阅读说明：本技术 可代谢的ph敏感聚合物囊泡 (Metabolizable pH-sensitive polymersome ) 是由 朱塞佩·巴塔利亚 亚历山德罗·波马 丹尼斯·赛钦 于 2019-04-11 设计创作，主要内容包括：本申请涉及pH敏感聚合物囊泡。聚合物囊泡能够在核内体的温和酸性pH下降解为可再次吸收的材料,这还使得避免脂质体一旦被内化在细胞内,有效载荷的脂质体过早降解。因此,该聚合物囊泡可用于治疗或预防疾病的方法中。(The present application relates to pH sensitive polymersomes. The polymersomes are capable of degrading to resorbable materials at the mildly acidic pH of endosomes, which also allows for the avoidance of premature degradation of the payload liposome once the liposome is internalized within the cell. Thus, the polymersome can be used in a method for treating or preventing a disease.)

1. A polymersome, comprising:

(a) a protein-rejecting polymer; and

(b) a pH sensitive biodegradable succinate polymer comprising pendant groups having a pKa of 4 to 7.

2. The polymersome of claim 1, wherein the protein-rejecting polymer is an acrylate polymer, a polypeptide, a polyester, or a polycarbonate.

3. The polymersome of claim 2, wherein the protein-rejecting polymer comprises units of formula (I)

Wherein R is^pIs hydrogen or methyl and A^pA radical of the formula (I

Wherein X^pIs C_1-6An alkylene group.

4. The polymersome of any one of claims 1 to 3, wherein the protein-rejecting polymer is poly (2- (methacryloyloxy) ethyl phosphorylcholine) or poly (ethylene oxide).

5. The polymersome of any one of claims 1 to 4, wherein the pendant group has a pKa of 5.5 to 7, preferably 6.2 to 7.

6. The polymersome of any one of claims 1 to 5, wherein the side group comprises an imidazole moiety.

7. The polymersome of claim 6, wherein the imidazole moiety is imidazole, methylimidazole, or dimethylimidazole.

8. The polymersome of any one of claims 1 to 7, wherein the pH sensitive biodegradable fumarate polymer is poly [ propylene 2- ((2- (2- (1H-imidazol-5-yl) acetamido) -3- (tert-butoxy) -3-oxopropyl) thio) succinate ].

9. The polymersome of any one of claims 1 to 8, further comprising a drug encapsulated within the polymersome.

10. A pharmaceutical composition comprising: a plurality of polymersomes according to any one of claims 1 to 10; and one or more pharmaceutically acceptable excipients or diluents.

11. Use of a polymersome according to any one of claims 1 to 10 as a medicament.

Technical Field

The present invention relates to metabolizable pH-sensitive polymersomes. The polymersomes are made from metabolizable materials classified as generally accepted safe as safe (GRAS) by the FDA and MHRA. Derivatives having imidazole rings have been used as the hydrophobic portion of amphiphilic block copolymers. An exemplary class of polymers, Polypropylene (((1H-imidazol-5-yl) acetamido) -3-oxopropyl) thio) succinate (Polypropylene (((1H-imidozol-5-yl) acetamido) -3-oxopropyl) thio) succinate, PPITS) and its derivatives are pH sensitive, hydrophobic at neutral pH, hydrophilic at mildly acidic pH, the hydrolysate of which will be metabolized by the liver or reabsorbed as a component of the Krebs cycle (Krebs cycle). Thus, the polymersomes are useful in methods of treating or alleviating the symptoms of diabetes, cancer, infectious and autoimmune diseases.

Background

Critical to effective and safe disease management is the need to deliver pharmacological doses of drugs to target sites with high efficiency using a route that will aid patient compliance.

Polymeric nanocarriers are being used to address key issues in drug delivery: the active substance is loaded at a sufficient dose to protect it from the surrounding environment in vivo and to provide a stable release to the target site without causing systemic toxicity.

Intracellular delivery of drugs has attracted increasing research interest, primarily due to their important role and function in several diseases. Subcellular targeting is critical for effective specific therapy and therefore specific obstacles must be overcome. The importance of organelle targeting increases when drugs are effective in treating or alleviating the symptoms of diseases such as cancer, alzheimer's disease, diabetes, infectious and autoimmune diseases. In particular, the intracellular environment contains compounds responsible for cell growth, proliferation, differentiation and death, and thus these compounds are promising drug targets. Thus, the target site may be distributed throughout the cytoplasm, nucleus, mitochondria, Endoplasmic Reticulum (ER) and golgi complex. However, low pH and enzyme-rich endosomes and lysosomes can lead to drug degradation or non-specific distribution. Thus, modulation of the size, charge, and surface composition of the nanomaterial may indicate an internalization pathway that enables the nanomaterial to escape lysosomes and interact with its target organelles. Indeed, the potential of nanomaterials to overcome this obstacle has led to the development of polymers such as poly [ propylene (1H-imidazol-5-yl) acetamido) -3-oxopropyl) thio) succinate ] (PPITS) and its derivatives, which are able to improve the bioavailability and intracellular delivery of drugs by exploiting their pH sensitivity.

Nanovesicles formed using PPITS and their derivatives are important because they enable drugs to safely and sustainably reach their target sites across physiological barriers. In fact, nanovesicles provide a stable biocompatible environment to encapsulate drugs, facilitating intracellular release and efficient absorption of the drugs. Nanovesicles also improve the duration of the therapeutic effect by driving the drug in a specific way towards the site of action and increasing the concentration of the drug in the pathological area, and minimize adverse reactions.

The pH sensitive smart polymer contains weakly acidic or weakly basic functional groups that accept or lose protons depending on the local pH. This stimulates degradation of the polymer at the endosomal level, provides intracellular release and avoids lysosomal degradation of the payload.

The development of such polymers has generally employed "click" chemistry methods, including alkyne-azide cycloaddition reactions, Diels-Alder reactions, thiol-ene addition reactions, and other coupling strategies such as Michael addition reactions, to increase the diversity of accessible macromolecular structures. Alkyne-azide cycloaddition reactions are one of the most extensively studied click reactions for the modification of preformed macromolecules and their synthesis. Diels-alder cycloaddition reactions have been used to construct a range of macromolecules whose thermo-sensitive reversibility provides a platform for the development of degradable nanocarriers for drug delivery.

The imidazole groups on the polymer exhibit pKa in the desired pH range for avoiding lysosomal degradation. However, the pKa value can also be further adjusted by modifying the position and nature of the different substituents around the imidazole ring. PH sensitivity is introduced by exploiting the double bond present in poly (propyl fumarate) (PPF) or that occurs after isomerization of poly (propyl maleate) (PPM), and conjugating t-butylcysteine by thiol-ene addition reaction on the original PPM. This is just one example of a possible conjugation chemistry involving double bonds within the polymer structure (e.g., diels-alder aldol condensation reactions). The cysteine N-terminus can be conjugated to imidazole-or methylimidazole-containing acids, including 1H-imidazole-1-acetic acid (pKa 6.8), 4-imidazoleacetic acid (pKa 6.6), dimethylimidazoleacetic acid (pKa 6.4), and methylimidazolic acid (pKa 6.2). This conjugation reaction can be carried out before or after the thiol-ene addition reaction by NHS chemistry or other types of conjugation reactions known to those skilled in the art. Similarly, the same cysteine derivative protected on the amino group (with a t-butoxycarbonyl moiety) and having a free carboxyl group may also be conjugated with the same imidazole moiety (but derived from a histamine derivative). In all cases, all of these structures are metabolite moieties of histidine metabolism and/or histamine catabolism, and they will adjust the final polymer pKa from 6.2 to 6.8. Furthermore, the number of functionalities can be adjusted by adding saturated monomers (e.g., succinic acid (in the case of polycondensation) or succinic anhydride (in the case of ring-opening copolymerization or ROOP)) to the polymerization mixture, thereby creating a range of saturated and unsaturated polyesters.

Modulating the chemistry makes it possible to create functionalized polymer vesicles with enhanced intracellular delivery, with the ability to escape premature lysosomal degradation and reach specific intracellular targets.

Disclosure of Invention

The present invention provides polymersomes comprising: (a) a protein-rejecting polymer; and (b) a pH sensitive biodegradable succinate polymer comprising pendant groups having a pKa of 4 to 7.

Polymersomes (vesicles formed from amphiphilic block copolymers) are the polymeric equivalent of liposomes. Due to the macromolecular nature of polymersomes, polymersomes are known to be more robust and stable than their lipid counterparts. In addition, the macromolecular nature of polymersomes also allows for very efficient modulation of membrane thickness. pH sensitive polymersomes have been previously developed and shown to be capable of delivering certain types of molecules to the cytosol of cells.

It has now been found that polymersomes comprising components (a) and (b) provide copolymers that are capable of self-assembling into robust polymersomes at physiological pH, which polymersomes are subsequently capable of breaking down into resorbable materials at mildly acidic intracellular pH (endosomes).

It has been found that components (a) and (b) within the polymer cells act synergistically. Component (a) is protein-repulsive and can selectively target receptors expressed by disease cells. Component (b) is a biodegradable polymer that releases molecules such as fumarates, thiosuccinate derivatives with or without t-butyl groups and imidazoles by in situ hydrolysis and/or enzymatic degradation (e.g., by lipases or esterases). The ability of the polymersomes of the invention to bind modulated intrinsic pH sensitivity significantly enhances intracellular drug delivery while also avoiding lysosomal degradation of the payload.

The present invention also provides a pharmaceutical composition comprising: a plurality of polymersomes of the invention; and one or more pharmaceutically acceptable excipients or diluents.

Drawings

Fig. 1 shows a transmission electron microscope image (50 nm scale) of PMPC-PPITS polymersomes as further described in the examples section;

FIG. 2 shows NMR characterization of PMPC-PPITS as further described in the examples section (peaks at 8.7 and 7.4 correspond to imidazole ring protons);

figure 3 shows the pH titration of PMPC-PPITS as further described in the examples section. The polymer was dissolved with 0.5N HCl at acidic pH. To the solution was slowly added 0.5N NaOH to pH 12.

Detailed Description

Polymer vesicles (Polymersome)

The polymersome of the present invention comprises: (a) a protein-rejecting polymer; and (b) a pH sensitive biodegradable succinate polymer comprising pendant groups having a pKa of 4 to 7.

Polymersomes are synthetic vesicles formed from amphiphilic block copolymers. In the past 15 years, polymersomes have received extensive research interest as multifunctional carriers due to their colloidal stability, tunable membrane properties, and ability to encapsulate or integrate other molecules (a representative review article, see J Control Release 2012161 (2)473-83, the contents of which are incorporated herein by reference in their entirety).

The polymersomes used in the present invention are typically self-assembled structures. The polymersomes typically comprise amphiphilic block copolymers. The amphiphilic block copolymer comprises a hydrophilic block and a hydrophobic block. In the polymersome of the present invention, the hydrophilic block generally comprises: a protein-rejecting polymer (a), typically comprising pendant phosphorylcholine groups; a hydrophobic block (b) which is a pH sensitive biodegradable succinate polymer comprising pendant groups with a pKa of 4 to 7, such as a polypropylene ((((1H-imidazol-5-yl) acetamido) -3-oxopropyl) thio) succinate (PPITS) polymer or derivative thereof.

Such polymersomes are capable of mimicking biological phospholipids. These polymers have much higher molecular weights than naturally occurring phospholipid-based surfactants, so they can assemble into more entangled membranes (j.am. chem. soc.2005,127,8757, the contents of which are incorporated herein by reference in their entirety), providing final structures with improved mechanical properties and colloidal stability. Furthermore, the flexible nature of the copolymer synthesis allows for the application of different compositions and functionalities over a wide range of molecular weights and hence film thicknesses. Thus, the use of these block copolymers as delivery vehicles provides significant advantages.

The polymersomes are typically substantially spherical. Polymersomes typically comprise a bilayer membrane. Bilayers are typically formed from two layers of amphipathic molecules that align to form a closed core, with the hydrophilic head group facing the exterior of the core and vesicle, and the hydrophilic tail group forming the interior of the membrane.

Typical (maximum) diameters of polymersomes are in the range of 50nm to 5000 nm. More typically, the diameter is in the range of 50nm to 1000 nm. Polymersomes with diameters in this range are commonly referred to as "nano-polymersomes" or "nanovesicles". The shape of the nano-polymersomes is preferably substantially spherical. Typically, the number average diameter of the nano-polymersomes vesicles is less than 300nm, preferably less than 250nm, most preferably less than 200nm or 150 nm. The thickness of the bilayer is typically from 2nm to 50nm, more typically from 5nm to 20 nm. These dimensions can be measured conventionally, for example, by using Transmission Electron Microscopy (TEM) and/or Small Angle X-ray Scattering (SAXS) (see, e.g., j.am. chem. soc. 12787572005, the contents of which are incorporated herein by reference in their entirety).

In aqueous solutions, there is usually an equilibrium between different types of structures, for example between polymersomes and micelles. Preferably at least 80 wt%, more preferably at least 90 wt% or 95 wt%, most preferably all of the structures in solution are present in the form of polymersomes. This can be achieved using the methods outlined herein.

After administration to a subject, polymersomes typically release, for example, fumarate and propylene glycol, thiosuccinate derivatives with or without a tert-butyl group, and imidazole in vivo. Typically, the polymersomes dissociate and, due to in situ hydrolysis and/or enzymatic degradation of polymer (b) (e.g., by lipases or esterases), fumarates and propylene glycol, thiosuccinate derivatives with or without tert-butyl groups, and imidazoles are released. When the fumarate backbone of the starting material is not fully saturated during functionalization (linking of pendant groups), then the polymersome releases the fumarate upon dissociation.

In a preferred embodiment, polymer (b) is or comprises polypropylene (((((1H-imidazol-5-yl) acetamido) -3-oxopropyl) thio) succinate or a derivative thereof. More preferably, polymer (b) is or comprises poly [ propene 2- ((2- (2- (1H-imidazol-5-yl) acetamido) -3- (tert-butoxy) -3-oxopropyl) thio) succinate ].

Poly [ propene (1H-imidazol-5-yl) acetamido) -3-oxopropyl) thio) succinate ] polymers or derivatives thereof, for example poly [ propene 2- ((2- (2- (1H-imidazol-5-yl) acetamido) -3- (tert-butoxy) -3-oxopropyl) thio) succinate ] are examples of polymers which hydrolyse to release fumarates, propylene glycol, thiosuccinate derivatives with or without tert-butyl groups and imidazoles. When the fumarate backbone of the starting material is not fully saturated during functionalization (linking of pendant groups), then the polymersome releases the fumarate upon dissociation and subsequent degradation.

If the polymersome comprises a drug encapsulated in the polymersome, the polymersome is also capable of releasing the drug in vivo upon administration to a subject. Also, often the polymersomes dissociate, releasing the drug.

As already explained, component (a) of the polymersome is a protein-rejecting polymer that, once injected, extends the circulating half-life of the nanoparticle. Component (b) is pH sensitive and releases e.g. fumarate and propylene glycol, thiosuccinate derivatives with or without tert-butyl groups and imidazole, typically by in situ hydrolysis and/or enzymatic degradation (e.g. by lipases or esterases). Thus, after the polymersome has been internalized within the cell, the encapsulated drug (if any) is released.

Dissociation of polymersomes can be promoted by a variety of mechanisms, but is typically promoted by hydrolysis of the hydrophobic block copolymer. Typically, ester linkages in the polymer backbone are hydrolyzed by in situ hydrolysis and/or enzymatic degradation (e.g., by lipases or esterases) releasing, for example, fumarate, propylene glycol (a metabolite that is rapidly metabolized by the liver (half-life 2 hours) to form lactate, acetate, and pyruvate), sulfosuccinate derivatives with or without a t-butyl group, and imidazole. This process is catalyzed by enzymes called esterases and acidic pH. Both cases are typical of the endolysosomal compartment, into which polymersomes migrate under phagocytosis.

Typically, polymer (b) comprises imidazole groups (typically as pendant groups), typically having a pKa in the range of from 6.4 to 7. Endocytosis involves a local pH drop experienced by the polymersome from about pH 7.4 to about pH 5-6. This pH drop is usually sufficient to trigger the disintegration of the polymersome.

pKa means the pH at which half of the pendant group is ionized. pKa can be determined by a variety of methods including pH titration followed by potentiometric titration, UV spectroscopy and Dynamic Light Scattering (DLS). The appropriate method should be chosen to measure pKa according to the copolymer being analyzed and its solubility in the test medium.

DLS is a particularly preferred method for measuring pKa. From copolymers (e.g., PMPC) in water as noted in J.Am.chem.Soc 200512717982-17983 (the contents of which are incorporated herein by reference in their entirety)₂₅-b-PDPA₂₀Copolymer) changes with pH. At a certain pH, the signal increases rapidly as the copolymer undergoes a transition from dissociating on the molecule to associating. The pKa was taken as the pH at the midpoint of this rapid increase. These experiments are further described in Biomacromolecules 2006,7,817-828, the contents of which are incorporated herein by reference in their entirety. In this reference, experiments were performed on micelles of block copolymers, but this technique can also be applied when the phase transition involves vesicle formation of polymersomes.

The pKa of the groups in the polymer is determined based on the polymer system (and is not assumed to be the same as the pKa of a similar moiety in a non-polymer system).

Typically, the pendant groups of the polymer (b) of the polymersome comprise cationizable moieties. Cationizable moieties are, for example, primary, secondary or tertiary amines or imidazole groups, which can be protonated at pH values in the range of less than 3 to 6.9. Alternatively, the group may be a phosphine. These cationizable moieties may be inherently present within the monomer unit or alternatively polymerized after conjugation by using a suitable conjugation strategy (e.g., thiol-ene addition reaction to the double bond of the fumarate backbone). Preferably, the cationizable moiety is imidazole.

Preferably, the pendant group has a pKa in the range of 4.0 to 6.9, more preferably in the range of 5.5 to 6.9. The polymersomes are accordingly capable of dissociating in this pH range.

The imidazole groups on the PPITS polymer or derivatives thereof exhibit pKa in the desired pH range for avoiding lysosomal degradation. However, the pKa value can also be further adjusted by modifying the position and nature of the different substituents around the imidazole ring. PH sensitivity is introduced by exploiting the double bond present in poly (propyl fumarate) (PPF) or that occurs after isomerization of poly (propyl maleate) (PPM), and conjugating t-butylcysteine by thiol-ene addition reaction on the original PPM. This is merely one example of the possible conjugation chemistries (e.g., diels-alder aldol condensation reactions) and conjugation motifs (e.g., amino acids, DNA, oligopeptides, intact proteins, drugs, etc.) that involve double bonds within the polymer structure. The double bonds within the polymer structure may be fully or partially functionalized. When the double bonds within the polymer structure are partially functionalized, a polymer is formed that contains both saturated (e.g., succinate) and unsaturated (e.g., fumarate) monomer units in the backbone. The cysteine N-terminus can be conjugated to imidazole-or methylimidazole-containing acids, including 1H-imidazole-1-acetic acid (pKa 6.8), 4-imidazoleacetic acid (pKa 6.6), dimethylimidazoleacetic acid (pKa 6.4), and methylimidazolic acid (pKa 6.2). This conjugation reaction can be carried out before or after the thiol-ene addition reaction by NHS chemistry or other types of conjugation reactions known to those skilled in the art. Similarly, the same cysteine derivative protected on the amino group (with a t-butoxycarbonyl moiety) and having a free carboxyl group may also be conjugated with the same imidazole moiety (but derived from a histamine derivative). In all cases, all of these structures are metabolite moieties of histidine metabolism and/or histamine catabolism, and they will adjust the final polymer pKa from 6.2 to 6.8. Furthermore, the number of functionalities can be adjusted by adding saturated monomers (e.g., succinic acid (in the case of polycondensation) or succinic anhydride (in the case of ring-opening copolymerization or ROOP)) to the polymerization mixture, thereby creating a range of saturated and unsaturated polyesters.

Preferably, the degree of polymerization of polymer (b) (the hydrophobic block of the polymersome) is at least 5, more preferably at least 10. Preferably, the degree of polymerization is no greater than 250, and even more preferably, no greater than 200. Preferably, the ratio of the degree of polymerization of the protein-repelling polymer (a) (hydrophilic block) to the polymer (b) (hydrophobic block) is in the range of 1: 2.5 to 1: within 8. All of these limitations promote the formation of polymersomes, rather than micelles.

The protein-rejecting polymer (a) may be based on polycondensates, for example polyesters, polyamides, polyanhydrides, polyurethanes, polyethers (including polyalkylene glycols, especially PEG), polyimines, polypeptides, polyureas, polyacetals and polysaccharides, but is preferably based on controlled radical polymerisation of ethylenically unsaturated monomers. The protein-rejecting polymer preferably comprises phosphorylcholine as a pendant group, in which case the pendant group may be present in the monomer and remain unchanged during the polymerization process. Alternatively, the pendant group of the monomer may be derivatized after polymerization to convert it to a phosphorylcholine group.

In one presently preferred embodiment, the protein-excluding polymer (a) is formed from ethylenically unsaturated monomers. Non-limiting examples of suitable ethylenically unsaturated monomers have the general formula (IV)

YBX(IV)，

Wherein:

y is an ethylenically unsaturated group selected from: h₂C＝CR-CO-A-、H₂C＝CR-C₆H₄-A¹-、H₂C＝CR-CH₂-A²-、R²O-CO-CR＝CR-CO-O-、RCH＝CH-CO-O-、RCH＝C(COOR²)CH₂-CO-O-、

A is-O-or NR¹；

A¹Is selected from the group consisting of a bond, (CH)₂)_LA²And (CH)₂)_LSO₃ ^-Wherein L is 1 to 12;

A²selected from the group consisting of a bond, -O-CO-, -CO-O, -CO-NR¹-、-NR¹-CO-、-O-CO-NR¹-and-NR¹-CO-O-。

R is hydrogen or C_1-4An alkyl group;

R¹is hydrogen, C_1-4Alkyl or BX;

R²is hydrogen or C_1-4An alkyl group;

b is a bond, or a linear or branched alkanediyl, alkyleneoxaalkylene, or alkylene (oligooxaalkylene) group optionally containing one or more fluoro substituents; and

x is a phosphorylcholine group, i.e. a group of the formula

In the monomers of the formula (IV), the ethylenically unsaturated group Y is preferably H₂C ═ CR-CO-A-. Such acrylic moieties are preferably methacrylic acid, i.e. wherein R is methyl; or acrylic acid, wherein R is hydrogen. Although the compound may be a (meth) acrylamide-based compound (wherein A is NR)¹) In this case R¹Preferably hydrogen, or less preferably methyl, and most preferably the compound is an ester, i.e. wherein a is O.

In the monomers of formula (IV), in particular where Y is the preferred (alkyl) acrylic group, B is most preferably an alkanediyl group. When some of the hydrogen atoms of the group may be substituted with fluorine atoms, it is preferred that B is an unsubstituted alkanediyl group, most preferably a straight chain group having from 2 to 6 carbon atoms.

A particularly preferred monomer of formula (IV) is 2-methacryloyloxyethyl-phosphorylcholine (MPC). Mixtures of the individual monomers of the above general formula can be used, as can mixtures with other monomers, for example with other hydrophilic monomers.

Preferably, the protein-rejecting polymer (a) is an acrylate polymer. Acrylate polymers are polymers formed from one or more acrylate monomers. The acrylate monomer is a monomer comprising an acrylate group (i.e., CH) that may be substituted or unsubstituted₂CH-COO —) and thus includes methacrylate monomers and other such monomers covered by formula (IV). Preferably, the protein-excluding polymer (a) is a polymer comprising pendant phosphorylcholine groups. Particularly preferably, the protein-excluding polymer (a) is an acrylate polymer comprising pendant phosphorylcholine groups. For example, the protein-rejecting polymer (a) may comprise units of formula (I)

Wherein R is^pIs hydrogen or methyl and A^pA radical of the formula (I

Wherein X^pIs C_1-6An alkylene group. In some cases, X^pMay be one or more amino acids, such as a peptide, dipeptide or oligopeptide.

Preferably, X^pIs C_2-6An alkylene group. Preferably, R^pIs methyl. In a particularly preferred embodiment, the phosphorylcholine polymer is poly (2-methacryloyloxy) ethyl phosphorylcholine.

Alternatively, the protein-rejecting polymer (a) may be a polypeptide, such as those described in Yakovlev and Deming, J.Am.chem.Soc.,2015,137(12), pp 4078-. For example, the protein-rejecting polymer (a) may comprise poly (L-phosphorylcholine serine) or poly (L-phosphorylcholine homoserine).

Alternatively, the protein-excluding polymer (a) may be coupled with polymers produced from epoxides, anhydrides, lactide and/or CO2 to produce polyesters and polycarbonates. Such as those described in Shyeni and Williams, chem.commun.,2015,51,6459, which is incorporated herein by reference in its entirety.

Typically, the pendant group on the pH sensitive biodegradable succinate polymer (b) has a pKa of 5.5 to 7, preferably 6.2 to 7.

Typically, the pH sensitive biodegradable succinate polymer (b) comprises succinate monomer units in the polymer backbone. The pH sensitive biodegradable polymer (b) may comprise a mixture of succinate and fumarate monomer units in the polymer backbone. As will be readily understood by the skilled person, such biodegradable polymers (b) may be obtained, for example, when starting from polymers comprising fumarate monomer units in the polymer backbone and when the functionalization to add pendant groups to these monomer units (which in each case results in the conversion of the fumarate monomer units to succinate monomer units having pendant groups) is partial but incomplete (i.e. not all fumarate monomer units in the reagent polymer are converted to succinate monomer units having pendant groups). For the avoidance of doubt, a "pH sensitive biodegradable succinate polymer comprising a pendant group having a pKa of 4 to 7" as defined herein includes: (i) a polymer comprised of succinate monomer units comprising a pendant group; and (ii) a polymer comprising both succinate and fumarate monomer units comprising a pendant group.

Preferably, the pH sensitive biodegradable succinate polymer (b) further comprises propylene monomers in the polymer backbone.

Typically, the pendant group on the pH sensitive biodegradable succinate polymer (b) comprises an imidazole moiety.

The imidazole moiety may be an imidazole or a substituted imidazole, for example an alkyl substituted imidazole. Imidazoles may be monosubstituted or polysubstituted. Preferably, the alkyl substituent is methyl or ethyl.

Typically, the imidazole moiety is imidazole, methylimidazole or dimethylimidazole.

Preferred pH sensitive biodegradable fumarate polymers (b) include those comprising poly (propylene (1H-imidazol-5-yl) acetamido) -3-oxopropyl) thio) succinate or a derivative thereof. Most preferably, polymer (b) is or comprises poly [ propene 2- ((2- (2- (1H-imidazol-5-yl) acetamido) -3- (tert-butoxy) -3-oxopropyl) thio) succinate ].

During degradation, for example, hydrolysis of the polymer (b) molecule, may release, for example, fumarate and propylene glycol (a metabolite that is rapidly metabolized by the liver (half-life of 2 hours) to form lactate, acetate and pyruvate), thiosuccinate derivatives with or without a tert-butyl group, and imidazole, by in situ hydrolysis and/or enzymatic degradation of the polymersome (e.g., by lipases or esterases).

Preferred polymers (b) comprise succinate and propylene monomers. The advantage of this polymer is that the by-product of its hydrolysis is propylene glycol, which is generally considered a safe material and has been approved for several clinical applications. Propylene glycol is metabolized by the liver to form lactate, acetate, and pyruvate. The unmetabolized drug is excreted mainly in the urine in the form of glucuronide conjugates, approximately 12% to 45% in an invariable manner.

More generally, one or both of polymers (a) and (b) may include comonomers, for example to provide functionality, control of hydrophobicity, control of pH sensitivity, pKa or pKb (as the case may be), control of temperature sensitivity, or as a general diluent. For example, comonomers that provide functionality may be used to provide conjugation of the pendant groups after polymerization and/or polymersome formation, to provide targeting moieties, or to provide conjugation between the bioactive molecule and the polymer. Alternatively, the functional groups may allow cross-linking of the polymer after formation of the polymersome to impart increased stability to the polymersome structure. Examples of suitable comonomers are compounds of the formula (VI)

Wherein

R¹⁸Selected from hydrogen, halogen, C_1-4Alkyl and group COOR²²Wherein R is²²Is hydrogen or C_1-4An alkyl group;

R¹⁹selected from hydrogen, halogen and C_1-4An alkyl group;

R²⁰selected from hydrogen, halogen, C_1-4Alkyl and group COOR²²Provided that R is¹⁸And R²⁰Not all being COOR²²(ii) a And

R²¹is composed of_1-10Alkyl radical, C_1-20Alkoxycarbonyl, mono (C)_1-10Alkyl) aminocarbonyl or di (C)_1-10Alkyl) aminocarbonyl, C_6-20Aryl (including alkaryl), C_7-20Aralkyl radical, C_6-20Aryloxycarbonyl group, C_1-20Aralkoxycarbonyl radical, C_6-20Arylaminocarbonyl group,C_7-20Aralkyl-amino, hydroxy or C_2-10Acyloxy groups, any of which may have one or more substituents selected from: halogen atoms, alkoxy, lower alkoxy, aryloxy, acyloxy, amido, amine (including monoalkylamino and dialkylamino groups where the alkyl group may be substituted and trialkylammonium), carboxy, sulfonyl, phosphoryl, phosphino (including monoalkylphosphine and dialkylphosphine and trialkylphosphonium), zwitterions, hydroxyl groups, vinyloxycarbonyl and other vinyl or allyl substituents, and reactive silyl or siloxy groups (e.g. trialkoxysilyl groups); or R²¹And R²⁰Or R²¹And R¹⁹May be taken together to form-CONR²³CO, wherein R²³Is C_1-20An alkyl group.

Preference is given to the radical R¹⁸、R¹⁹、R²⁰And R²¹Is a halogen or, more preferably, a hydrogen atom. Preferably, R¹⁸And R¹⁹Are all hydrogen atoms. Particularly preferably, the compound of the formula X is a styrene or acrylic compound. In the styrene compound, R²¹Represents an aryl group, in particular a substituted aryl group, wherein the substituent is an aminoalkyl group, a carboxylate group or a sulfonate group. When the comonomer is an acrylic compound, R²¹Is an alkoxycarbonyl, alkylaminocarbonyl or aryloxycarbonyl group. Most preferably, in such compounds, R²¹Is C optionally substituted by hydroxy_1-20-an alkoxycarbonyl group. The acrylic compound is usually methacrylic acid, in which case R²⁰Is methyl.

Preferably, the comonomer is a non-ionic comonomer, such as (alkyl) acrylic acid C_1-24Alkyl esters or C_1-24Alkyl (alkyl) -acrylamides, monohydroxy (alkyl) acrylates-C_1-6Alkyl esters or dihydroxy-C (alk) acrylates_1-6Alkyl esters or monohydroxy-C_1-6Alkyl (alkyl) -acrylamides or dihydroxy-C_1-6Alkyl (alkyl) -acrylamides, (alkyl) acrylic acid oligo (C)_2-3Alkoxy) C_2-18Alkyl esters or oligo (C)_2-3Alkoxy) C_2-18Alkyl (alkyl) -acrylamides, styrene, vinyl acetate or N-vinyl lactams.

For optimal nanovesicle formation, the block copolymer should have a controlled molecular weight. Each block preferably has a molecular weight that is controlled within a narrow band, i.e., has a narrow polydispersity. The polydispersity of the molecular weight should, for example, preferably be less than 2.0, more preferably less than 1.5, for example in the range from 1.1 to 1.4.

In a preferred embodiment of the invention, the polymeric vesicles comprise poly (2-methacryloyloxy) ethyl phosphorylcholine and polypropylene ((((1H-imidazol-5-yl) acetamido) -3-oxopropyl) thio) succinate (PPITS) or a derivative thereof. In this preferred embodiment, the polymersome typically comprises a block copolymer, wherein one block comprises poly (2-methacryloyloxy) ethyl phosphorylcholine and the other block comprises polypropylene ((((1H-imidazol-5-yl) acetamido) -3-oxopropyl) thio) succinate (PPITS) or a derivative thereof. In an exemplary embodiment, the polymersome comprises a block copolymer comprising a block of poly (2-methacryloyloxy) ethyl phosphorylcholine and a block of polypropylene ((((1H-imidazol-5-yl) acetamido) -3-oxopropyl) thio) succinate (PPITS) or a derivative thereof.

In a particularly preferred embodiment of the invention, the polymeric vesicles comprise poly (2-methacryloyloxy) ethylphosphorylcholine and poly [ propene 2- ((2- (2- (1H-imidazol-5-yl) acetamido) -3- (tert-butoxy) -3-oxopropyl) thio) succinate ]. In this particularly preferred embodiment, the polymersome typically comprises a block copolymer, wherein one block comprises poly (2-methacryloyloxy) ethylphosphorylcholine and the other block comprises poly [ propene 2- ((2- (2- (1H-imidazol-5-yl) acetamido) -3- (tert-butoxy) -3-oxopropyl) thio) succinate ]. In an exemplary embodiment, the polymersome comprises a block copolymer comprising a block of poly (2-methacryloyloxy) ethylphosphorylcholine and a block of poly [ propene 2- ((2- (2- (1H-imidazol-5-yl) acetamido) -3- (tert-butoxy) -3-oxopropyl) thio) succinate ].

The block copolymer may be a simple A-B block copolymer or may also be an A-B-A or B-A-B block linear triblock copolymer or (A)₂B or A (B)₂Star copolymer (wherein A is phosphorylcholine polymer containing block, B is fumarate polymer containing block). The block copolymer may also be an A-B-C, A-C-B or B-A-C linear triblock copolymer or an ABC star copolymer, where C is a different type of block. The C block may, for example, include functional, e.g., crosslinking or ionic groups, to allow the copolymer to react, e.g., in the novel composition. Crosslinking reactions, particularly of copolymers of the A-C-B type, can confer useful stability to the polymersome. Crosslinking can be covalent in nature and can sometimes be electrostatic. Crosslinking may involve the addition of a separate reagent to attach the functional group, for example using a bifunctional alkylating agent to attach two amino groups. Alternatively, the block copolymer may be a star-shaped molecule with a hydrophilic or hydrophobic core, or may be a comb-like polymer with a hydrophilic backbone (blocks) and hydrophobic side chains, or vice versa. Such polymers may be formed, for example, by random copolymerization of monounsaturated macromers with monomers.

Further details of suitable methods for polymerizing monomers can be found in WO 03/074090, the contents of which are incorporated herein by reference in their entirety.

Methods for polymerizing the monomers are living radical polymerization, functionalized NCA (N-carboxy anhydride) polymerization, polycondensation and Ring Opening Polymerization (ROP), and copolymerization (rock). In all cases, effective post-polymerization modification can be carried out by using appropriate monomers with appropriate motifs which are suitably protected in the case of poor compatibility with the polymerization process, as is well known to the person skilled in the art. Living radical polymerization has been found to provide polymers of monomers having a polydispersity (molecular weight) of less than 1.5 as judged by gel permeation chromatography. The polydispersity of the or each block is preferably in the range of from 1.2 to 1.4. The polymersomes can be loaded using a pH-varying system, electroporation or membrane hydration. During a pH-changing system, the polymer is dispersed in the aqueous liquid in an ionized form, wherein the polymer is dissolved at a relatively high concentration without forming polymersomes. The pH is then changed so that some or all of the ionized groups are deprotonated so that they are in a non-ionic form. At the second pH, the hydrophobicity of the block increases and polymersomes form spontaneously.

The method of forming polymersomes encapsulating a drug in the core may involve the steps of: (i) dispersing an amphiphilic copolymer in an organic medium; (ii) (ii) adding a phosphate buffer to the composition formed in step (i) by means of a syringe pump; (iii) adding the drug to the aqueous phase if the drug is hydrophilic or to the organic phase if the drug is hydrophobic; and (iv) at the end of the addition, excess PBS is added to the formulation and residual organic solvent is removed by dialysis against sterile phosphate buffer using a dialysis tube.

The method preferably comprises a preliminary step in which the amphiphilic copolymer is dispersed in an organic solvent in a reaction vessel, and then a phosphate buffer is added with a syringe pump to form polymersomes.

Step (ii), the solvent is changed and the pH is increased above the pKa value of the poly [ propene (1H-imidazol-5-yl) acetamido) -3-oxopropyl) thio) succinate ] (PPITS) group.

Another method of forming polymersomes encapsulating a drug in the core may involve the steps of: (i) dispersing the amphiphilic copolymer and the hydrophobic and/or amphiphilic drug (when needed) in an organic solvent in a reaction vessel; (ii) evaporating the solvent to form a film inside the reaction vessel; and (iii) rehydrating the membrane with an aqueous solution optionally comprising a solubilized hydrophilic drug.

In more detail, polymersomes are typically prepared by dissolving the copolymer in an organic solvent (e.g., a 2: 1 chloroform: methanol mixture) in a glass container. If a hydrophobic or amphiphilic drug is to be encapsulated, it may be added with the copolymer. The solvent can be evaporated under vacuum to deposit a copolymeric film on the vessel walls. The membrane is then rehydrated with an aqueous solution, for example using a phosphate buffer. If a hydrophilic drug is to be encapsulated, it may be included in an aqueous solution. The pH of the resulting suspension was lowered to a pH of about 2 to dissolve the membrane, and then the pH was slowly raised to about 6. The polymer hydrates at neutral pH allowing the drug to encapsulate. The dispersion may then be sonicated and extruded, for example using a bench top extruder. The packaging efficiency can be calculated using UV spectroscopy and HPLC chromatography using techniques well known in the art. Another method of forming polymersomes with encapsulated drugs may involve simple electroporation of the drug and polymersome in water. For example, the drug may be contacted in solid form with an aqueous dispersion of polymersomes, and an electric field applied to allow pores to form in the membrane of the polymersomes. Soluble drug molecules can then enter the polymersome through the pores. This is followed by a membrane self-repair process, in which the active molecules are continuously embedded inside the polymersome.

Alternatively, a drug dissolved in an organic solvent may be emulsified into an aqueous dispersion of polymersomes, thereby incorporating the solvent and drug into the core of the vesicles, followed by evaporation of the solvent from the system.

The polymersome used in the present invention may be formed of two or more different block copolymers. In this embodiment, in the method of forming polymersomes, a mixture of two or more block copolymers is used.

For example, in the above method, 0.01% to 10% (w/w) of the drug is mixed with the copolymer.

Encapsulated drug

The polymersome of the present invention may comprise a drug encapsulated within the polymersome.

It will be appreciated that the polymersomes of the invention are capable of being degraded in vivo by in situ hydrolysis and/or enzymatic degradation (e.g. by lipases or esterases) into resorbable materials such as fumarates, thiosuccinate derivatives with or without a tert-butyl group and imidazoles. Polymersomes may also provide drug encapsulation. For the avoidance of doubt, it is also possible to encapsulate a plurality of different drugs in a single polymersome, or to provide a plurality of polymersome each containing a particular encapsulated drug.

Non-limiting examples of specific drugs that can be encapsulated include: 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, ginsenoside-Rd, granulocyte colony stimulating factor, Tat-NR2B9c, magnesium, albumin, paracetamol, aspirin, choline, and magnesium salicylate, 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, and baicalin), Corticosteroid, alemtuzumab, interferon beta-1 b, fingolimod, glatiramer acetate, natalizumab, plegridy, pegylated interferon beta-1 a, teriflunomide, methotrexate, sulfasalazine, leflunomide, adalimumab, etanercept, golimumab, ustekinum, uitlizumab, azathioprine, cyclosporine, infliximab, golimumab, cetuximab, hydroxychloroquine, methotrexate, azathioprine, mycophenolate mofetil, vitamin A acid, hydroxyurea, isotretinoin, mycophenolate mofetil, sulfasalazine, 6-thioguanine, calcipotriol, calcitriol, tacalcitol, tacrolimus, pimecrolimus, anthratriphenol, engamostine, bendamustine, carmustine, chlorambucil, cyclophosphamide, dacarbazine, ifosfamide, melphalan, cabazine, valcabazitaxel, leuprolide, lexolone, lefraxidine, lexapride, lefraxidine, leflunomide, lexaglipium, lexapride, lexapri, Streptozotocin, temozolomide, capecitabine, 5-fluorouracil, fludarabine, gemcitabine, 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, bentuximab, cetuximab, crizotinib, denosumab, erlotinib, gefitinib, imatinib, interferon, pririma, lapatinib, panitumumab, pertuzumab, rituximab, sunitinib, sorafenib, trastuzumab, temsirolimumab, trelimumab, vemurafenib, Clodronate, ibandronic acid, disodium pamidronate, zoledronic acid, anastrozole, abiraterone, bexarotene, bicalutamide, buserelin, cyproterone, degarelix, exemestane, flutamide, folinic acid, fulvestrant, goserelin, lanreotide, lenalidomide, letrozole, leuprorelin, medroxyprogesterone, megestrol, mesna, octreotide, diethylstilbestrol, tamoxifen, and thalidomide. For the avoidance of doubt, the encapsulated drug may also be a fumarate (fumarate ester) to supplement the action of the fumarate ester resulting from degradation of the polymer in vivo.

Pharmaceutical composition

The polymersomes of the present invention may be formulated into pharmaceutical compositions using conventional techniques known in the art. For example, pharmaceutical compositions have been used to formulate polymersomes or drug-containing liposomes.

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

Typical pH of aqueous pharmaceutical compositions is 7.0 to 7.6, preferably 7.2 to 7.4. Pharmaceutically acceptable buffers may be used to achieve the desired pH. The pharmaceutical compositions may be in the form of a sterile, aqueous, isotonic saline solution.

Typically, the pharmaceutical composition is an injectable composition, e.g. the pharmaceutical composition is suitable for intravenous delivery, e.g. the pharmaceutical composition is suitable for infusion.

Examples

Synthetic vesicles are made using amphiphilic copolymers made from poly [ propylene 2- ((2- (2- (1H-imidazol-5-yl) acetamido) -3- (tert-butoxy) -3-oxopropyl) thio) succinate ] (a PPITS derivative; hereinafter referred to as "PPITS") as the hydrophobic block and poly (2- (methacryloyloxy) ethylphosphorylcholine) (PMPC) as the hydrophilic block.

A new synthetic route was developed to produce diblock PMPC-PPITS copolymers and its assembly into vesicles was investigated. The results show that PMPC-PPITS copolymers degrade by hydrolysis and/or enzymatic degradation in situ (e.g., by lipase or esterase) to fumarate (due to incomplete functionalization of polyfumarate feedstock) and propylene glycol (a metabolite that is rapidly metabolized by the liver (half-life 2 hours) to form lactate, acetate and pyruvate), thiosuccinate derivatives with or without a tert-butyl group, and imidazole. It was thus demonstrated that PMPC-PPITS formed polymersomes that degraded into resorbable materials.

Production of polymersomes

Synthesis of PMPC-PPF

Examples of the Ring-opening copolymerization step (Synthesis of PPM)

0.020mmol SalenCrCl (or other suitable catalyst; e.g., zinc diphenyl) and 4.0mmol maleic anhydride were placed in a vial equipped with a stir bar under a controlled dry atmosphere. The appropriate solvent (toluene, 0.50mL) was added followed by 4.0mmol of propylene oxide (epoxide). The sealed vial was placed in an aluminum heating block preheated to the desired temperature (45 ℃). After the reaction became viscous, use¹H-NMR spectroscopic analysis was performed to determine the monomer conversion. The viscous reaction mixture was then dissolved in a minimum amount of dichloromethane and precipitated into an excess of hexane. This process is repeated (in the case of PPM, using diethyl ether as non-solvent) until all residual monomers are removed. After the polymer was washed, the material was redissolved in THF and passed through an ion exchange column to remove the metal catalyst. After this further purification step, the material was collected and dried in vacuo.

Isomerization step examples (from PPM to PPF)

For one-pot methodAt the end of the polymerization reaction 0.1 equivalent of diethylamine was added directly to the polymer mixture and the polymer was dissolved in CDCl₃In (1). For the two-step process, the separated polymer sample was dissolved in CDCl₃Then 0.1 equivalent of diethylamine was added. For both steps, the solution is stirred and passed¹H-NMR spectroscopy was used to check the progress of the isomerization. After completion of the reaction, all volatiles were removed under vacuum. The polymer is subsequently redissolved in CH₂Cl₂And precipitated into hexane. The polymer was then dried in vacuo and the isomerization was confirmed to be complete by 1H NMR spectroscopy.

Polycondensation reaction example (direct Synthesis of PPF)

The polypropylene fumarate is ZnCl₂Is a catalyst and is synthesized by the ester exchange polycondensation reaction of diethyl fumarate and propylene glycol by using hydroquinone as a free radical inhibitor. In a typical two-step polymerization reaction, 14.98mL diethyl fumarate and 20mL propylene glycol were added to a two-necked round bottom flask and the reagents were stirred under a stream of nitrogen for 30 min. Then, 200mg of ZnCl was added₂And 124mg of hydroquinone were added to the reaction mixture and the reagents were heated to 150 ℃ overnight under a stream of nitrogen. Then, the nitrogen flow was stopped and the vacuum was applied for 6 hours while stirring at 170 ℃. By comparison with CHCl₃、CHCl₃The product was purified by exhaustive dialysis (3.5 kDa cut-off) of/MeOH followed by MeOH (at least three changes of each solvent).

Example for the Synthesis of PPF ATRP macroinitiator

A solution of PPF (1 eq) and triethylamine (3 eq) in dry THF was cooled slightly in an ice-water bath. Then, a minimum amount of anhydrous THF solution of 2-bromoisobutyryl bromide (4 equivalents) was added dropwise to the reaction mixture. The solution was warmed to room temperature and stirred for 48 h. The mixture was poured into water and dialyzed extensively against water with a 1000kDa MWCO membrane, then freeze-dried.

Example of Synthesis of PMPC-PPF by ATRP (atom transfer radical polymerization)

PPF macroinitiator (1 equivalent) and MPC monomer (5 equivalents) were weighed into a round bottom flask and dissolved in 2: 1(v/v) in toluene/isopropanol. Then, 2 equivalents of N, N, N ', N' -pentamethyldiethylenetriamine were added and N was used under stirring₂The reaction mixture was purged for 1 hour. 2 equivalents of Cu (I) Br were then weighed and added under a stream of nitrogen, the reaction was sealed under a nitrogen atmosphere and kept stirring at 35 ℃ for 48 h. After 48h, the reaction was quenched with 2: 1(v/v) CHCl₃Diluted with MeOH and filtered over silica to remove oxidized catalyst. The product was concentrated by rotary evaporation and further concentrated by evaporation with respect to 2: 1v/v CHCl₃the/MeOH mixture (2 changes), MeOH (3 changes), then dialysis (3.5 KDa cut-off) against Milli-Q water (at least 4 changes) thoroughly. The product was recovered by freeze drying. The product is then isolated by freeze-drying and by¹And H-NMR characterization.

Example of Synthesis of PMPC-PPITS by ATRP

PPF-PMPC (1 eq) was dissolved in 2mL of 2: 1(v/v) CHCl₃In MeOH, and tert-butyl (2- (1H-imidazol-5-yl) acetyl) cysteine (3 equivalents) dissolved in 20mL MeOH was added. The reaction mixture was irradiated with a UV lamp at λ 365nm for 6h with stirring. The product was then purified by exhaustive dialysis (3.5 KDa cut-off) against MeOH (3 changes), then acidic Milli-Q water (pH 3, at least 2 changes), and finally neutral Milli-Q water (at least 4 changes). The product was recovered by freeze drying. The product is then isolated by freeze-drying and by¹H-NMR and GPC characterization.

Polymer vesicle preparation

Polymersomes are prepared by solvent inversion, which is a technique known in the art. Preparation was carried out under sterile conditions in a biosafety cabinet (KS15-Thermo Fisher Scientific). Before any manipulations, the polymer stock solution (0.5mL, 10mg/mL to 80mg/mL in 2: 1v/v DMF/MeOH) was filtered on a sterile 0.22 μm sterile filter. Then, 0.1M PBS (1.15mL) pH 7.4 was added at a flow rate of 1. mu.L/min to 100. mu.L/min by a syringe pump under vigorous stirring to form polymersomes. At the end of the addition, excess PBS (1.85mL) was added to the formulation and the residual organic solvent was removed by dialysis against 0.1M PBS at sterile pH 7.4 for 48h (4-6 changes/day) using a dialysis tube (MWCO ═ 1000) at 25 ℃. The resulting polymersome preparation was then further purified by sonication at 0 ℃ for 15 minutes and centrifugation at 1000g for 20 minutes. The concentration of the solution was assessed gravimetrically, while the size and shape of the polymersomes were characterized by DLS and TEM analysis.

The polymersomes were purified from aggregates and micelles by Gel Permeation Chromatography (GPC) using sepharose 4B as substrate. Dynamic Light Scattering (DLS) was used to assess the particle size distribution of polymersomes by means of a Malvern Zetasizer Nano ZS laser diffuser equipped with a He-Ne4 35633 nm laser. Polymersomes were diluted in filtered PBS in 1ml disposable cuvettes and experiments were performed with n-3 averages at a set angle of 173 °. The morphology of the polymersomes in filtered PBS was also evaluated using Transmission Electron Microscopy (TEM). The samples were mounted on a discharged carbon coated grid by dipping the grid into the polymer solution for 60 seconds, followed by staining with 0.75% (w/w) Phosphotungstic acid (PTA) for 5 seconds. The grid was then washed with PBS, dried under vacuum, and evaluated by JEOL microscopy using a voltage tension of 100 kV.

PMPC-PPITS degradation assay

Degradation studies were performed by incubating the polymersomes with a suitable mixture of enzymes (e.g., from porcine and/or human origin), which qualitatively and in terms of enzyme concentration mimics the conditions of exposure to endosomes. Characterization of degradation can be assessed by Field Flow Fractionation (FFF) and Dynamic Light Scattering (DLS), which allows tracking the transition from vesicles to micellar structures. In addition, GPC and HPLC analysis were also performed to assess the molecular weight changes produced by enzymatic digestion. The identity of the metabolites obtained after degradation was also confirmed by HPLC-MS.