Warkany syndrome 2, Weissenbacher-Zweyrmiller syndrome, Weyer's ulnar ray / oligodactyly syndrome, Williams syndrome, Wilson disease, Woodhouse-Sakati syndrome, Wolf-Hirschhorn syndrome, Xeroderma pigmentosum, X- linked intellectual disability and macroorchidism (fragile X syndrome), X-linked spinal- bulbar muscle atrophy (spinal and bulbar muscular atrophy), Xpl 1.2 duplication syndrome, X-linked severe combined immunodeficiency (X-SCID), X-linked sideroblastic anemia (XLSA), 47, XXX (triple X syndrome), XXXX syndrome (48, XXXX), XXXXX syndrome (49, XXXXX), XXXXY syndrome (49,XXXXY), XYY syndrome (47,XYY), XXYY syndrome (48,XXYY), XYYY syndrome (48,XYYY), XXXY syndrome (48,XXXY), XYYYY syndrome (49,XYYYY) and Zellweger syndrome.

[0313] Medical uses and methods of treatment, of course, involve the administration of a therapeutically effective amount of the polymersome. A therapeutically effective amount of the polymersomes is administered to a patient. A typical dose is from 0.001 to 1000 mg, measured as a weight of the drug, according to the activity of the specific drug, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration. Preferably, daily dosage levels are from 0.001 mg to 4000 mg.

[0314] The present invention further provides a method of treating or preventing a disorder that comprises administering a therapeutically effective amount of a nanoparticle or microparticle of the invention to a patient in need thereof. For example, the present invention provides a method of treating or preventing a disorder selected from any disorder specified in this disclosure, the drug being a drug that is capable of treating or preventing the said disorder, such as a brain disorder, an immune and / or inflammatory disorder, a cancer, or a genetic disease. The present invention still further provides the use of a nanoparticle or microparticle of the present invention in the manufacture of a medicament for use in a method of treating or preventing a disorder as identified above.

[0315] The present invention further provides a vaccine comprising a polymersome according to the invention, and one or more pharmaceutically acceptable excipients or diluents. In a particularly preferred embodiment, the polymersome comprises a cargo, optionally within the polymersome comprises an mRNA that encodes an antigen, and an mRNA-binding protein. An antigen is any agent that causes the immune system of an animal body to produce an immune response, e.g. bacteria, viruses or pollen. Typically, after administration of the vaccine to a human or animal recipient, the human or animal recipient transcribes the mRNA within the vaccine to produce the antigen. The production of this antigen in vivo induces the memory function of the adaptive immune system towards the specific antigen. Preferably, the vaccine is a vaccine against infectious disease (e.g. HIV, influenza, or a coronavirus such as the common cold or COVID-19), or a cancer vaccine.

[0316] In all aspects of the present invention, the polymersomes may further comprise a label or imaging agent (e.g., encapsulated therein and / or attached to the surface of the particles). For instance, the label / imaging agent could be a dye. A dye for imaging refers to any substance that is used as a label, or that enhances specific structures in any imaging technique. An imaging agent, hence, includes optical an imaging agent, magnetic resonance imaging agent, radioisotope, and contrast agent. Examples, without limitation, of optical imaging agents are an acridine dye, a coumarin dye, a rhodamine dye, a xanthene dye, a cyanine dye, a pyrene dye, Texas Red, Alexa Fluor® dye, BOD IP Y® DYE, Fluorescein, Oregon Green® dye, and Rhodamine Green™ dye, which are commercially available or readily prepared by methods known to those skilled in the art. Examples of imaging agents appropriate for the present invention include, but are not limited to, transition metals and radioactive transition metals chelated to chelating agents for instance DTPA (diethylene triamine pentaacetic acid), DOTA (1,4,7, 10-tetraazacyclododeane- 1,4,7-tetraacetic acid) and NOTA (l,4,7-Triazacyclononane-l,4,7-triacetic acid).

[0317] Further, in all aspects of the present invention, the polymersomes may further comprise a targeting unit, i.e. antibodies, peptides, proteins etc. (e.g. encapsulated therein and / or attached to the surface of the particles). A targeting unit is any chemical structure that functionally interacts with a binding site to cause a physical association between the agent and a surface, e.g., a cell surface. The term targeting unit embraces any molecule (e.g. a naturally occurring molecule, or a chemically / physically modified variant thereof) that is capable of binding to a binding site on the target surface. The binding site could be, but not exclusively, also be capable of internalisation (e.g. endosome formation), also referred to as receptor-mediated endocytosis. The targeting unit may possess an endosomal membrane translocation function, in which case separate targeting unit and translocation domain components need not be present in an agent of the present invention.

[0318] The publications, patent publications and other patent documents cited herein are entirely incorporated by reference. Herein, any reference to a term in the singular also encompasses its plural. Where the term “comprising”, “comprise” or “comprises” is used, said term may substituted by “consisting of’, “consist of’ or “consists of’ respectively, or by “consisting essentially of’, “consist essentially of’ or “consists essentially of’ respectively. In particular, any reference to a hydrophobic block “comprising” a particular type of monomer unit throughout this specification can be substituted by a reference to that hydrophobic block “consisting of’ that particular type of monomer unit. Any reference to a hydrophilic block “comprising” a particular type of monomer unit throughout this specification can likewise be substituted by a reference to that hydrophilic block “consisting of’ that particular type of monomer unit. Any reference to a linker moiety “comprising” a particular type of monomer unit throughout this specification can likewise be substituted by a reference to that linker moiety “consisting of’ that particular type of monomer unit. Any reference to a numerical range or single numerical value also includes values that are about that range or single value. Any reference to alginate encompasses any physiologically acceptable salt thereof unless otherwise indicated. Unless otherwise indicated, any % value is based on the relative weight of the component or components in question.

[0319] Examples

[0320] The present invention is illustrated by the following examples. However, these examples do not limit the scope of the invention.

[0321] Example 1: Comparison of properties of polymers with and without linker moieties, and containing different amounts of free carboxylate The following block copolymers were prepared:

[0322] (1) PEG(45)-OCO-PLA(150) comprising HOOC-PLA as impurity;

[0323] (2) PEG(45)-OCO-PLA(157) comprising trace HOOC-PLA; and

[0324] (3) PEG(45)-NHCO-PCL(2)-PLA(150) comprising trace HOOC-PCL-PLA.

[0325] Polymer (1) was synthesised through the organobase-catalyzed (1,8- diazabicyclo(5.4.0)undec-7-ene; DBU) ring-opening polymerisation (ROP) of D,L-lactide, with hydroxyl-terminated PEG (PEG-OH) used as the initiator. The reaction was conducted using a Schlenk line, with moisture control that was not rigorously maintained. The synthesised polymer underwent no further purification apart from dialysis. As a result, a PEG-OCO-PLA polymer was formed, characterised by an ester bond linking PEG and PLA, along with a certain amount of carboxylic acid-functionalized PLA (HOOC- PLA). The latter is generated through water-initiated ROP and the hydrolysis-induced ester cleavage between PEG and PLA.

[0326] Polymer (2) was synthesised through tin(II) 2-ethylhexanoate-catalyzed ROP of Delactide, with PEG-OH as the initiator. The reaction was carried out under improved conditions to remove moisture, and the polymer solution was passed through an amine- functionalized silica gel column to eliminate HOOC-PLA. As a result, a polymer consisting primarily of PEG-OCO-PLA, with a significantly reduced amount of HOOC- PLA, was produced.

[0327] Polymer (3) was synthesised through tin(II) 2-ethylhexanoate-catalyzed ROP of 8- caprolactone and D,L-lactide, added sequentially, with amine-terminated PEG (PEG-NH2) as the initiator. The synthetic process was further optimised to remove water and moisture. As with polymer (2), the polymer solution was passed through an amine- functionalized silica gel column to eliminate carboxylic acid-functionalized polymers. The polymers prepared in this method are composed of PEG-NHCO-PCL-PLA, characterised by an amide bond and a short PCL chain (2 monomer units) between PEG and PLA, with a trace amount of HOOC-PCL-PLA.

[0328] Polymers (1), (2) and (3) were then self-assembled into polymersomes (“Batch 1”, “Batch 2” and “Batch 3”, respectively) by a solvent injection method. The polymer was first dissolved in dimethylformamide (DMF) as organic solvent. Using an injection system, the organic solution was injected at 50 pL / min into a phosphate-buffered saline (PBS) solution at pH 7.4 under constant stirring. After self-assembly of polymersomes, any remaining organic solvent was removed by dialysis against PBS solution at pH 7.4 for 2 hours at 4°C and / or size-exclusion chromatography in PBS solution at pH 7.4.

[0329] The particle size distribution of the polymersomes was measured via dynamic light scattering (DLS). A Malvern Zetasizer Nano ZS laser light scatterer equipped with a He- Ne 4mW 633 nm laser was used. Polymersomes were diluted in filtered milliQ water in 1 mL disposable cuvettes, and experiments were an average of n=3 runs at a set angle of 173°.

[0330] The results of the DLS experiments are shown in Figs. 2-4 for polymersome batches 1 to 3, respectively. The DLS measurements suggest the formation of spherical structures. This feature implies that the copolymers assemble into membranes that in turn form into spherical polymersomes. Figs. 2(a), 3(a) and 4(a) show the particle size distributions by number, whilst Figs. 2(b), 3(b) and 4(b) show the particle size distributions by intensity. These distributions demonstrate that all the polymersomes have an average diameter of approximately 100 nm, which is considered to be a desirable particle size for in vivo applications of the polymersomes. In Figs. 2(c), 3(c) and 4(c) a correlogram of the DLS data is plotted. Dynamic light scattering measures the correlation coefficient from intensity traces performed using an integrated digital correlator. As can been seen the measured correlation curves have all similar decayed time to baseline, confirming that the size of the polymersomes in solution in all the samples is very similar.

[0331] Polymersomes in filtered milliQ water were also assessed for morphology using transmission electron microscopy (TEM). Samples were mounted on glow-discharged carbon coated grids by submerging the grids into the polymersome solution for 60 seconds, followed by staining for 5 seconds using 0.5% (w / w) phosphotungstic acid (PTA) and dried under vacuum and assessed via a JEOL microscope using 100 kV voltage tension.

[0332] The resulting TEM micrographs are shown (for each polymersome sample, respectively) in Figs. 2(d), 3(d) and 4(d). Transmission electron microscopy (TEM) was used to confirm the vesicular structure. All the samples appeared spherical, with varied diameters, in agreement with DLS measurements, and moreover support vesicle formation. The zeta potential of the polymersomes was then determined over a 30-day period with storage in phosphate-buffered saline (PBS) at 5°C, using the electrophoretic light scattering (ELS) method, which involves measuring the velocity of nanoparticles moving towards the electrode in the presence of an external electric field. Fig. 5 illustrates variation in the zeta potential of the polymersomes formed from polymers (1) to (3).

[0333] The zeta potential of Batch 1 measured a negative charge below -12 mV immediately after preparation. The zeta potential decreased over the following 30 days. Without wishing to be bound by any particular theory, the high initial negative potential is believed to be associated with significant amounts of HOOC-PLA polymer present in the polymersomes (as the carboxylic acid groups can dissociate to form carboxylate anions, contributing to the negative surface charge on the polymersome). It is believed that more HOOC-PLA is generated through the hydrolysis of the ester bond between PEG and PLA blocks over time, resulting in the measured zeta potential becoming more negative over time.

[0334] The formulation of Batch 2 exhibited a zeta potential around -2 mV on the day of polymersome preparation, indicating a significantly lower level of HOOC-PLA. This reduction (compared to Batch 1) is attributed to improved moisture control and the purification process involving amine-functionalized silica gel. However, the surface charge of Batch 2 polymersomes did decrease significantly over the 30-day period, reaching -12 mV after 30 days. This suggests that HOOC-PLA is generated through the hydrolysis of the ester bond between PEG and PLA blocks over time.

[0335] In Batch 3, the polymersomes exhibited a zeta potential of around 0 mV on the day of preparation. Due to the high stability of the amide bond and the presence of a short-chain PCL between the PEG and PLA blocks, the formulation of Batch 3 showed a better zeta potential than those of Batch 1 and Batch 2 at all tested time points. The values were maintained above -4 mV at 10 days and around -10 mV at 30 days.

[0336] Example 2: Investigating the effect of varying the number of monomer units in the linker moiety

[0337] The following block copolymers were prepared:

[0338] (a) PEG(45)-O-PCL(20)-PLA(149);

[0339] (b) PEG(45)-NH-PCL(2)-PLA(150); and (c) PEG(45)-NH-PCL(7)-PLA(105).

[0340] These triblock copolymers were synthesised via the ring-opening polymerisation (ROP) of 8-caprolactone (s-CL) and D,L-lactide in two steps. Typically, (a-methoxy, co-hydroxy)- terminated poly(ethylene glycol) (MeO-PEG-OH) and s-CL along with molecular sieves were weighed out into a vial with a magnetic stirrer, added to anhydrous toluene and sealed with a rubber septum, stirring in 65 °C oil bath to remove water. Tin(II) 2- ethylhexanoate Sn(Oct)2 was added into another vial with a stirrer bar and sealed with a rubber septum, and dried under vacuum. The PEG and s-CL solution was transferred to the vial of Sn(Oct)2 via evacuated syringe with PTFE filter, then placed in a 100°C oil bath. After the reaction, the polymer solution was added to citric acid, then filtered through an amino-functionalised silica column. APEG-PCL diblock copolymer was isolated by precipitation in diethyl ether and pentane. The purified PEG-PCL was dissolved in toluene and dried under argon with molecular sieves at 65°C. D,L-lactide and tin(II) 2-ethylhexanoate Sn(Oct)2 were added into another vial with a stirrer bar and sealed with a rubber septum and dried with Schlenk line. The PEG-PCL solution was transferred to the vial of Sn(Oct)2 and D,L-lactide via evacuated syringe with PTFE filter, then placed in a 100°C oil bath. When the reaction was complete, the polymer solution was added to citric acid, then filtered through an amino-functionalised silica column. The final PEG- PCL-PLA triblock polymer was isolated by precipitation in diethyl ether and pentane.

[0341] Polymers (a), (b) and (c) were then self-assembled into polymersomes (“Batch A”, “Batch B” and “Batch C”, respectively) by a solvent injection method. The polymer was first dissolved in dimethylformamide (DMF) as organic solvent. Using an injection system, the organic solution was injected at 50 pL / min into a phosphate-buffered saline (PBS) solution at pH 7.4 under constant stirring. After self-assembly of polymersomes, any remaining organic solvent was removed by dialysis against PBS solution at pH 7.4 for 2 hours at 4°C and / or size-exclusion chromatography in PBS solution at pH 7.4.

[0342] The particle size distribution of the polymersomes was measured via dynamic light scattering (DLS). A Malvern Zetasizer Nano ZS laser light scatterer equipped with a He- Ne 4mW 633 nm laser was used. Polymersomes were diluted in filtered milliQ water in 1 mL disposable cuvettes, and experiments were an average of n=3 runs at a set angle of 173°. The results of the DLS experiments are shown in Figs. 6-8 for polymersome batches A to C, respectively. The DLS measurements suggest the formation of spherical structures. This feature implies that the copolymers assemble into membranes that in turn form into spherical polymersomes. Figs. 6(a), 7(a) and 8(a) show the particle size distributions by number, whilst Figs. 6(b), 7(b) and 8(b) show the particle size distributions by intensity. These distributions demonstrate that all the polymersomes have an average diameter of approximately 100 nm, which is considered to be a desirable particle size for in vivo applications of the polymersomes. In Figs. 6(c), 7(c) and 8(c) a correlogram of the DLS data is plotted. Dynamic light scattering measures the correlation coefficient from intensity traces performed using an integrated digital correlator. As can been seen the measured correlation curves have all similar decayed time to baseline, confirming that the size of the polymersomes in solution in all the samples is very similar.

[0343] Polymersomes in filtered milliQ water were also assessed for morphology using transmission electron microscopy (TEM). Samples were mounted on glow-discharged carbon coated grids by submerging the grids into the polymersome solution for 60 seconds, followed by staining for 5 seconds using 0.5% (w / w) phosphotungstic acid (PTA) and dried under vacuum and assessed via a JEOL microscope using 100 kV voltage tension.

[0344] The resulting TEM micrographs are shown (for each polymersome sample, respectively) in Figs. 6(d), 7(d) and 8(d). Transmission electron microscopy (TEM) was used to confirm the vesicular structure. All the samples appeared spherical, with varied diameters, in agreement with DLS measurements, and moreover support vesicle formation.

[0345] The zeta potential of the polymersomes was then determined over a 30-day period with storage in phosphate-buffered saline (PBS) at 5°C, using the electrophoretic light scattering (ELS) method, which involves measuring the velocity of nanoparticles moving towards the electrode in the presence of an external electric field. Fig. 9 illustrates variation in the zeta potential of the polymersomes formed from polymers (a) to (c).

[0346] Batch B in this example is the same as Batch 3 in Example 1 described above. Although Example 1 and Fig. 5 show that this batch of polymersomes has improved properties over polymersomes which do not contain the PCL linker moiety, in Fig. 9 we can see that this particular batch shows the most instability in zeta potential over time. Although the polymersomes exhibited a zeta potential of around 0 mV on the day of preparation, by the end of the 30-day experiment the zeta potential had decreased to around -10 mV.

[0347] Batch C displayed a somewhat improved stability compared to Batch B. This batch was prepared from triblock copolymers having a higher number of monomer units of PCL in the linker moiety / increased ratio of Mwof the linker moiety compared to the hydrophobic block. Thus, on the day of preparation this batch showed a zeta potential of around -8 mV, but this did not decrease as significantly over the course of the 30-day experiment as for Batch B; after 30 days, the zeta potential of Batch C was around -12 mV. This suggests that the copolymers of Batch C, having a larger number of monomer units in the linker moiety, are less susceptible to hydrolysis than the copolymers used to make Batch B.

[0348] Batch A however showed a significant improvement in stability compared to both Batches B and C. This batch was prepared from triblock copolymers having a higher number of monomer units of PCL in the linker moiety / increased ratio of Mwof the linker moiety to the hydrophobic block. Thus, on the day of preparation of the polymersomes, the zeta potential for Batch A is around -7 mV (less negative than for Batch C). Significantly, the zeta potential does not deteriorate at all over the 30-day period, with a measured potential of around -5 mV on day 30. This suggests that the longer length of the PCL linker moiety has resulted in a triblock copolymer that, when self-assembled into polymersomes, is surprisingly more resistant to hydrolysis.

Claims

CLAIMS1. A triblock copolymer comprising a hydrophilic block, a linker moiety and a hydrophobic block, wherein the hydrophilic block is covalently bonded to the hydrophobic block via the linker moiety, and wherein:(a) the hydrophobic block is a polyester, and the linker moiety consists of from 1 to 50 monomer units of:(i) an ester monomer which comprises a longer linear chain of carbon atoms, from the carbonyl carbon to the hydroxy- substituted carbon within the monomer, than the ester monomer which makes up the polyester of the hydrophobic block;(ii) an amide monomer;(iii) an ether monomer; or(iv) a thioether monomer; or(b) the hydrophobic block is a polyamide, and the linker moiety consists of from 1 to 50 monomer units of:(i) an amide monomer which comprises a longer linear chain of carbon atoms, from the carbonyl carbon to the amino-substituted carbon within the monomer, than the amide monomer which makes up the polyamide of the hydrophobic block;(ii) an ether monomer; or(iii) a thioether monomer.

2. A triblock copolymer according to claim 1, wherein:(1) the hydrophobic block is a polyester, wherein each ester monomer unit in the polyester comprises from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy- substituted carbon, and the linker moiety consists of from 1 to 50 monomer units of an ester monomer comprising more than 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy-substituted carbon; or(2) the hydrophobic block is a polyester, preferably wherein each ester monomer unit in the polyester comprises from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy- substituted carbon, and the linker moiety83consists of from 1 to 50 monomer units of an amide monomer, preferably an amide monomer comprising from 2 to 4 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon; or(3) the hydrophobic block is a polyamide, wherein each amide monomer unit in the polyamide comprises 2 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon, and the linker moiety consists of from 1 to 50 monomer units of an amide monomer comprising more than 2 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon; or(4) the hydrophobic block is a polyamide, wherein each amide monomer unit in the polyamide comprises 3 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon, and the linker moiety consists of from 1 to 50 monomer units of an amide monomer comprising more than 3 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon.

3. A triblock copolymer according to claim 1 or claim 2, wherein the linker moiety is of formula (A) or formula (B) or formula (C):wherein, in formula (A): m is an integer from 4 to 40;84n is an integer from 1 to 50; each R1is independently selected from hydrogen, substituted or unsubstituted Ci-Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, substituted or unsubstituted Ci-Ce aminoalkyl, substituted or unsubstituted Ci-Ce haloalkyl, substituted or unsubstituted Ci-Ce haloalkoxy, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C4-C16 cycloalkylalkyl, substituted or unsubstituted C2- Ce alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted Ce-Cio aryl, substituted or unsubstituted C7-C16 aralkyl, substituted or unsubstituted C4-C8 heterocycyl, substituted or unsubstituted C5-C14 heterocycloalkyl, substituted or unsubstituted C5- C10 heteroaryl, substituted or unsubstituted Ce-Ci6 heteroaralkyl, halo, hydroxy, amino, cyano or azido; each R2is independently selected from hydrogen, substituted or unsubstituted Ci-Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, substituted or unsubstituted Ci-Ce aminoalkyl, substituted or unsubstituted Ci-Ce haloalkyl, substituted or unsubstituted Ci-Ce haloalkoxy, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C4-C16 cycloalkylalkyl, substituted or unsubstituted C2- Ce alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted Ce-Cio aryl, substituted or unsubstituted C7-C16 aralkyl, substituted or unsubstituted C4-C8 heterocyclyl, substituted or unsubstituted C5-C14 heterocycloalkyl, substituted or unsubstituted C5- C10 heteroaryl, substituted or unsubstituted Ce-Ci6 heteroaralkyl, halo, hydroxy, amino, cyano or azido;* represents the point of attachment to the hydrophilic block; and A represents the point of attachment to the hydrophobic block; and in formula (B): p is an integer from 1 to 40, preferably from 2 to 40; q is an integer from 1 to 50; each R3is independently selected from hydrogen, substituted or unsubstituted Ci-Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy,substituted or unsubstituted Ci-Ce aminoalkyl, substituted or unsubstituted Ci-Ce haloalkyl, substituted or unsubstituted Ci-Ce haloalkoxy, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C4-C16 cycloalkylalkyl, substituted or unsubstituted C2- Ce alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted Ce-Cio aryl, substituted or unsubstituted C7-C16 aralkyl, substituted or unsubstituted C4-C8 heterocyclyl, substituted or unsubstituted C5-C14 heterocycloalkyl, substituted or unsubstituted C5- C10 heteroaryl, substituted or unsubstituted Ce-Ci6 heteroaralkyl, halo, hydroxy, amino, cyano or azido; each R4is independently selected from hydrogen, substituted or unsubstituted Ci-Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, substituted or unsubstituted Ci-Ce aminoalkyl, substituted or unsubstituted Ci-Ce haloalkyl, substituted or unsubstituted Ci-Ce haloalkoxy, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C4-C16 cycloalkylalkyl, substituted or unsubstituted C2- Ce alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted Ce-Cio aryl, substituted or unsubstituted C7-C16 aralkyl, substituted or unsubstituted C4-C8 heterocyclyl, substituted or unsubstituted C5-C14 heterocycloalkyl, substituted or unsubstituted C5- C10 heteroaryl, substituted or unsubstituted Ce-Ci6 heteroaralkyl, halo, hydroxy, amino, cyano or azido; each R5is independently selected from hydrogen or unsubstituted Ci-Ce alkyl;* represents the point of attachment to the hydrophilic block; and A represents the point of attachment to the hydrophobic block; and in formula (C):- t is an integer from 1 to 40 and u is an integer from 0 to 40, provided that (t+u) is an integer from 1 to 40; v is an integer from 1 to 50;X is O or S;each R6is independently selected from hydrogen, substituted or unsubstituted Ci-Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, substituted or unsubstituted Ci-Ce aminoalkyl, substituted or unsubstituted Ci-Ce haloalkyl, substituted or unsubstituted Ci-Ce haloalkoxy, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C4-C16 cycloalkylalkyl, substituted or unsubstituted C2- Ce alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted Ce-Cio aryl, substituted or unsubstituted C7-C16 aralkyl, substituted or unsubstituted C4-C8 heterocyclyl, substituted or unsubstituted C5-C14 heterocycloalkyl, substituted or unsubstituted C5- C10 heteroaryl, substituted or unsubstituted Ce-Ci6 heteroaralkyl, halo, hydroxy, amino, cyano or azido; each R7is independently selected from hydrogen, substituted or unsubstituted Ci-Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, substituted or unsubstituted Ci-Ce aminoalkyl, substituted or unsubstituted Ci-Ce haloalkyl, substituted or unsubstituted Ci-Ce haloalkoxy, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C4-C16 cycloalkylalkyl, substituted or unsubstituted C2- Ce alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted Ce-Cio aryl, substituted or unsubstituted C7-C16 aralkyl, substituted or unsubstituted C4-C8 heterocyclyl, substituted or unsubstituted C5-C14 heterocycloalkyl, substituted or unsubstituted C5- C10 heteroaryl, substituted or unsubstituted Ce-Ci6 heteroaralkyl, halo, hydroxy, amino, cyano or azido; each R8is independently selected from hydrogen, substituted or unsubstituted Ci-Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, substituted or unsubstituted Ci-Ce aminoalkyl, substituted or unsubstituted Ci-Ce haloalkyl, substituted or unsubstituted Ci-Ce haloalkoxy, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C4-C16 cycloalkylalkyl, substituted or unsubstituted C2- Ce alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted Ce-Cio aryl, substituted or unsubstituted C7-C16 aralkyl, substituted or unsubstituted C4-C8 heterocyclyl, substituted orunsubstituted C5-C14 heterocycloalkyl, substituted or unsubstituted C5- C10 heteroaryl, substituted or unsubstituted Ce-Ci6 heteroaralkyl, halo, hydroxy, amino, cyano or azido; each R9is independently selected from hydrogen, substituted or unsubstituted Ci-Ce alkyl, substituted or unsubstituted Ci-Ce alkoxy, substituted or unsubstituted Ci-Ce aminoalkyl, substituted or unsubstituted Ci-Ce haloalkyl, substituted or unsubstituted Ci-Ce haloalkoxy, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C4-C16 cycloalkylalkyl, substituted or unsubstituted C2- Ce alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted Ce-Cio aryl, substituted or unsubstituted C7-C16 aralkyl, substituted or unsubstituted C4-C8 heterocyclyl, substituted or unsubstituted C5-C14 heterocycloalkyl, substituted or unsubstituted C5- C10 heteroaryl, substituted or unsubstituted Ce-Ci6 heteroaralkyl, halo, hydroxy, amino, cyano or azido;* represents the point of attachment to the hydrophilic block; and A represents the point of attachment to the hydrophobic block.

4. A triblock copolymer according to claim 3, wherein:(a) n is from 2 to 40, preferably wherein n is from 3 to 30, and more preferably where n is from 5 to 25; or(b) q is from 2 to 40, preferably wherein q is from 3 to 30, and more preferably where q is from 5 to 25; or(c) v is from 2 to 40, preferably wherein v is from 3 to 30, and more preferably where v is from 5 to 25.

6. A triblock copolymer according to claim 4 or claim 5, wherein:(a) each R1is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, and preferably wherein each R1is hydrogen; and / or(b) each R2is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, and preferably wherein each R2is hydrogen; and / or(c) each R3is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, and preferably wherein each R3is hydrogen; and / or(d) each R4is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl, and preferably wherein each R4is hydrogen; and / or(e) each R5is hydrogen; and / or(f) each R6is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl; and / or(g) each R7is hydrogen; and / or(h) each R8is independently selected from hydrogen, Ci-Ce alkyl, phenyl or benzyl; and / or(i) each R9is hydrogen.

6. A triblock copolymer according to any one of claims 3 to 5, wherein:(1) the hydrophobic block is a polyester, wherein each ester monomer unit in the polyester comprises from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy-substituted carbon, the linker moiety is of formula (A), and m is an integer from 4 to 30, preferably from 4 to 20, and more preferably from 5 to 10; or(2) the hydrophobic block is a polyester, preferably wherein each ester monomer unit in the polyester comprises from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy- substituted carbon, the linker moiety is of formula (B), and p is an integer from 2 to 30, preferably from 2 to 5; or(3) the hydrophobic block is a polyamide, wherein each amide monomer unit in the polyamide comprises 2 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon, the linker moiety is of formula (B), and p is an integer from 3 to 30, preferably from 3 to 5; or(4) the hydrophobic block is a polyamide, wherein each amide monomer unit in the polyamide comprises 3 carbon atoms in a linear chain from the carbonyl carbon to the amino-substituted carbon, the linker moiety is of formula (B), and p is an integer from 4 to 30, preferably 4 or 5.

7. A triblock copolymer according to any one of claims 3 to 6, wherein the hydrophobic block is a polyester, wherein each ester monomer unit in the polyester comprises from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon tothe hydroxy-substituted carbon, the linker moiety is of formula (A), m is 5, each R1is hydrogen and each R2is hydrogen.

8. A triblock copolymer according to any one of claims 1 to 7, wherein the hydrophilic block is a polyether, poly(oligo(ethylene glycol methyl ether methacrylate) (POEGMA), poly(2 -methacryloyloxyethyl phosphorylcholine) (PMPC), poly(vinyl pyrrolidine) (PVP), or polysarcosine, preferably wherein the hydrophilic block is a polyether, optionally wherein the polyether is poly(ethylene glycol), amine-terminated poly(ethylene glycol), polypropylene glycol) or amine- terminated polypropylene glycol).

9. A triblock copolymer according to any one of claims 1 to 8, wherein:(a) the hydrophilic block is a polyether which is covalently bound to the linker moiety through a terminal -OH group; or(b) the hydrophilic block is a polyether which is covalently bound to the linker moiety through a terminal -NH2 group.

10. A triblock copolymer according to any one of claims 1 to 9, wherein the hydrophilic block is a polyether having a weight-average molecular weight of from 44 to 2,500 Da, and preferably from 1,800 to 2,200 Da.

11. A triblock copolymer according to any one of claims 1 to 10, wherein the hydrophobic block is:(a) a polyester selected from poly(lactic acid) and poly(glycolic acid); and / or(b) a polyester having a weight-average molecular weight of from 5,000 to 20,000 Da, and preferably of from 7,500 to 14,000 Da.

12. A triblock copolymer according to any one of claims 1 to 11, wherein the hydrophilic block is polypthylene glycol) or amine-terminated polypthylene glycol) and the hydrophobic block is poly(lactic acid), preferably wherein:(i) the polypthylene glycol) or amine-terminated polypthylene glycol) has a weight-average molecular weight of from 44 to 2,500 Da, preferably from 1,800 to 2,200 Da; and / or(ii) the poly(lactic acid) has a weight-average molecular weight of from 5,000 to 20,000 Da, preferably from 7,500 to 14,000 Da; and / or(iii) the linker moiety is of formula (A), m is 5, each R1is hydrogen and each R2is hydrogen.

13. A triblock copolymer according to any one of claims 1 to 12, wherein the ratio of the weight-average molecular weight of the hydrophilic block to the weightaverage molecular weight of the hydrophobic block is from 1 :9 to 1 :3, preferably from 1 :6 to 1 :4.

14. A triblock copolymer comprising a hydrophilic block, a linker moiety and a hydrophobic block, wherein: the hydrophilic block is covalently bonded to the hydrophobic block via the linker moiety; the hydrophobic block is a polyester or a polyamide; and the linker moiety consists of from 1 to 50 monomer units, wherein the bond between adjacent monomer units in the linker moiety and / or the bond between the linker moiety and the hydrophilic block is less susceptible to hydrolysis than the bond between adjacent monomer units in the hydrophobic block; optionally wherein the triblock copolymer has any of the further features of claims 2 to 13.

15. A polymersome comprising the triblock copolymer of any one of claims 1 to 14.

16. A polymersome according to claim 15, wherein:(a) the polymersome has a polydispersity index from 0.01 to 0.2, preferably from 0.01 to 0.1, as measured by dynamic light scattering; and / or(c) the z-average diameter of the polymersome particles as measured by dynamic light scattering is from 50 to 120 nm.

17. A polymersome which comprises:(a) a triblock copolymer as defined in any one of claims 1 to 14; and(b) a diblock copolymer comprising a hydrophilic block that is directly covalently bonded to a hydrophobic block, wherein the hydrophobic block is (i) a polyester, preferably wherein each ester monomer unit in the polyester comprises from 1 to 4 carbon atoms in a linear chain from the carbonyl carbon to the hydroxy- substituted carbon, or (ii) a polyamide, preferably wherein each amide monomer unit in the polyamide comprises 2 or 3 carbon atoms, more preferably 2 carbon atoms, in a linear chain from the carbonyl carbon to the amino-substituted carbon; wherein the ratio of triblock copolymer (a) to diblock copolymer (b) in the polymersome is from 1 :1 to 100: 1, preferably wherein the hydrophobic block of triblock copolymer (a) is the same as the hydrophobic block of diblock copolymer (b), and / or the hydrophilic block of triblock copolymer (a) is the same as the hydrophilic block of diblock copolymer (b).

18. A polymersome according to any one of claims 15 to 17, wherein the polymersome is for binding to the surface of a cell and comprises (a) a polymer brush and (b) at least a first ligand type on its external surface, wherein said first ligand type is capable of binding to a first receptor type on said cell surface, optionally wherein the polymersome comprises 2 to 1000 ligands, and preferably from 5 to 200 ligands, of the first ligand type; further optionally wherein the polymersome further comprises at least a second ligand type on its external surface, wherein said second ligand type is capable of binding to a second receptor type on said cell surface, optionally wherein the polymersome comprises from 2 to 1000 ligands, and preferably from 5 to 200 ligands, of the second ligand type; and preferably wherein each ligand on the external surface of the polymersome is covalently bound to the hydrophilic block of the copolymer, optionally via a linker.

19. A polymersome according to any one of claims 15 to 18, wherein the polymersome comprises a cargo encapsulated within the polymersome, optionally wherein saidcargo is a drug, an antibody or a nucleic acid, preferably wherein the drug is selected from a neuroprotectant, an immunomodulatory drug, a non-steroidal antiinflammatory drug (NSAID), a corticosteroid, a disease-modifying antirheumatic drug (DMARD), an immunosuppressant, a TNF-alpha inhibitor and an anti-cancer drug, optionally wherein said cargo comprises a nucleic acid and a nucleic acid-binding protein, preferably wherein:(i) said nucleic acid is a DNA and said nucleic acid-binding protein is a DNA- binding protein, preferably wherein the nucleic acid is plasmid DNA, and the nucleic acid-binding protein is a histone, more preferably histone Hl; or(ii) said nucleic acid is an RNA and said nucleic acid-binding protein is an RNA-binding protein; or(iii) said nucleic acid is a PNA and said nucleic-acid binding protein is a PNA- binding protein.

20. A pharmaceutical composition comprising a plurality of the polymersomes according to any one of claims 14 to 19, and one or more pharmaceutically acceptable excipients or diluents.

21. A polymersome according to any one of claims 14 to 19, or a pharmaceutical composition according to claim 20, for use in the treatment of a disease, optionally wherein the disease is cancer, an infectious disease, or a disorder of the nervous system.

22. A method of treating cancer, an infectious disease or a disorder of the nervous system in a human patient, wherein said method comprises administration of a polymersome according to any one of claims 14 to 19, or a pharmaceutical composition according to claim 20, to a patient in need thereof.

23. Use of a polymersome according to any one of claims 14 to 19, or a pharmaceutical composition according to claim 20, for the manufacture of a medicament for thetreatment of cancer, an infectious disease, or a disorder of a nervous system in a patient.

24. A vaccine comprising a polymersome according to any one of claims 14 to 19, and one or more pharmaceutically acceptable excipients or diluents, preferably wherein the polymersome comprises an mRNA cargo encapsulated within the polymersome.

25. A method of preparing a polymersome according to any one of claims 14 to 19, wherein said method comprises:(i) dissolving a triblock copolymer according to any one of claims 1 to 13 in an organic solvent having a polarity index of greater than 3.9; and(ii) adding the resulting polymer solution to an aqueous solution.94

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