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WO2017199023A1 - Polymersomes - Google Patents

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Publication number
WO2017199023A1
WO2017199023A1 PCT/GB2017/051374 GB2017051374W WO2017199023A1 WO 2017199023 A1 WO2017199023 A1 WO 2017199023A1 GB 2017051374 W GB2017051374 W GB 2017051374W WO 2017199023 A1 WO2017199023 A1 WO 2017199023A1
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Prior art keywords
polymersomes
tubular
cells
polymersome
composition
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Inventor
Giuseppe Battaglia
Loris Rizzello
Denis CECCHIN
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UCL Business Ltd
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UCL Business Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1273—Polymersomes; Liposomes with polymerisable or polymerised bilayer-forming substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to therapeutic polymersomes.
  • the compositions of the invention comprise a high proportion of tubular polymersomes. Methods of producing such compositions and therapeutic applications of such compositions are also described.
  • nanoparticles may represent a new tool for tailoring, and possibly probing, cellular pathways, because such nanomaterials coexist in the same nano-universe as the molecular machineries of cells, where nanoscale interactions take place.
  • nanotoxicology general toxicity of nanomaterials
  • Polymersomes (vesicles formed from amphiphilic block copolymers) are the polymeric equivalent of liposomes. They are known to be much more robust and stable than their lipid counterparts due to their macromolecular nature. In addition, their macromolecular nature also allows a very effective tuning of the membrane thickness. Polymersomes that are sensitive to pH have previously been developed and shown to be capable of delivering certain types of molecules to the cell cytosol. Selective targeting of tissues of therapeutic significance is also possible.
  • polymersome technologies include those where the polymersome incorporates an encapsulated active agent (the polymersome functioning as a delivery system for the active agent) and those where the polymersome itself constitutes an active agent, for example by way the substances formed when it degrades in vivo.
  • Polymersome technology has the potential to enhance selectivity and efficacy in treating a broad range of pathological conditions. Nonetheless, improvements in the potency and selectivity of polymersome therapeutics would be desirable. For example, further polymersome compositions having anti-cancer activity would be desirable.
  • polymersomes have a substantial impact on their interaction with biological materials, including on interactions that are of direct therapeutic significance, for example in cancer therapy.
  • Shaped polymersomes have been developed that demonstrate surprising and beneficial therapeutic properties.
  • a production method for obtaining such polymersomes has been found.
  • the resulting compositions have surprisingly been found to be capable of suppressing the replication activity of tumor cells.
  • the present invention provides a method of producing a tubular polymersome composition, the method comprising the steps of: providing a mixed polymersome composition comprising a mixture of tubular polymersomes and non-tubular polymersomes; subjecting the mixed polymersome composition to density gradient centrifugation in a centrifuge; and isolating a tubular polymersome composition from the centrifuge.
  • the present invention also provides a tubular polymersome composition, wherein the percentage of tubular polymersomes in the total population of polymersomes is at least 50%, as well as a tubular polymersome composition obtainable by the method of the present invention.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: the tubular polymersome composition of the present invention; and one or more pharmaceutically acceptable excipients or diluents.
  • the present invention still further provides a tubular polymersome composition of the present invention for use as a medicament.
  • the present invention also provides a tubular polymersome composition of the present invention for use in the treatment of cancer.
  • the present invention additionally provides a method of treating cancer, the method comprising administering a therapeutically effective amount of the tubular polymersome composition of the present invention to the subject.
  • the present invention further provides use of a tubular polymersome composition of the present invention in the manufacture of a medicament for use in the treatment of cancer.
  • Figure 1 shows TEM characterisations of spherical (A) and tubular (B) polymersomes, after a sucrose-based density gradient centrifugation, as explained in more detail in Example 1.
  • Figure 2 shows viability assays of FaDu, HeLa, and FIDF cells incubated with spherical polymersomes (A), and tubes (B), as explained in more detail in Example 1. 3 different polymersomes/tubes concentrations and 3 time points (24h, 48h, and 96h) were tested.
  • Control + 2.5% DMSO.
  • Figure 3 shows FtPLC-based quantification of polymersomes uptake in FaDu, HeLa, and FIDF cells: (A) Comparison between the total mass of up taken spheres and tubes in the different cells (value of polymer present in each cell culture); and (B) Normalisation of polymer/cell, during time; as explained in more detail in Example 1.
  • Figure 4 shows confocal investigations of uptake of spherical polymersomes (A) and tubes (B) in FaDu cells, as explained in more detail in Example 1.
  • Figure 5 shows a screenshot of the Matlab based software for the quantification of NDI and MM, as explained in more detail in Example 1.
  • the software is able to discriminate cells with one, two, or more nuclei, as well as bi-nucleated cells with a micronucleus.
  • Figure 6 shows: (A) NDI quantification for FaDu (left side), HeLa (centre), and HDF (right side), after incubation with spheres and tubes; and (B) Micronucleus assay (MM) for addressing the presence of DNA damage; as explained in more detail in Example 1.
  • Control + H202 20 uM. *: p ⁇ 0.05, **: p ⁇ 0.01, ***: p ⁇ 0.005.
  • Figure 7 shows a Trypan blue-based proliferation assay for FaDu, HeLa, and HDF cells, incubated with spherical polymersomes (upward pointing triangles), and tubes (downward pointing triangles), as explained in more detail in Example 1.
  • Control + 2.5% DMSO.
  • Figure 8 shows real Time qPCR for quantifying the expression of 10 different genes involved in replication activity (p21, p53), oxidative stress (SOD1, CAT), detoxification metabolism (CYPlAl, CYPIBI), Unfolded Protein Response - UPR (ATF4, ATF6), and general shock (HSP27, HSP70), as explained in more detail in Example 1.
  • White histograms genes expression for cells treated with spherical polymersomes; black histograms: genes expression for cells treated with tubes.
  • Figure 9 shows a caspase 3/7 assay for analysing the activation of extrinsic apoptosis, as explained in more detail in Example 1.
  • Figure 10 shows the combinatorial effect of nanoparticles shape and anticancer drug, as discussed in more detail in Example 2. FaDu, HeLa, and FIDF cells were incubated with free DOXO, or with DOXO encapsulated in spheres and tubes, having the same final dose of anticancer drug. T-test was applied with *p ⁇ 0.05 or **p ⁇ 0.01.
  • Polymersomes are synthetic vesicles formed from amphiphilic block copolymers. Over the last fifteen 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 J Control Release 2012 161(2) 473-83, the contents of which are herein incorporated by reference in their entirety).
  • polymersomes that can suitably be used in the present invention
  • this disclosure does not imply that the products and methods of the invention can only be put into practice using polymersomes having the specifically exemplified chemical compositions.
  • the polymersome used in the present invention is typically a self -assembled structure.
  • the polymersome comprises an amphiphilic block copolymer.
  • the amphiphilic block copolymer comprises a hydrophilic block and a hydrophobic block.
  • Such polymersomes are able to mimic biological phospholipids.
  • Molecular weights of these polymers are at least 5 times higher than naturally-occurring phospholipid-based surfactants such that they can assemble into more entangled membranes (J. Am. Chem. Soc. 2005, 127, 8757, the contents of which are herein incorporated by reference in their entirety), providing a final structure with improved mechanical properties and colloidal stability.
  • the flexible nature of the copolymer synthesis allows the application of different compositions and functionalities over a wide range of molecular weights and consequently of membrane thicknesses.
  • these block copolymers offers significant advantages.
  • Polymersomes typically comprise a bilayered membrane.
  • the bilayer is generally formed from two layers of amphiphilic molecules, which align 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.
  • a typical (largest) diameter of a polymersome is in the range 50 to 50,000 nm (for instance 50 to 5000 nm). More typically, the diameter is in the range 50 to 2000 nm. Polymersomes having a diameter in this range are normally termed “nanopolymersomes" or “nanovesicles”.
  • the thickness of the bilayer is generally between 2 to 50 nm, more typically 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, J. Am. Chem. Soc. 127 8757 2005, the contents of which are herein incorporated by reference in their entirety).
  • TEM Transmission Electron Microscopy
  • SAXS Small Angle X-ray Scattering
  • 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. This can be achieved using the methods outlined herein.
  • the polymersome may be capable of dissociating and releasing any contents after it has been internalised within a cell. Dissociation may be promoted by a variety of mechanisms, but is typically promoted by pH sensitivity of the block copolymer. It is thus preferred that the hydrophilic or the hydrophobic block of the amphiphilic copolymer, preferably the hydrophobic block, has a pendant group with a pKa in the range 3.0 to 6.9. The process of endocytosis induces a reduction in the local pH experienced by the polymersome from around pH 7.4 to around pH 5-6. This pH drop is sufficient to trigger disintegration of the polymersome and release of any internalised content.
  • pKa is meant the pH where half of the pendant (side) groups are ionised.
  • pKa can be determined by a variety of methods including pH titration followed by potentiometric titration, UV spectroscopy and Dynamic Light Scattering (DLS). An appropriate method should be selected to measure the pKa according to the copolymer which is being analysed and its solubility in the test media.
  • DLS is a particularly preferred method for measuring pKa.
  • the DLS signal from a copolymer such as PMPC25-&-PDPA20 copolymer, in water varies with pH. At a certain pH the signal rapidly increases as the copolymer undergoes a transition from being molecularly deassociated to associated. The pKa is taken as the pH of the mid-point of this rapid increase.
  • the pKa of a group in a polymer is determined on the basis of a polymer system (and not assumed to be the same as the pKas of similar moieties in non-polymeric systems).
  • the hydrophobic block of the polymersome comprises pendant cationisable moieties as pendant groups.
  • Cationisable moieties are, for instance, primary, secondary or tertiary amines, capable of being protonated at pHs below a value in the range 3 to 6.9.
  • the group may be a phosphine.
  • the pKa of the pendant groups is in the range 4.0 to 6.9, more preferably 5.5 to 6.9.
  • the polymersomes are correspondingly capable of disassociating in such pH ranges.
  • the hydrophobic block of the polymersome has a degree of polymerisation of at least 50, more preferably at least 70.
  • the degree of polymerisation of the hydrophobic block is no more than 250, even more preferably, no more than 200.
  • the degree of polymerisation of the hydrophilic block is at least 15, more preferably at least 20. It is preferred that the ratio of the degree of polymerisation of the hydrophilic to hydrophobic block is in the range 1 :2.5 to 1 :8. All of these limitations promote polymersome, rather than micelle formation.
  • the hydrophilic block may be based on condensation polymers, such as polyesters, polyamides, polyanhydrides, polyurethanes, polyethers (including polyalkylene glycols, especially PEG), polyimines, polypeptides, polypeptoids, polyureas, polyacetals and polysaccharides, but preferably the hydrophilic block is based on a radical polymerised addition polymer of ethylenically unsaturated monomers.
  • the hydrophilic block may have zwitterionic pendant groups, in which case the zwitterionic pendant groups may be present in the monomers and remain unchanged in the polymerisation process. It is alternatively possible to derivatise a functional pendant group of a monomer to render it zwitterionic after polymerisation.
  • the hydrophilic block is formed from ethylenically-unsaturated zwitterionic monomers.
  • ethylenically unsaturated zwitterionic monomers have the general formula (I)
  • H 2 C CR-C 6 H 4 -A 1 -
  • H 2 C CR-CH 2 -A 2 -
  • R 2 0-CO-CR CR-CO-0-
  • RCH CH-CO-0-
  • RCH C(COOR 2 )CH 2 -CO-0-
  • A is -O- or NR 1 ;
  • a 1 is selected from a bond, (CH 2 )LA" 2 and (CH 2 )LS0 3 - in which L is 1 to 12;
  • a 2 is selected from a bond, -0-, -0-CO-, -CO-O, -CO- R 1 -, - R ⁇ CO-, -O-CO- R 1 - and - R ! -CO-O-;
  • R is hydrogen or C 1-4 alkyl
  • R 1 is hydrogen, C 1-4 alkyl or BX;
  • R 2 is hydrogen or C 1-4 alkyl
  • B is a bond, or a straight or branched alkanediyl, alkylene oxaalkylene, or alkylene
  • X is a zwitterionic group.
  • X is an ammonium, phosphonium, or sulphonium phosphate or phosphonate ester zwitterionic group, more preferably a group of the general formula (II)
  • the moieties A 3 and A 4 which are the same or different, are -0-, -S-, - H- or a valence bond, preferably -0-, and W + is a group comprising an ammonium, phosphonium or sulphonium cationic group and a group linking the anionic and cationic moieties which is preferably a Ci-12-alkanediyl group.
  • W + is a group of formula -W ⁇ N + R ⁇ , -W ⁇ P + R ⁇ , -W ⁇ S + R 4 ! or -W ⁇ He in which:
  • W 1 is alkanediyl of 1 or more, preferably 2-6 carbon atoms optionally containing one or more ethylenically unsaturated double or triple bonds, disubstituted-aryl (arylene), alkylene arylene, arylene alkylene, or alkylene aryl alkylene, cycloalkanediyl, alkylene cycloalkyl, cycloalkyl alkylene or alkylene cycloalkyl alkylene, which group W 1 optionally contains one or more fluorine substituents and/or one or more functional groups;
  • the groups R 3 are the same or different and each is hydrogen or alkyl of 1 to 4 carbon atoms, preferably methyl, or aryl, such as phenyl, or two of the groups R 3 together with the nitrogen atom to which they are attached form an aliphatic heterocyclic ring containing from 5 to 7 atoms, or two or more of the groups R 3 together with the nitrogen atom to which they
  • Het is an aromatic nitrogen-, phosphorus- or sulphur-, preferably nitrogen-, containing ring, for example pyridine.
  • Monomers in which X is of the general formula in which W + is W 1 N + R 3 3 may be made as described in WO-A-9301221, the contents of which are herein incorporated by reference in their entirety.
  • Phosphonium and sulphonium analogues are described in WO-A-9520407 and WO-A-9416749, the contents of both of which are herein incorporated by reference in their entirety.
  • groups R 5 are the same or different and each is hydrogen or C 1-4 alkyl, and m is from 1 to 4.
  • the groups R 5 are preferably the same, for example they are preferably all methyl.
  • X may have the general formula (IV)
  • a 5 is a bond, -0-, -S- or -NH- (preferably -0-);
  • R 6 is a bond or alkanediyl, -C(0)-alkanediyl- or -C(0) H-alkanediyl- (wherein R 6 is preferably alkanediyl; and wherein alkanediyl is preferably C 1-6 alkanediyl);
  • W 2 is SR 7 , PR 7 2 or NR 7 2 , wherein the or each group R 7 is hydrogen or alkyl of 1 to 4 carbon atoms or the two groups R 7 together with the heteroatom to which they are attached form a heterocyclic ring of 5 to 7 atoms;
  • R 8 is alkanediyl of 1 to 20, preferably 1 to 10, more preferably 1 to 6 carbon atoms;
  • a 6 is a bond, NH, S or O, preferably O;
  • R 9 is a hydroxyl, C 1-12 alkyl, C 1-12 alkoxy, C7-18 aralkyl, C7-18 aralkoxy, C 6 -i8 aryl or C 6 -i8 aryloxy group.
  • Monomers comprising a group of the general formula IV may be made by methods as described in JP-B-03-031718, the content of which is herein incorporated by reference in its entirety, in which an amino substituted monomer is reacted with a phospholane.
  • a 5 is a bond
  • R 6 is a C2-6 alkanediyl
  • W 2 is NR 7 2 : each R 7 is CM alkyl
  • R 8 is C 2-6 alkanediyl
  • a 6 is O
  • R 9 is Ci-4 alkoxy.
  • X may be a zwitterion in which the anion comprises a sulphate, sulphonate or carboxylate group.
  • sulphobetaine group of the general formula (V)
  • the groups R 10 are the same or different and each is hydrogen or C 1-4 alkyl and s is from 2 to 4.
  • the groups R 10 are the same. It is also preferable that at least one of the groups R 10 is methyl, and more preferable that the groups R 10 are both methyl.
  • s is 2 or 3, more preferably 3.
  • Another example of a zwitterionic group having a carboxylate group is an amino acid moiety in which the alpha carbon atom (to which an amine group and the carboxylic acid group are attached) is joined through a linker group to the backbone of the biocompatible polymer.
  • Such groups may, for example, be represented by the general formula (VI)
  • a 7 is a bond, -0-, -S- or - H- (preferably -0-); R 11 is a bond or alkanediyl,
  • alkanediyl is preferably C 1-6 alkanediyl; wherein R 11 is preferably alkanediyl); and the groups R 12 are the same or different and each is hydrogen or alkyl of 1 to 4 carbon atoms, preferably methyl, or two or three of the groups R 12 , together with the nitrogen to which they are attached, form a heterocyclic ring of from 5 to 7 atoms, or the three group R 12 together with the nitrogen atom to which they are attached form a fused ring heterocyclic structure containing from 5 to 7 atoms in each ring.
  • Another example of a zwitterion having a carboxylate group is a carboxy betaine
  • R 13 -N + (R 13 )2(CH2) r COO " in which the R 13 groups are the same or different and each is hydrogen or Ri-4 alkyl and r is 2 to 6, preferably 2 or 3.
  • Such acrylic moieties are preferably methacrylic, that is in which R is methyl, or acrylic, in which R is hydrogen.
  • the compounds may be (meth)acrylamido compounds (in which A is R 1 ), in which case R 1 is preferably hydrogen, or less preferably, methyl, most preferably the compounds are esters, that is in which A is O.
  • B is most preferably an alkanediyl group. Whilst some of the hydrogen atoms of such group may be substituted by fluorine atoms, preferably B is an unsubstituted alkanediyl group, most preferably a straight chain group having 2 to 6 carbon atoms.
  • a particularly preferred zwitterionic monomer is 2-methacryloyloxyethyl-phosphorylcholine (MPC). Mixtures of zwitterionic monomers each having the above general formula may be used, as can mixtures of other hydrophilic monomers described herein.
  • the hydrophilic block is formed from ethylenically- unsaturated monomers that comprise a polyalkylene glycol side chain (e.g., a PEG side chain).
  • the ethylenically-unsaturated monomers may have the general formula (I)
  • H 2 C CR-C 6 H 4 -A 1 -
  • H 2 C CR-CH 2 -A 2 -
  • R 2 0-CO-CR CR-CO-0-
  • RCH CH-CO-0-
  • RCH C(COOR 2 )CH 2 -CO-0-
  • A is -O- or R 1 ;
  • a 1 is selected from a bond, (CH 2 ) L A 2 and (CH 2 ) L S03 " in which L is 1 to 12;
  • a 2 is selected from a bond, -0-, -0-CO-, -CO-O, -CO- R 1 -, - R ⁇ CO-, -O-CO- R 1 - and R ! -CO-O-;
  • R is hydrogen or C 1-4 alkyl
  • R 1 is hydrogen, C 1-4 alkyl or BX;
  • R 2 is hydrogen or C 1-4 alkyl
  • B is a bond, or a straight or branched alkanediyl, alkylene oxaalkylene, or alkylene (oligooxalkylene) group, optionally containing one or more fluorine substituents;
  • X is a polyalkylene glycol side chain.
  • the polyalkylene glycol side chain may have the formula -[0(CH2) n ] P OR24 in which n is from 1 to 6, p is from 1 to 100 and R24 is hydrogen or C 1-6 alkyl.
  • n is 2 (i.e., the side chain is a polyethylene glycol side chain).
  • p is from 1 to 50, more preferably from 5 to 20.
  • R24 is hydrogen or methyl, most preferably hydrogen.
  • Typical number average molecular weights of the monomers may be in the range 25 to 1000, preferably 50 to 800.
  • the number molecular average molecular weight of the monomers may be from 200 to 800.
  • a particularly preferred hydrophilic block of this nature is formed from oligo(ethylene glycol) methacrylate
  • OEGMA (OEGMA) monomers.
  • the hydrophilic block comprises a phosphorylcholine polymer.
  • a phosphorylcholine polymer is a polymer that comprises one or more phosphorylcholine groups.
  • the hydrophilic block comprises a phosphorylcholine polymer and the hydrophobic block comprises a pendant group with a pKa in the range 3.0 to 6.9.
  • the hydrophobic block may be formed of polymers such as polyethers (including
  • polyalkylene glycols polyesters, polyamides, polyanhydrides, polyurethanes, poiyimines, polypeptides, polypeptoids, polyureas, polyacetals, or polysiloxanes.
  • a suitable hydrophobic block is polyalkylene oxide, usually polypropylene oxide, that is the same type of block as has been used in the well-studied Pluronic/Poloxamer based systems.
  • One type of highly hydrophobic block is poly(dimethylsiloxane).
  • the type of polymer forming the hydrophobic block is the same as that forming the hydrophilic block.
  • the polymer is formed by radical polymerisation of ethylenically unsaturated monomers.
  • Suitable monomers from which the hydrophobic block may be formed have the general formula (VII)
  • R 14 CH C(COOR 16 )CH 2 -CO-0-
  • a 8 is -O- or - R 15 -;
  • a 9 is selected from a bond, (CH 2 ) q A 10 and (CH 2 ) q S03 " in which q is 1 to 12;
  • a 10 is selected from a bond, -0-, -0-CO-, -CO-0-, -CO- R 15 -, - R 15 -CO-,
  • R 14 is hydrogen or C 1-4 alkyl
  • R 15 is hydrogen, C 1-4 alkyl or 1 Q
  • R 16 is hydrogen or C 1-4 alkyl
  • B 1 is a bond, or a straight or branched alkanediyl, alkylene oxaalkylene, or alkylene
  • Q is a cationic or cationisable group of the formula - R 17 P , -PR 17 P and SR 17 r , in which p is 2 or 3, r is 1 or 2, the groups R 17 are the same or different and each is selected from the group consisting of hydrogen, C 1-24 alkyl and aryl, or two of the groups R 17 together with the heteroatom to which they are attached from a 5 to 7 membered heterocyclic ring or three R 17 groups together with the heteroatom to which they are attached form a 5 to 7 membered heteroaromatic ring, either of which rings may be fused to another 5 to 7 membered saturated or unsaturated ring, and any of the R groups may be substituted by amino or hydroxyl groups or halogen atoms; wherein if p is 3, at least one of the groups R 17 is hydrogen.
  • Preferred groups B 1 are alkanediyl, usually with linear alkyl chains and preferably having 2 to 12 carbon atoms, such as 2 or 3 carbon atoms.
  • Q is R 17 2 where R 17 is Ci-12-alkyl.
  • R 17 is Ci-12-alkyl.
  • both R 17 s are the same.
  • Particularly useful results have been achieved where the groups R 17 are C 1-4 alkyl, especially ethyl, methyl or isopropyl.
  • Either or both the hydrophobic and hydrophilic blocks may include comonomers, for instance to provide functionality, control over hydrophobicity, control over pH sensitivity, pKa or pKb as the case may be, control over temperature sensitivity or as general diluents.
  • comonomers providing functionality may be useful to provide conjugation of pendant groups following polymerisation and/or polymersome formation, to targeting moieties, or to provide for conjugation between the biologically active molecule and the polymer.
  • functional groups may allow for crosslinking of the polymer following polymersome formation, to confer increased stability on the polymersome structure.
  • suitable comonomers are compounds of the general formula (VIII)
  • R is selected from hydrogen, halogen, C 1-4 alkyl and groups COOR in which R is hydrogen or C 1-4 alkyl;
  • R 19 is selected from hydrogen, halogen and C 1-4 alkyl
  • R 20 is selected from hydrogen, halogen, C 1-4 alkyl and groups COOR 22 provided that R 18 and R 20 are not both COOR 22 ;
  • R 21 is a Ci-10 alkyl, a C 1-20 alkoxycarbonyl, a mono-or di-(C 1-10 alkyl)amino carbonyl, a C 6 - 2 o aryl (including alkaryl) a C7-20 aralkyl, a C 6 - 2 o aryloxycarbonyl, a Ci-2o-aralkyloxycarbonyl, a C 6 -2o arylamino carbonyl, a C7-20 aralkyl-amino, a hydroxyl or a C2-10 acyloxy group, any of which may have one or more substituents selected from halogen atoms, alkoxy, oligo-alkoxy, aryloxy, acyloxy, acylamino, amine (including mono and di- alkyl amino and thalkylammonium in which the alkyl groups may be substituted), carboxyl, sulphonyl, phosphoryl, phosphino
  • R 18 , R 19 , R 20 and R 21 are halogen or, more preferably, hydrogen atoms.
  • R 18 and R 19 are both hydrogen atoms.
  • compound of general formula VIII is a styrene or acrylic compound.
  • R 21 represents an aryl group, especially a substituted aryl group in which the substituent is an amino alkyl group, a carboxylate or a sulphonate group.
  • the comonomer is an acrylic type compound
  • R 21 is an alkoxycarbonyl, an alkyl amino carbonyl, or an aryloxy carbonyl group.
  • R 21 is a C1-20- alkoxy carbonyl group, optionally having a hydroxy substituent.
  • Acrylic compounds are generally methacrylic in which case R 20 is methyl.
  • the comonomer is a non-ionic comonomer, such as a C 1-24 alkyl(alk)-acrylate or - acrylamide, mono- or di- hydroxy-Ci-6-alkyl(alk)-acrylate, or acrylamide, oligo(C2-3 alkoxy) C2-i8-alkyl (alk)-acrylate, or -acrylamide, styrene, vinylacetate or N-vinyllactam.
  • a non-ionic comonomer such as a C 1-24 alkyl(alk)-acrylate or - acrylamide, mono- or di- hydroxy-Ci-6-alkyl(alk)-acrylate, or acrylamide, oligo(C2-3 alkoxy) C2-i8-alkyl (alk)-acrylate, or -acrylamide, styrene, vinylacetate or N-vinyllactam.
  • the block copolymers should have controlled molecular weights. It is preferable for each of the blocks to have molecular weight controlled within a narrow band, that is, to have a narrow polydispersity.
  • the polydispersity of molecular weight should, for instance, be preferably less than 2.0, more preferably less than 1.5, for instance in the range 1.1 to 1.4.
  • the blocks should be selected so that they have the requisite pKa value.
  • the monomer from which the hydrophobic block is formed is 2-(diisopropylamino)ethyl methacrylate (DP A) or 2-(diethylamino)ethyl methacrylate (DEA).
  • the hydrophilic block is PMPC or poly(oligo (ethylene glycol) methacrylate) (POEGMA).
  • the copolymer is a PMPC-6-PDPA block copolymer or a POEGMA-PDPA block copolymer.
  • the block copolymer has general formula PMPC m -£-PDPA n or POEGMA m - PDPAn, wherein m is in the range from 2 to 500, or from 15 to 30 (for instance 25), and n is from 6 to 2000 or from 70 to 180, preferably from 100 to 160, more preferably from 120 to 160.
  • the block copolymer may have the general formula POEGMA m -PDPA n where m is from 15 to 30 and n is from 100 to 160 (i.e. a block copolymer comprising a block derived from m OEGMA monomers joined to a block derived from n DPA monomers).
  • the hydrophobic block is not formed from 2-(dimethyl)ethyl methacrylate (DMA) monomers.
  • the block copolymer may be a simple A-B block copolymer, or may be an A-B-A or B-A-B block linear triblock copolymer or a (A) 2 B or A(B) 2 star copolymers (where A is the hydrophilic block and B is the hydrophobic block). It may also be an A-B-C, A-C-B or B-A- C block linear triblock copolymers or a ABC star copolymers (blocks linked together by the same end), where C is a different type of block.
  • C blocks may, for instance, comprise functional, e.g. cross-linking or ionic groups, to allow for reactions of the copolymer, for instance in the novel compositions.
  • Crosslinking reactions especially of A-C-B type copolymers may confer useful stability on polymersomes.
  • Cross-linking may be covalent, or sometimes, electrostatic in nature.
  • Cross-linking may involve addition of a separate reagent to link functional groups, such as using a difunctional alkylating agent to link two amino groups.
  • the block copolymer may alternatively be a star type molecule with hydrophilic or hydrophobic core, or may be a comb polymer having a hydrophilic backbone (block) and hydrophobic pendant blocks or vice versa.
  • Such polymers may be formed for instance by the random copolymerisation of monounsaturated macromers and monomers.
  • Exemplary methods that can be used for polymerising the monomers are atom-transfer radical polymerisation (ATRP) (see, e.g., an exemplary method described in Journal of the American Chemical Society 127, 17982-17983), living radical polymerisation process, functional NCA (N-carboxyanhydride) polymerisation with efficient postpolymerization modification and ring opening polymerisation (ROP).
  • ATRP atom-transfer radical polymerisation
  • functional NCA N-carboxyanhydride
  • ROP ring opening polymerisation
  • 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 1.2 to 1.4 for the or each block are preferred.
  • the polymersomes may be loaded using a pH change system, electroporation or film hydration.
  • polymer 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.
  • a method of forming polymersomes with an encapsulated material (e.g. an encapsulated drug) in the core may involve the following steps: (i) dispersing the amphiphilic copolymer in an aqueous medium; (ii) acidifying the composition formed in step (i); (iii) adding the material to be encapsulated to the acidified composition; and (iv) raising the pH to around neutral to encapsulate the material.
  • steps may involve the following steps: (i) dispersing the amphiphilic copolymer in an aqueous medium; (ii) acidifying the composition formed in step (i); (iii) adding the material to be encapsulated to the acidified composition; and (iv) raising the pH to around neutral to encapsulate the material.
  • 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.
  • Step (ii), of acidifying the composition typically reduces the pH to a value below the pKa of the pendant group.
  • Another method of forming polymersomes with an encapsulated material in the core may involve the following steps: (i) dispersing the amphiphilic copolymer, and when needed the material to be encapsulated, in an organic solvent 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 material to be encapsulated.
  • polymersomes are typically prepared by dissolving copolymer in an organic solvent, such as a 2: 1 chloroform:methanol mix in a glass container. If a hydrophobic or amphiphilic material is to be encapsulated, it can be added with the copolymer. Solvent can be evaporated under vacuum leaving a copolymeric film deposited on the walls of the container. The film is then re-hydrated with an aqueous solution, for instance using phosphate buffer saline. If a hydrophilic material is to be encapsulated, it can be included in the aqueous solution. The pH of the resultant suspension is decreased to a pH of around 2, to solubilise the film, and then increased slowly to a pH or around 6.
  • an organic solvent such as a 2: 1 chloroform:methanol mix in a glass container.
  • the polymer hydration at neutral pH allows the encapsulation of the material.
  • the dispersion may then be sonicated and extruded, for instance using a bench top extruder. UV spectroscopy and HPLC chromatography may be used to calculate the encapsulation efficiency, using techniques well known in the art.
  • 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 drug 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.
  • 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.
  • the polymersomes used in the invention may be formed from two or more different block copolymers.
  • a mixture of the two or more block copolymers is used.
  • 0.01% to 10% (w/w) of material to be encapsulated is mixed with copolymer in the methods described above.
  • a composition that comprises a mixture of polymersomes of different shapes.
  • a mixed polymersome composition typically comprises a mixture of tubular and non-tubular polymersomes.
  • Non-tubular polymersomes include substantially spherical polymersomes.
  • the mixed polymersome composition may, for example, be one in which the percentage of tubular polymersomes in the total population of polymersomes is less than 50%, preferably less than 40% and more preferably still less than 30%.
  • a tubular polymersome is a polymersome that comprises at least a tubular portion.
  • tubular portion can be used interchangeably herein with “elongated portion”.
  • the tubular polymersome may consist substantially of the tubular portion or alternatively the tubular portion may form only part of the tubular polymersome, with other parts of the tubular polymersome being non-tubular in shape.
  • a skilled person would readily recognize a polymersome having a tubular portion and would not have any difficulty in distinguishing it from a polymersome lacking a tubular portion.
  • tubular polymersome includes a toroidal polymersome.
  • a toroidal polymersome is a tubular polymersome having two ends that are connected to each other (as in a torus or "donut").
  • a tubular polymersome can be unbranched or branched.
  • a branched tubular polymersome is a polymersome that comprises one or more branching points with one or more arms extending from the or each branching point.
  • a branched tubular polymersome is clearly non-spherical in shape because a sphere does not comprise branching points or arms.
  • An unbranched tubular polymersome is also non-spherical in shape in view of its
  • Methods of quantitatively determining whether a polymersome is tubular include determining (a) its aspect ratio (either in 3D or more typically from a 2D projection of the polymersome in, for example, a TEM image) and (b) its sphericity or circularity (most typically the circularity of a 2D projection of the polymersome in, for example, a TEM image).
  • a tubular polymersome has an aspect ratio of less than 1, typically 0.95 or less, preferably 0.9 or less, and more preferably 0.8 or less (wherein aspect ratio is the minimum Feret diameter divided by the maximum Feret diameter of the polymersome).
  • a sphere has an aspect ratio of 1.
  • Polymersomes are of course three-dimensional structures. Maximum and minimum Feret diameters of such a structure are defined as the maximum and minimum distances, respectively, between two parallel planes restricting the structure perpendicular to that direction. However, it will be appreciated that visualization techniques, such as TEM, typically project a polymersome in 2D rather than directly showing its 3D shape. Thus, the aspect ratio as defined herein is typically the aspect ratio of such a 2D projection of the polymersome. Furthermore, the maximum and minimum Feret diameters are the maximum and minimum distances, respectively, between two parallel tangential lines restricting the 2D projection of the polymersome perpendicular to that direction.
  • an unbranched tubular polymersome has a sphericity of 0.9 or less, more preferably 0.8 or less (wherein sphericity is the ratio of the surface area of a sphere with the same volume as the unbranched tubular polymersome to the surface area of the unbranched tubular polymersome).
  • an unbranched tubular polymersome is one whose 2D projection, for example in a TEM image, has a circularity of 0.75 or less, more preferably 0.7 or less.
  • Circularity, fare is defined herein as fcirc — p 2 in which A is area and P is perimeter.
  • a 2D projection of a tubular polymersome may underestimate the extent to which it is non- spherical/non-circular in view of its projection onto the 2D surface.
  • the projection of a perfect cylinder end-to-end onto a 2D surface is a circle.
  • the characterisations provided herein take this factor into account.
  • the percentage of tubular polymersomes in the total population of polymersomes is specified to be at least a certain percentage (e.g. at least 50%, 70%, 80%) or 90%) account has been taken that some polymersomes that are in fact tubular may nonetheless be ascribed to be non-tubular owing to the measurement method employed.
  • Figure 1 shows illustrative TEM images of two different polymersome compositions (A) and (B) (two separate images are provided for each composition).
  • the composition clearly mainly comprises non-tubular polymersomes (in particular, spherical polymersomes).
  • the composition clearly mainly comprises tubular polymersomes, specifically in the form of a mixture of unbranched tubular polymersomes and branched tubular polymersomes.
  • the percentage of tubular polymersomes in the total population of polymersomes in the tubular polymersome composition is greater than the percentage of tubular polymersomes in the total population of polymersomes in the mixed polymersome composition.
  • the percentage of tubular polymersomes in the total population of polymersomes is typically at least 50% (e.g. at least 70%, 80% or 90%). These percentages can readily be determined by a skilled person.
  • the percentage can be determined using TEM.
  • Preferably one or more TEM images of the composition are obtained, such that the total number of polymersomes visible in the one or more images is at least 20 (more preferably at least 30, more preferably still at least 50).
  • the number of tubular polymersomes i.e., the total number of branched tubular
  • polymersomes and unbranched tubular polymersomes can in one embodiment be determined by visual inspection of the one or more TEM images.
  • polymersomes in the total population of polymersomes can thus easily be determined.
  • the number of tubular polymersomes can be determined by calculating the aspect ratio of each polymersome in the one of more images. The percentage of tubular polymersomes in the total population of polymersomes can again then easily be determined.
  • the number of tubular polymersomes can be determined by determining, first, the number of branched polymersomes and second, by determining the number of polymersomes amongst those remaining that have a circularity of 0.75 or less, more preferably 0.7 or less (i.e. those polymersomes that constitute unbranched tubular polymersomes). The percentage of tubular polymersomes in the total population of polymersomes can again then easily be determined.
  • the total population of polymersomes is considered to be the total number of polymersomes that are one of: (a) branched polymersomes; (b) unbranched tubular polymersomes; and (c) substantially spherical polymersomes.
  • Substantially spherical polymersomes are for example those whose 2D projection (e.g. in a TEM image) have a circularity of greater than 0.9 and/or an aspect ratio of greater than 0.95.
  • the percentage of tubular polymersomes in the total population of polymersomes is typically at least 50%.
  • the percentage of tubular polymersomes in the total population of polymersomes is at least 70%, more preferably at least 80% and more preferably still at least 90%.
  • the percentage of tubular polymersomes in the total population of polymersomes may even be as high as 95% or more.
  • the tubular polymersome composition of the present invention is obtainable by carrying out the production method of the present invention.
  • the method is based on density gradient centrifugation and in particular comprises subjecting a mixed polymersome composition (e.g. a conventionally known and conventionally prepared mixed polymersome composition) to density gradient centrifugation.
  • a mixed polymersome composition e.g. a conventionally known and conventionally prepared mixed polymersome composition
  • Density gradient centrifugation is itself a well known and established technique. For example, this techniques is well known for use in separating cells parts after lysis according to their density. It has also previously been used for the purification of gold nanoparticles and carbon nanotubes with different sizes and shapes. It has not, however, previously been applied to polymersome compositions, e.g. to obtain a tubular polymersome composition.
  • the density gradient centrifugation is preferably sucrose gradient centrifugation.
  • sucrose gradient centrifugation solutions of successively increasing sucrose concentration (e.g. in PBS) are layered in a centrifuge (e.g. from most dense to least dense). The mixed
  • polymersome composition is then deposited as a top layer, and centrifugation is then effected.
  • centrifugation conditions e.g. RCF and time of centrifugation
  • RCF and time of centrifugation can readily be adjusted to those skilled in the art depending on the chemical composition of the mixed polymersome composition.
  • An illustrative example is provided in the Examples section of this
  • the centrifugation is carried out in a centrifuge. Subjecting the polymersome composition to density gradient centrifugation gives rise to separate layers of polymersome compositions having different compositions in the centrifuge.
  • the tubular polymersome composition can be isolated from the centrifuge, e.g. as one of the layers formed by the centrifugation.
  • the tubular polymersomes optionally comprise one or both of a targeting moiety and an encapsulated material.
  • the tubular polymersome optionally comprises a targeting moiety on its external surface.
  • a targeting moiety on its external surface is meant that the targeting moiety 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 polymersome).
  • the targeting moiety is adapted to enable the polymersome to bind to a target.
  • the targeting moiety 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 be 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.
  • the targeting moiety can be any moiety that binds specifically to the target.
  • a wide range of substances can be used as targeting moieties, e.g. to target receptors.
  • the targeting moiety is a moiety that is attached to the external surface of the polymersome.
  • suitable targeting moieties include antibodies, antibody fragments, aptamers, oligonucleotides, small molecules, peptides and carbohydrates. Peptide, antibody and antibody fragment targeting moieties are particularly preferred.
  • any such moiety can be used as a targeting moiety 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.
  • a targeting moiety is a targeting moiety that is adapted to enable the polymersome to bind to a cancer cell.
  • targeting moieties include proteins (mainly antibodies and their fragments), peptides, nucleic acids (aptamers), small molecules, vitamins and carbohydrates. It will be appreciated that targeting of the polymersomes of the present invention to cancer cells is of particular interest in view of their capacity to decrease the replication activity of such cells.
  • the targeting moiety can be attached to the external surface of the polymersome using routine techniques, for example by adapting well known methods for attaching targeting moieties 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.
  • the targeting moiety can be attached by reacting a suitable functional group on the targeting moiety (including but not limited to an amine group, a carboxyl group and a thiol 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.
  • a peptide targeting moiety 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).
  • a 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.
  • the targeting moiety may be attached directly to the external surface of the polymersome or it may be attached via a chemical spacer.
  • the targeting moiety may also be a pendant group of a polymer comprised by the polymersome (i.e. at least one of the copolymers forming the polymersome itself). Clearly in this embodiment it is not necessary to undertake separate synthetic steps to attach the targeting moiety to the copolymer or the resulting polymersome.
  • the tubular polymersome also optionally comprises an encapsulated material.
  • Suitable encapsulated materials include anti-cancer drugs.
  • tubular polymersome composition of the present invention can be formulated as a pharmaceutical composition using routine techniques known in the art.
  • pharmaceutical compositions already utilised for the formulation of polymersomes or drug- containing liposomes can be adapted to incorporate the tubular polymersome composition of the present invention.
  • the pharmaceutical composition 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).
  • 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.
  • the pharmaceutical composition is an injectable composition, e.g. it is suitable for intravenous delivery, for example it is suitable for infusion.
  • tubular polymersomes of the present invention are particularly useful for treating cancer. As illustrated in the Example, it has surprisingly been found that tubular polymersomes are capable of selectively reducing the replication activity of tumor cells compared with non tumor cells. This reduction of tumor cell division may be related to an up-regulation of the tumor suppressor proteins p53 and p21. Specifically, tumor cell death is believed to occur via activation of the caspase 3/7 extrinsic pathway of apoptosis, suggesting that the tubular polymersomes lead to the activation of the cell death receptor.
  • Cancers particularly suitable for treatment according to the present invention include those susceptible to amelioration by: up-regulation of the tumor suppressor proteins p53 and p21; and/or activation of the caspase 3/7 extrinsic pathway of apoptosis.
  • cancers that can be treated 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,
  • a therapeutically effective amount of the tubular polymersomes is administered to a patient.
  • a typical dose is from 0.0001 to 1000 mg, measured as a weight of the tubular polymersomes, according to 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. This dose may for instance be administered once daily.
  • daily dosage levels are from 0.0001 mg to 4000 mg.
  • tubular polymersomes may be administered from 0.0001 to 1000 mg/kg, in total. Typically, from 0.01 to 100 mg/kg of the tubular polymersomes may be administered.
  • tubular polymersomes Preferably, from 0.01 to 50 mg/kg or from 1.0 to 20 mg/kg of the tubular polymersomes may administered. These are typically daily doses of the tubular polymersomes.
  • PMPC-PDPA phosphorylcholine-poly(2-(diisopropylamino)ethyl methaciylate)
  • a method was developed for isolating the two population of polymersomes (namely, spheres and tubes), based on density gradient centrifugation. This enabled work to be carried out with highly pure samples in terms of shape distribution.
  • a cytome assay was then carried out, useful for quantifying the replication activity of cells, through the quantification of the nuclear division index (NDI), with the aim to understand any possible different interaction of polymersomes and tubes with the replication machinery of the cells.
  • NDI nuclear division index
  • all the investigations were carried out on two immortalised cell lines, namely the HeLa and FaDu cells, as well as on primary (non tumor) human dermal fibroblast (HDF).
  • the polymersomes were formed by means of film rehydration. Briefly, an organic solution of PMPC-PDPA was placed in a glass vial, and allowed to dry to deposit a polymeric film on the internal surface of the vials. The film was then rehydrated with a PBS solution, enabling the formation of both vesicular polymersomes and tubes (see the experimental section for more details). During this step, the copolymer will create different shaped structures that are strongly dependent on the copolymer/water ratio. This is a complex process from a kinetical viewpoint, and usually leads to the formation of different metastable phases such as spheres, multilamellar aggregates, and tubular polymersomes. Consequently, a sample will inevitably contain a mixture of both spheres and tubes.
  • Figure 3 A shows the total mass of spherical polymers present in the different cell cultures over time, quantified by means of HPLC. It is evident that the uptake of polymersomes follows an increasing trend in all the cells, especially between 48 and 96 hours of treatment, where the engulfment process is much more pronounced. The only difference here is the final quantity of the up taken material, so that FaDu cells resulted to be the most effective in the endocytosis of spherical structures after 96 hours of incubation, with an average polymer quantity of 7 ⁇ g (figure 3 A, left). The polymersomes uptake was quantified to be c.a. 4 ⁇ g for the HeLa cells, and about 2 ⁇ g in the primary fibroblasts after 4 days treatments.
  • FaDu cells were incubated with rhodamine B- encapsulated polymersomes (in the same way as for the previous HPLC quantification), and after 96 hours the cells were stained with calcein green, with the double aim to check for viability and internalisation.
  • the confocal images in Figure 4 demonstrate that all the spherical polymersomes are effectively engulfed within the cells (Figure 4A), and that they are spread throughout the cytosol. On the other side, most of the tubular structures were found to be attached outside of the cells, and only small amount of material engulfed (Figure 4B).
  • cytokinesis-block micronucleus cytome assay This is a method for quantifying the (eventual) cytostatic effects of compounds, as well as DNA damage, and general cytotoxicity.
  • Cyt-B cytochalasin-B
  • NDI Nuclear Division Index
  • Fadu, HeLa and HDF cells were first synchronised by serum starvation, in order to force them into the stationary phase of the cell cycle (Go), and then treated with both spherical polymersomes and tubular vesicles for 24h at a final concentration of 0.5 mg/mL.
  • Cell cycle synchronisation is a crucial optimisation step in this technique, as the NDI will be otherwise significantly affected.
  • Cyt-B was added in the cell culture, and cells were finally fixed and analysed by means of confocal microscopy. For each set of experiments, more than 3000 cells were analysed in order to have an optimal and correct representation of the NDI (see experimental section for details).
  • Figure 5 is a representative picture of the ability of the software to recognise, distinguish, and count the cells having one or more nuclei, scanned by means of confocal microscopy.
  • Figure 6A shows the NDI found in all the tested cells.
  • the NDI for the control (untreated) cells displayed a value of -1.65, while the same cells treated with both spherical vesicles and tubes had a lower NDI of -1.55.
  • the positive control (cells treated with H2O2) revealed an increase value up to 1.8.
  • the trend was found to be quite similar also in HeLa cells.
  • the NDI in untreated cells displayed a value of -1.7 while the positive control was -1.8.
  • the treatment with polymersomes did not induce any statistically significant shift in the NDI (with respect to the control), while this was quite remarkable in
  • FaDu were in the range of - lxlO 5 cells, while HeLa grew up to a maximum of 1.8xl0 5 between 24 and 96 hours of incubation. This confirms the intrinsic cytostatic activity of these nanostructures. At the same time, the number of cells contacting spherical polymersomes are completely similar to the control ( ⁇ 3xl0 5 and ⁇ 6xl0 5 cells for FaDu and HeLa, respectively), confirming that such vesicles do not possess the ability of inhibit cell growth during time.
  • polymersomes-treated HDF behaved similar to the control ( ⁇ 1.4xl0 5 ) in the first 1 day.
  • NDI and MM are morphological assays, so that the conclusions came from macroscopic observations, which are represented in this cases by the variation in the number of multi -nucleated cells (for the NDI) and the presence of micronuclei (MM).
  • MM micronuclei
  • the p53 gene was slightly down regulated only in the presence of tubes, while spherical polymersomes did not induce any significant effect over the regulation of p21. All the other tested genes did not display remarkable regulations in the two conditions. Also in HeLa cells, the treatment with both vesicles and tubes resulted in a significant over expression of p21 (-1.5 times), as in the case of FaDu. However, also found was an important up regulation of p53 upon treatment with tubes, while spherical vesicles were not effective towards p53 regulation. In addition, a slight over expression of the CAT gene was observed when HeLa cells contacted tubular structures. Again, spherical polymersomes did not induce any effect on CAT gene regulation.
  • caspase activation As a final consideration, it was investigated whether this activation of p21 and p53, together with the consequent decrease in the replication activity of cells, may be related to caspase activation, which are markers of apoptosis.
  • caspase 3/7 and 9 the activity of caspase 3/7 and 9 was investigated.
  • Caspase 3/7 are known to become active as a function of the binding of external ligands to cell death receptors, which then become active in the pathway known as extrinsic apoptosis.
  • the caspase 9 undergoes activation upon intracellular signalling, related to the stress-related release of cytochrome c from mythocondria, a process known as intrinsic apoptosis.
  • Figure 9 shows the activation of capsize 3/7.
  • cyclin- dependent kinase inhibitor p21 acts as both sensor and actuator multiple anti-proliferative signals.
  • the treatment with spherical vesicles and tubes led to a significant up-regulation of p21 FaDu cells, while p53 was slightly down-regulated upon treatment with tubes.
  • This outcome was interesting. It should be considered that usually p21 and p53 are part of a similar pathways, so that activation of p21 usually induce the up- regulation of p53.
  • p21 and p53 may act also in independent ways.
  • the present data suggest that the treatment with both spherical and tubular nanovesicles leads to the activation of the alternative pathway of p21, independently of p53.
  • This situation is different in HeLa cells.
  • these cells up-regulate both p53 and p21 upon treatment with tubes (in this case spherical polymersomes do not lead to p53 activation, thus similarly to the case of FaDu).
  • the CAT gene codifying for the catalase (enzyme important for detoxification from oxidative stress), was found to be slightly activated in HeLa, suggesting that those cells might sense a stronger stress upon incubation with tubes.
  • caspase 3/7 and caspase 9 were investigated upon incubating cells with spheres and tubes, to further be able to distinguish between the potential activation of extrinsic (caspase 3/7) or intrinsic (caspase 9) pathways of apoptosis. It was observed that the treatment with tubes in both HeLa and FaDu cells induces a light activation of caspase 3/7 ( Figure 9), while spherical polymersomes did not have detectable effects on cells. As expected, caspase 3/7 are not regulated in primary fibroblast in any of the condition tested.
  • the tubes-related caspase 3/7 activity in tumor cells confirms the hypothesis that the slowing down of cell division is related to the activation of p21, which in turns slightly promotes the apoptotic pathway.
  • the analyses of caspase 9 confirmed that an intrinsic apoptosis does not occur in all the experimental conditions.
  • the extrinsic pathway of apoptosis is strictly related to the activation of extracellular death receptors, and in our evidences tubular structures are thus most likely to interact with them.
  • PMPC25-PDPA70 was prepared by atom-transfer radical polymerisation (ATRP) as described in Journal of the American Chemical Society 127, 17982-17983, 2005.
  • ATRP atom-transfer radical polymerisation
  • the initiator was mixed with 25 equivalents of MPC, dissolved ethanol, degassed and
  • TEM analysis was performed using a FEI Tecnai G2 Spirit electron microscope and/or a JEOL 2100 operating at 200 kV equipped with a CCD camera Orius SC2001 from Gatan.. Copper grids were glow discharged and the sample was adsorbed onto the grid. The sample was then stained with 0.75wt% phosphotungstic acid (PTA) raised to pH 7.4 with NaOH.
  • PTA phosphotungstic acid
  • FIDF Primary human dermal fibroblasts
  • HeLa ovarian cancer cells
  • FaDu oral carcinoma cells
  • FIDF, HeLa, and FaDu cells were cultured and maintained using Dulbecco's Modified Eagle Medium (DMEM) (Sigma-Aldrich ® ) containing: 10 (v/v) fetal calf serum, 2 111M L-glutamine, 100 mg/ml streptomycin and 100 lU/ml penicillin (Sigma-Aldrich " ).
  • DMEM Dulbecco's Modified Eagle Medium
  • 10 v/v
  • fetal calf serum 10 (v/v) fetal calf serum
  • 2 111M L-glutamine 100 mg/ml streptomycin and 100 lU/ml penicillin
  • Cells were cultured at 37 C/95% air/5% CO?.
  • Cells were periodically sub-cultured using Trypsin-EDTA solution 0.25% (Sigma-Aldrich
  • the Thiazolyl Blue Tetrazolium Blue (M I T, Sigma) method was used. Briefly, cells were seeded at a concentration of 5 x 10 3 cells/well in a 96 well plate O.N.. Increasing concentrations of polymersomes and tubes were then added in the growth media, namely 0.1, 0.5, and 1 mg/mL, for periods of 24, 48, and 96 hours. The medium growth was then removed and an acidified solution of isopropanol was added to dissolve the water- insoluble MTT formazan. The solubilised blue crystals were measured col ori metrically at 570 nm (plate reader ELx800, BioTek).
  • cells were seeded at a concentration of 8 x 10 3 cells/well in a six well plate, and then incubated with both polymersomes and tubes at a concentration of 0.5 mg/mL for 24, 48, and 96 hours. Cells were then detached with a Trypsin-EDTA solution 0.25%, and counted with an automated cell counter (TC20, Bio-Rad).
  • FaDu cells were seeded in the same glass bottom dishes, incubated with either polymersomes or tubes, and finally stained with Calcein (Life Technologies) for vital staining.
  • Cell imaging for both NDI/MNi and uptake was carried out with confocal microscope (Leica TCS SP8), and imaging quantification was carried out with an ad hoc designed Matlab script. For the scoring of DI and MM, the guidelines of Fenech were adopted.
  • RT-PCR Reverse transcription polymerase chain reaction
  • RNA concentration was measured with NanoDrop spectrophotometer (Thermo).
  • cDNA Complementary DNA
  • Thermo NanoDrop spectrophotometer
  • cDNA Complementary DNA
  • Quantitative analysis was assessed with QuantiTect SYBR Green RT-qPCR Kit (Qiagen).
  • the amplification process was done in 20 ⁇ ⁇ , using the following steps: 95°C for 5 min to make active the DNA Polymerase, followed by 40 cycles of 95°C (10 s) for denaturation, and 60°C (30 s) for combined annealing and extension for all primers. Melting curve was also acquired, to analyse the sample quality, from 55°C to 99°C, by increasing of l°C/min. Data were analysed via AACt value. 2 " ⁇ was calculated as follows: Ct GAPDH;
  • AACt ACt(treated) -ACt(control). The genes expression was analysed using the following list of primers:
  • CYP1A1 For: GAAGCAGCTGGATGAGAACG
  • CYP1B1 For: CTCGAGTGCAGGCAGAATTG
  • HSP70 For: CCTCAGTCTGATGGCTCCAG
  • HSP27 For: CCAAGTTTCCTCCTCCCTGT
  • the luminesce-based Caspase- Glo® 3/7 Assay Systems Promega
  • the Caspase-Glo® 9 Assay Systems Promega
  • Cells were seeded at a concentration of 8 x 10 3 cells per well in a 96-well plate, and incubated with polymersomes and tubes (0.5 mg/mL) for 24 hours.
  • the caspase solution was then directly added to the media to have a 1 : 1 final ratio, and the luminescence was measured (Varian Cary Eclipse).
  • cells were seeded to a final concentration of 5 x 10 3 cells/well in a 96 well plate overnight. 0.1, 0.5 and 2.5 ug/mL of Doxorubicin were then inoculated, either as a free DOXO or DOXO encapsulated within spherical or tubular polymersomes, to cancer (i.e., HeLa and FaDu) and non-cancer primary (HDF) cells.
  • cancer i.e., HeLa and FaDu
  • HDF non-cancer primary

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  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
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Abstract

La présente invention concerne des polymersomes thérapeutiques. Les compositions de l'invention comprennent une proportion élevée de polymersomes tubulaires. L'invention concerne également des procédés de production de ces compositions et des applications thérapeutiques de ces compositions.
PCT/GB2017/051374 2016-05-18 2017-05-17 Polymersomes Ceased WO2017199023A1 (fr)

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WO2020144467A1 (fr) 2019-01-07 2020-07-16 Ucl Business Ltd Polymersomes fonctionnalisés dotés de multiples ligands
WO2020225538A1 (fr) 2019-05-03 2020-11-12 Ucl Business Ltd Production de nanoparticules et de microparticules
US10874611B2 (en) 2016-02-25 2020-12-29 Ucl Business Ltd Chemotactic, drug-containing polymersomes
US10881613B2 (en) 2016-03-17 2021-01-05 Ucl Business Ltd Fumarate polymersomes
WO2023094810A1 (fr) 2021-11-24 2023-06-01 Ucl Business Ltd Polymersomes pour l'élimination de protéines amyloïdes bêta et/ou tau
WO2024147020A1 (fr) 2023-01-06 2024-07-11 Vianautis Bio Limited Polymersomes pour l'administration de charges d'acide nucléique

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10874611B2 (en) 2016-02-25 2020-12-29 Ucl Business Ltd Chemotactic, drug-containing polymersomes
US10881613B2 (en) 2016-03-17 2021-01-05 Ucl Business Ltd Fumarate polymersomes
WO2020144467A1 (fr) 2019-01-07 2020-07-16 Ucl Business Ltd Polymersomes fonctionnalisés dotés de multiples ligands
US12257344B2 (en) 2019-01-07 2025-03-25 Ucl Business Ltd Polymersomes functionalised with multiple ligands
WO2020225538A1 (fr) 2019-05-03 2020-11-12 Ucl Business Ltd Production de nanoparticules et de microparticules
WO2023094810A1 (fr) 2021-11-24 2023-06-01 Ucl Business Ltd Polymersomes pour l'élimination de protéines amyloïdes bêta et/ou tau
WO2024147020A1 (fr) 2023-01-06 2024-07-11 Vianautis Bio Limited Polymersomes pour l'administration de charges d'acide nucléique

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