Polymersomes with methacrylate

Abstract

The present invention relates to drug delivery, more specifically, to polymersomes used as a drug delivery systems. The present invention also relates to polymersomes comprising a composition for use in treating, for example, ocular disorders and pulmonary disorders.

Description

[0001] Title of the Invention

[0002] POLYMERSOMES WITH METHACRYLATE

[0003] Field of the Invention

[0004] The present invention relates to drug delivery, more specifically, to polymersomes used as a drug delivery systems. The present invention also relates to polymersomes comprising a composition for use in treating, for example, ocular disorders and pulmonary disorders.

[0005] Background to the Invention

[0006] In 2019, the World Health Organizations ‘world report on vision’ determined that at least 2.2 billion people are blind or visually impaired, a number which is set to increase given the aging population. The priority for effective new treatments remains high and has led to the expanding use of biologies, however these newer drug compounds are compounding the problem of ocular drug delivery.

[0007] Ocular drug delivery (ODD) presents a major ongoing challenge for clinicians worldwide. Effective delivery, particularly to the posterior segment of the eye, is hindered by the presence of barriers, both precorneal and corneal. Precorneal factors include the conjunctiva and the presence of tears. The corneal barriers present selectivity of the layers to either hydrophobic or hydrophilic molecules, the electrostatic state of the drug molecules or the size of the drug molecules or transporting vehicle with efflux transporters such as P-glycoproteins also hinder the entry of substances through the epithelial layers.

[0008] To circumvent these barriers, various methods have been employed to increase drug delivery and concentration of drug at target sites in the eye. Examples include the use of contact lenses, microcannulation, intravitreal injections (I Vis), solutions, implants and nanoparticles (NPs). However, delivery of drugs to the posterior segment of the eye still remains a difficult task.

[0009] Thus, there is a clear unmet need for improved ocular drug delivery systems that circumvent the aforementioned challenges, as well as reduce the possibility of infection and reduce cost. Also, current therapies such as I Vis can only be delivered by a trained health care specialist within a clinical setting. The present invention addresses these needs.

[0010] The present invention investigates the potential of polymersomes to enable ocular delivery using various model drug compounds and enhanced through the addition of a cationic charge. This will be achieved through several experiments using both in-vitro human corneal epithelial cells as well as ex-vivo and in-vivo murine models, ex-vivo bovine models and bovine vitreous humour. Polymersomes have not been investigated as potential ocular drug delivery systems until now. In addition to the delivery of therapeutic compounds in ocular disorders, polymersomes may also be useful in the delivery of drug compounds for pulmonary disease. Pulmonary disorders include chronic obstructive pulmonary disease (COPD), lung cancers, asthma, infections like pulmonary tuberculosis (TB), cystic fibrosis, pneumonia, and idiopathic pulmonary fibrosis (IPF).

[0011] Conventional drug treatment offers many limitations such as elimination by clearance mechanisms, ingestion by the alveolar macrophages, rapid absorption into the blood, enzymatic degradation, and low control over the deposition rate and location, thus resulting in reduced patient response.

[0012] Polymersomes offer a unique solution to this, as they can be tailored to specific needs, including the protection of drug cargo from degradation as well as the ability to control size, and thus deposition rate and location.

[0013] Summary of the Invention

[0014] According to a first aspect of the present invention, there is provided a polymersome comprising: a) a hydrophobic monomer; and b) a hydrophilic monomer.

[0015] Optionally, the hydrophilic monomer may be polyethylene glycol (PEG), polyethylene glycol (PEG) methacrylate, polyethylene oxide, poly(isocyano-L-alanine-L-lanine, polyacrylic acid, poly(methyloxazoline), poly(4-vinyl pyridine), poly-L-glutamic acid, poly(N e-2-(2-(2- methoxyethoxyl)ethoxy)acetyl-L-lysine, poly(y-benzyl L-glutamate), or dextran.

[0016] Optionally, the hydrophilic monomer has a molecular weight of 500-20,000. Optionally, the hydrophilic monomer has a molecular weight of 1 ,000-15,000. Optionally, the hydrophilic monomer has a molecular weight of 5,000-10,000. Optionally, the hydrophilic monomer has a molecular weight of 5000.

[0017] Optionally, the polyethylene glycol may be methoxypoly(ethelyne glycol) 5000.

[0018] Preferably, the hydrophilic monomer is polyethylene glycol (PEG) methacrylate.

[0019] Further preferably, the polyethylene glycol (PEG) methacrylate is defined as:

[0020] Optionally, the polyethylene glycol (PEG) methacrylate has an average molecular weight of 500 Da. Optionally, the hydrophobic monomer may be the decyl methacrylate, polylactide, poly(lactic acid), poly(ethylethylene), polybutadiene, polycaprolactone, polypropylene sulfide, polystyrene, poly-L- leucine, polyester, poly(butylene oxide), poly(isobutylene), polystyrene-b-poly(isocyanoalanine(2- thiophene-3-ylethyl)amide, poly(2-nitrophenylalanine), poly(y-methyl-L-caprolactone), poly(trimethylene carbonate) or hyaluronan.

[0021] Preferably, the hydrophobic monomer is decyl methacrylate.

[0022] Further preferably, the decyl methacrylate is defined as:

[0023] Preferably, there provided a polymersome comprising: a) a hydrophobic monomer; and b) a hydrophilic monomer wherein the hydrophilic monomer is polyethylene glycol (PEG) methacrylate and the hydrophobic monomer is decyl methacrylate.

[0024] Further preferably, there provided a polymersome comprising: a) a hydrophobic monomer; and b) a hydrophilic monomer. wherein the hydrophilic monomer is polyethylene glycol (PEG) methacrylate as defined by: and the hydrophobic monomer is decyl methacrylate as dined by: Optionally, the polymersome is known as C10:PEG.

[0025] Optionally, the molar ratio of hydrophilic:hydrophobic monomer is about 1: 1. Optionally, the molar ratio of hydrophilic:hydrophobic monomer is about 2:1. Optionally, the molar ratio of hydrophilic:hydrophobic monomer is about 3:1. Optionally, the molar ratio of hydrophilic: hydrophobic monomer is about 3:2. Optionally, the molar ratio of hydrophilic:hydrophobic monomer is about 4:1. Optionally, the molar ratio of hydrophilic:hydrophobic monomer is about 4:2. Optionally, the molar ratio of hydrophilic:hydrophobic monomer is about 4:3. Optionally, the molar ratio of hydrophilic:hydrophobic monomer is about 5:1. Optionally, the molar ratio of hydrophilic: hydrophobic monomer is about 5:2. Optionally, the molar ratio of hydrophilic:hydrophobic monomer is about 5:3. Optionally, the molar ratio of hydrophilic:hydrophobic monomer is about 5:4. Optionally, the molar ratio of hydrophilic:hydrophobic monomer is about 6:1. Optionally, the molar ratio of hydrophilic:hydrophobic monomer is about 6:2. Optionally, the molar ratio of hydrophilic: hydrophobic monomer is about 6:3. Optionally, the molar ratio of hydrophilic:hydrophobic monomer is about 6:4. Optionally, the molar ratio of hydrophilic:hydrophobic monomer is about 6:5.

[0026] Preferably, the molar ratio of hydrophilic:hydrophobic monomer is about 5:3.

[0027] Optionally, the polymersome further comprises: c) cholesteryl.

[0028] Optionally, the cholesteryl is:

[0029] Optionally, the polymersome further comprises cholesteryl for enhanced uptake.

[0030] Optionally, the polymersome is known as C10:PEG:Chol.

[0031] Preferably, there provided a polymersome comprising: a) a hydrophobic monomer; b) a hydrophilic monomer; and c) cholesteryl, wherein the hydrophilic monomer is polyethylene glycol (PEG) methacrylate and the hydrophobic monomer is decyl methacrylate. Further preferably, there provided a polymersome comprising: a) a hydrophobic monomer; b) a hydrophilic monomer; and c) cholesteryl, wherein the hydrophilic monomer is polyethylene glycol (PEG) methacrylate as defined by: the hydrophobic monomer is decyl methacrylate as dined by: and cholesteryl defined by:

[0032] Optionally, the polymersome further comprises: d) a quaternary amine monomer.

[0033] Preferably, the quaternary amine monomer is 2-hydroxy-dimethyl[(10-(2-methylprop-2- enoyloxy)decyl]ammonium.

[0034] Preferably, the quaternary amine monomer is quaternary amine monomer defined by: Optionally, the quaternary amine monomer includes tripeptides with lysine as the positive charge on the decyl methacrylate.

[0035] Optionally, the quaternary amine monomer ensures a permanent cationic feature.

[0036] Optionally, the quaternary amine monomer ensures a permanent cationic feature, known as 010: PEG: cat.

[0037] Preferably, there provided a polymersome comprising: a) a hydrophobic monomer; b) a hydrophilic monomer; c) cholesteryl; and d) a quaternary amine monomer, wherein the hydrophilic monomer is polyethylene glycol (PEG) methacrylate and the hydrophobic monomer is decyl methacrylate.

[0038] Preferably, there provided a polymersome comprising: a) a hydrophobic monomer; b) a hydrophilic monomer; c) cholesteryl; and d) a quaternary amine monomer, wherein the hydrophilic monomer is polyethylene glycol (PEG) methacrylate and the hydrophobic monomer is decyl methacrylate.

[0039] Further preferably, there provided a polymersome comprising: a) a hydrophobic monomer; b) a hydrophilic monomer; c) cholesteryl; and d) a quaternary amine monomer, wherein the hydrophilic monomer is polyethylene glycol (PEG) methacrylate as defined by: the hydrophobic monomer is decyl methacrylate as dined by: cholesteryl defined by: and the quaternary amine monomer is 2-hydroxy-dimethyl[(10-(2-methylprop-2- enoyloxy)decyl]ammonium defined by:

[0040] Optionally, the polymersome further comprises: e) a fluorescent component.

[0041] Further preferably, the fluorescent component is bodipy as defined by:

[0042] Preferably, there provided a polymersome comprising: a) a hydrophobic monomer; b) a hydrophilic monomer; c) cholesteryl; d) a quaternary amine monomer; and e) a fluorescent component wherein the hydrophilic monomer is polyethylene glycol (PEG) methacrylate and the hydrophobic monomer is decyl methacrylate.

[0043] Further preferably, there provided a polymersome comprising: a) a hydrophobic monomer; b) a hydrophilic monomer; c) cholesteryl; d) a quaternary amine monomer; and e) a fluorescent component wherein the hydrophilic monomer is polyethylene glycol (PEG) methacrylate, the hydrophobic monomer is decyl methacrylate and the fluorescent component is bodipy.

[0044] Even further preferably, there provided a polymersome comprising: a) a hydrophobic monomer; b) a hydrophilic monomer; c) cholesteryl; d) a quaternary amine monomer; and e) a fluorescent component wherein the hydrophilic monomer is polyethylene glycol (PEG) methacrylate as defined by: the hydrophobic monomer is decyl methacrylate as dined by: cholesteryl defined by: the quaternary amine monomer is 2-hydroxy-dimethyl[(10-(2-methylprop-2- enoyloxy)decyl]ammonium defined by: and the fluorescent component is bodipy as defined by:

[0045] Optionally, the fluorescent component is covalently linked within the polymersome.

[0046] Optionally, the quaternary amine monomer and the fluorescent component are covalently linked to a decyl chain containing methacrylate for a new carbon-carbon bond through polymerisation.

[0047] Optionally, the polymersome has a diameter of 50 - 450 nm. Optionally, the polymersome has a diameter of 75 - 400 nm. Optionally, the polymersome has a diameter of 100 - 350 nm. Optionally, the polymersome has a diameter of 150 - 400 nm. Optionally, the polymersome has a diameter of 200 - 400 nm.

[0048] Preferably, the polymersome has a diameter of 100 - 300 nm.

[0049] According to a second aspect of the present invention, there is provided a polymersome according to the first aspect of the present invention further comprising a composition for use as a medicament. Optionally, the composition comprises a monoclonal antibody (mAb). Optionally, the composition comprises an oligonucleotide. Optionally, the composition comprises siRNAs. Optionally, the composition comprises miRNA. Optionally, the composition comprises steroids. Optionally, the composition comprises antibiotics. Optionally, the composition comprises anti-inflammatories.

[0050] Optionally, the composition comprises any one or more of monoclonal antibodies (mAb), oligonucleotides, siRNAs, miRNA, steroids, antibiotics and anti-inflammatories

[0051] According to a third aspect of the present invention, there is provided a polymersome according to the first aspect of the present invention further comprising a composition for use in treating an ocular disorder.

[0052] Optionally, the composition comprises a monoclonal antibody (mAb). Optionally, the composition comprises an oligonucleotide. Optionally, the composition comprises siRNAs. Optionally, the composition comprises miRNA. Optionally, the composition comprises steroids. Optionally, the composition comprises antibiotics. Optionally, the composition comprises anti-inflammatories.

[0053] Optionally, the composition comprises any one or more of monoclonal antibodies (mAb), oligonucleotides, siRNAs, miRNA, steroids, antibiotics and anti-inflammatories.

[0054] Optionally, the ocular disorder may be selected form any one or more of age-related macular degeneration (AMD), macular oedema, retinopathy, diabetic retinopathy, glaucoma, ocular melanoma, proliferative vitreoretinopathy and uveal melanoma.

[0055] According to a fourth aspect of the present invention, there is provided a polymersome according to the first aspect of the present invention further comprising a composition for treating an ocular disorder.

[0056] Optionally, the composition comprises a monoclonal antibody (mAb). Optionally, the composition comprises an oligonucleotide. Optionally, the composition comprises siRNAs. Optionally, the composition comprises miRNA. Optionally, the composition comprises steroids. Optionally, the composition comprises antibiotics. Optionally, the composition comprises anti-inflammatories.

[0057] Optionally, the composition comprises any one or more of monoclonal antibodies (mAb), oligonucleotides, siRNAs, miRNA, steroids, antibiotics and anti-inflammatories.

[0058] Optionally, the ocular disorder may be selected form any one or more of age-related macular degeneration (AMD), macular oedema, retinopathy, diabetic retinopathy, glaucoma, ocular melanoma, proliferative vitreoretinopathy and uvleal melanoma. According to a fifth aspect of the present invention, there is provided a polymersome according to the first aspect of the present invention further comprising a composition for use in treating a pulmonary disorder.

[0059] Optionally, the composition comprises a monoclonal antibody (mAb). Optionally, the composition comprises an oligonucleotide. Optionally, the composition comprises siRNAs. Optionally, the composition comprises miRNA. Optionally, the composition comprises steroids. Optionally, the composition comprises antibiotics. Optionally, the composition comprises anti-inflammatories.

[0060] Optionally, the composition comprises any one or more of monoclonal antibodies (mAb), oligonucleotides, siRNAs, miRNA, steroids, antibiotics and anti-inflammatories.

[0061] Optionally, the pulmonary disorder may be selected form any one or more of chronic obstructive pulmonary disease (COPD), lung cancers, asthma, infections like pulmonary tuberculosis (TB), cystic fibrosis, pneumonia, and idiopathic pulmonary fibrosis (IPF).

[0062] According to a sixth aspect of the present invention, there is provided a polymersome according to the first aspect of the present invention further comprising a composition for treating a pulmonary disorder.

[0063] Optionally, the composition comprises a monoclonal antibody (mAb). Optionally, the composition comprises an oligonucleotide. Optionally, the composition comprises siRNAs. Optionally, the composition comprises miRNA. Optionally, the composition comprises steroids. Optionally, the composition comprises antibiotics. Optionally, the composition comprises anti-inflammatories.

[0064] Optionally, the composition comprises any one or more of monoclonal antibodies (mAb), oligonucleotides, siRNAs, miRNA, steroids, antibiotics and anti-inflammatories.

[0065] Optionally, the pulmonary disorder may be selected form any one or more of chronic obstructive pulmonary disease (COPD), lung cancers, asthma, infections like pulmonary tuberculosis (TB), cystic fibrosis, pneumonia, and idiopathic pulmonary fibrosis (IPF).

[0066] According to a seventh aspect of the present invention, there is provided a method of enhancing uptake of a composition to a region of a subject by using a polymersome according to the first aspect of the present invention.

[0067] Preferably, said region is an eye or the lung.

[0068] Optionally, the method includes the addition of a viscosity enhancer to prolong contact with the region of the subject. Optionally, the viscosity enhancer is selected from any one of more of carboxy methyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, hydroxy ethyl cellulose, cellulose, hydromellose, xanthan gum, starch, dextrin.

[0069] Optionally, the viscosity enhancer is added at a concentration of 1-10 % w / v. Optionally, the viscosity enhancer is added at a concentration of 2-9 % w / v. Optionally, the viscosity enhancer is added at a concentration of 3-8 % w / v. Optionally, the viscosity enhancer is added at a concentration of 4-7 % w / v. Optionally, the viscosity enhancer is added at a concentration of 4-5 % w / v.

[0070] Preferably, the viscosity enhancer is added at a concentration of 5% w / v.

[0071] Alternatively or additionally, the method includes the addition of a permeation enhancers. Including cyclodextrins, chelating agent such as EDTA, crown ethers, surfactants or cell penetrating peptides.

[0072] Brief Description of the Drawings

[0073] Figure 1 illustrates synthesis of polymers C10:PEG (A), C10:PEG:Chol (B) and C10:PEG:cat (C);

[0074] Figure 2 illustrates 1 H NMR spectra for decyl methacrylate;

[0075] Figure 3 illustrates 1 H NMR spectra for cholesteryl;

[0076] Figure 4 illustrates 1 H NMR spectra for a quaternary amine monomer;

[0077] Figure 5 illustrates 1 H NMR spectra for bodipy;

[0078] Figure s illustrates uptake in human corneal epithelial cells using both neutral and cationic polymersomes in comparison to an identical concentration of the free drug (fitc-cm-dextran);

[0079] Figure 7 illustrates fluorescent microscopy of murine eyes;

[0080] Figure 8 illustrates representative images of healthy mice;

[0081] Figure 9 illustrates individual components of ex-vivo model chamber depicting how they are assembled (A) and a diagram of ex-vivo study set up (B);

[0082] Figure 10 illustrates fluorescent emission from fluorescein collected from the infusion fluid following external application with and without the polymersomal delivery mechanism; and

[0083] Figure 11 illustrates Images of 100 L of 1.25mg / mL of Fitc-CM-dextran through bovine vitreous humour over a 4-hour period; and Figure 12 illustrates the comparison between efficacies of polymersomes encapsulating a monoclonal antibody (mab) compared with electroporation of the same mab.

[0084] Examples

[0085] The invention will now be described with reference to the following non-limiting examples.

[0086] Example 1

[0087] Synthesis of polymers C10:PEG, C10:PEG:Chol and C10:PEG:cat

[0088] The molar ratio of hydrophilic: hydrophobic monomers was kept consistent at 5:3 regardless of additional components added, with the corresponding hydrophobic / philic monomer adjusted to compensate for any addition.

[0089] Methacrylate monomers: were added to a Carius reaction vessel together with: in anhydrous tetrahydrofuran to allow for the final polymers as described in Table 1. The free radical generator 1 ,10 Azobis( cyclohexanecarbonitrile) (AICN, approximately 0.3 molar ratio) was also added to the vessel. After three consecutive freeze- pump-thaw cycles the reaction vessel was sealed under vacuum at 80 °C for 72 h. Hexane (60 ml) was added and the contents centrifuged at 6000 rpm for 5 min, the supernatant was discarded and the resulting pellet re-dissolved in THF:hexane 90:10. This was repeated 3 times. The resulting pellet was dried under vacuum to leave the resultant amphiphilic co-polymers as an oil.

[0090] Table 1. Individual molar ratios used in the preparation of polymers C10:PEG, C10:PEG:Chol and

[0091] C10: PEG: cat

[0092] A schematic representation of the polymers prepared are shown in Figure 1.

[0093] Figure 1A shows polymer C10:PEG, Figure 1 B shows polymer C10:PEG:Chol and Figure 1C shows polymer C10:PEG:cat displaying the hydrophilic polyethylene glycol (PEG), fluorescent bodipy, quaternary amine, hydrophobic decyl and cholesteryl (chol).

[0094] The 1H NMR characterisation of each monomer is shown in Figures 2-5. Figure 2 shows the 1 H NMR spectra for decyl methacrylate (CDCI3, 500 MHz): 5 = 6.10 (s, 1H), 5.57 (s, 1 H), 4.14 (t, J = 7.0 Hz, 2H), 1.95 (s, 3H), 1.68 (t, J = 7.0 Hz, 2H), 1.26 (m, 14H), 0.88 (t, J = 7.0 Hz, 3H) ppm.

[0095] Figure 3 shows the 1 H NMR spectra for cholesteryl (CDCI3, 500 MHz): 5 = 6.59 (s, 1H), 5.73 (s, 1 H), 5.36 (s, 1 H), 5.00 (s, 1 H), 4.48 (s, 1H), 3.43 (s, 2H), 3.36 (s, 2H), 2.29 (m, 2H)

[0096] Figure 4 shows the 1H NMR spectra for a quaternary amine monomer (CDCI3, 500 MHz): 5 = 6.10 (s, 1H), 5.55 (s, 1H), 4.15 (t, J = 6.5 Hz, 4H), 3.75 (s, 2H), 3.53 (t, J = 8.5 Hz, 2H), 3.38 (s, 6H), 1.95 (s, 2H), 1.78-1.60 (m, 8H), 1.42-1.20 (m, 9H) ppm.

[0097] Figure 5 shows the 1 H NMR spectra for bodipy; (CDCI3, 500 MHz): 5 = 8.19 (d, J = 8.0 Hz, 2H), 7.41 (d, J = 8.5 Hz, 2H), 6.10 (s, 1H), 5.99 (s, 2H), 5.54 (s, 1H), 4.36 (t, J = 7.0 Hz, 2H), 4.14 (t, J = 6.5 Hz, 2H), 2.56 (s, 6H), 1.94 (s, 3H), 1.81 (m, 2H), 1.68 (m, 2H), 1.47 (m, 2H), 1.37 (s, 6H), 1.33 (m, 10H) ppm.

[0098] Example 2

[0099] Formulation of Polymersomes

[0100] Initially 500 l polymeric solution in chloroform (5mg / ml) was added to a 5ml round bottom flask and the solution was evaporated to dryness using rotory evaporation to obtain a thin film of polymer. 1 ml chloroform was added and the solution sonicated for 15 mins. A further 500 l of polymeric solution in PBS (5mg / ml) was added followed by the addition of 500 I of PBS and the resulting emulsion was further sonicated for 30 minutes before rotary-evaporation at 35°C to remove the chloroform and obtain the 1ml of polymersomal colloidal suspension (5mg / ml).

[0101] The process was repeated with the addition of various hydrophilic compounds for encapsulation. The encapsulated compound was added after the thin film was produced and evaporated to dryness a second time before the addition of chloroform. Once the formulations were prepared the resulting nanoparticles were characterised using DLS.

[0102] Polymersomes varied in size between 100 - 300 nm with a polydispersity index (PDI) <0.5. The zeta potential of C10:PEG:cat polymersomes was determined to be 8.2+ / - 6.3. Example 3

[0103] Quantification of polymersomal cellular uptake using Human Corneal Epithelial cells

[0104] 5mg / ml polymersomes in water were prepared using the reverse phase evaporation method with 100ug / ml Fitc-CM-dextran encapsulated into the polymersomes. The polymers used were C10:PEG:cat for positive polymersomes and C10:PEG:Chol for neutral polymersomes. Human corneal epithelial cells were cultured in high glucose DMEM media supplemented with 10% FBS, 2% pen / strep and 2% non-essential amino acids) and seeded at a density of 10,000 cells per cell into a 96 well plate. The polymersomes were sterile filtered using 0.45pm filtered syringe and 80pl solution was added to each well after removing the respective amount ensuring the overall volume remained at 200pl per well. Cells were incubated at 37°C for 24hrs, after which all the medium was removed, and cells were washed twice with PBS. Fluorescence of cells was measured in 100pl PBS at Ex 485nm and Em 520nm using the ELISA plate reader. Experiment was repeated two times.

[0105] Polymersomes prepared using both the neutral and positive polymer, were assessed for cellular uptake within Human corneal epithelial (HCE) cells. The fluorescent dye Fitc-CM-dextran was chosen as a biological mimic due to its anionic nature and large molecular size (4kDa) as this is similar to that of biological peptides. The results such in Figure 6, display a fourfold increase in cellular uptake of polymersomal drug when compared to the non encapsulated drug.

[0106] Example 4

[0107] Ex-vivo murine study

[0108] 5mg / ml C10:PEG:cat polymer were formulated into polymersomes using the reverse phase evaporation method described. Murine eyes were collected post-mortem and immediately submerged in PBS before being dipped in the polymersome solution for 1 hour. After which they were removed and dipped in 4% formaldehyde for 30 mins. Then they were dipped in 30% Sucrose solution overnight. Then frozen in OCT blocks and stored in -80 until required for analysis. Frozen blocks were then cryo-sectioned and fixed onto glass slides where they were stored at -80°C without any further treatment. Slides were removed from the freezer immediately before imaging. A few drops of fluoroshield having DAPI was added onto the slides and covered using a coverslip and imaged. For DAPI exposure was 1 sec and gain 1.2x, for FITC exposure was 2sec and gain 2x.

[0109] Polymer C10:PEG:cat was used in the ex-vivo murine study. As this polymer contains the fluorescent bodipy within the backbone of the structure, no imaging compound was required in order to visualise uptake of the polymersome. Murine eyes were excised post mortem and submerged within a solution containing the polymersome. The eyes were then frozen, sliced and fixed using paraformaldehyde before being visualised using a fluorescent microscope. The resulting images are displayed in Figure 7. Figure 7A and Figure 7D display the cornea and retina with dapi stain respectively, dapi is a nuclei stain known to cross the ocular membranes, and therefore used as a guide Figure 7B and Figure 7E display the cornea and retina of the control experiment, as expected there a small amount of inherent green fluorescence is apparent from these images. Finally, Figure 7C and Figure 7D display the cornea and retina of the polymersome containing solution following 1 hr of incubation. The results display a significant increase in the both corneal and retinal uptake of the polymersome.

[0110] Example 5

[0111] In-vivo murine study

[0112] 5mg / ml Polymersomes were prepared in deionised water using the cationic polymer C10:PEG:chol by the reverse phase evaporation method with a porphyrin sulphonate dye (50ul / ml PS) for imaging. 1ml of these PS were freeze dried along with similar concentration for free dye solution without polymer. At the time of study, these formulations were reconstituted in 50ul of 1.5% Sodium CMC in water. NOD-SCID mice were anesthetised by IP Hypnorm-Hypnovel injection at 0 hrs (1 OOul), 3 hrs (50ul) and 6 hrs (50ul) before imaging. 5u I drop was added to mouse eye accordingly and imaged at 0 mins, 30 mins, 1 hr, 3 hrs, 6hrs and 24 hrs using a Perkin Elmer I VI S spectrum in vivo imaging with an excitation wavelength of 430nm and emission collected above 600nm.

[0113] Polymersomes were prepared containing an imaging agent (bodipy) in a 8% CMC solution. A 5 L volume of solution containing 0.5mgs polymer and 3.3 g bodipy was applied to the eye of the mouse. Figure 8 displays representative images of healthy mice that were administered a formulation containing polymersome loaded with bodipy on right eye with the left receiving no solution.

[0114] Images recorded 15 minutes and 3 hours after application and display clearly an enhanced fluorescence from the polymersomal formulations.

[0115] Example 6

[0116] Ex-vivo bovine study

[0117] A bovine ex vivo organ culture model was used to assess transfer of the polymersome suspension across the anterior of the eye. Bovine eyes were used on the day of collection, the anterior segment of the eye was removed and fixed to a bespoke chamber with perfusion culture medium infused with a syringe pump at a constant infusion rate of 2.5 pl_ / min. The exterior of the eye was then subjected to 100 l of a C10:PEG polymersomal suspension containing 5mg / ml polymer and 0.5mg / ml Fluoresceine. The perfusion media was collected over a period of 24hrs and analysed using spectroscopy, excitation 490nm, emission 520nm. Using a bespoke ex-vivo bovine model, as depicted in Figure 9, a solution of either free fluorescein or encapsulated fluorescein was applied to the exterior of the eye. The chamber was perfused with culture media at a constant rate of 2.5pl / min and this was collected and analysed for fluorescein present.

[0118] Figure 10 shows the emission of fluorescein collected over a period of 4 hrs with the inset recording of the pooled fluorescein emission from 4 - 24 hrs.

[0119] In order to confirm transport across the posterior section of the eye of the polymersome, the infusion fluid was also analysed by dynamic light scattering with the particles confirmed as present within this fluid.

[0120] Example 7

[0121] Ex-vivo bovine vitreous humour (VH) study

[0122] Bovine eyes were collected and the vitreous humour removed within 3 hrs. The VH was distributed into scintillation vials in 20 ml portions and used immediately. The vials were subjected to either free fitc-CM-dextran (1.25mg / mL) or polymersomal fitc-CM-dextran, at the same concentration, using the polymer C10:PEG at a final concentration of 5 mg / mL. 100 L of either solution was added carefully to the top of the vitreous humour, and maintained on an orbital shaker at 50 rpm at 37°C. The fluorescence of the fitc-CM-dextran was monitored using a handheld UV lamp at 354 nm and images recorded on a Samsung galaxy android phone.

[0123] In addition to movement across the exterior barriers of the eye, we were also interested to establish movement of our polymersomes within the interior and specifically through the vitreous humour. We have successfully shown our novel drug delivery systems to move through a bovine vitreous humour (VH) at an increased rate when compared to the free drug (Figure 11 ). When analysing the rate of movement through the VH, the free drug only managed a rate of 1.7 mm / hr, whereas the same drug encapsulated within our neutral polymersome travelled at a rate of 7mm / hr.

[0124] Figure 11 shows images of 100 L of 1.25mg / mL of Fitc-CM-dextran through bovine vitreous humour over a 4-hour period. The vials are free fitc-CM-dextran and neutral Ps at polymer concentration of 5 mg / mL. The experiment was carried out on an orbital shaker at 50 rpm and maintained at 37°C Example 8

[0125] Comparison study with electroporation as a control for cellular uptake

[0126] Polymersomes were prepared using C10:PEG and formulated to contain either the antibodies IgG, AB1 , AB2 or AB3 (antibody AB of undisclosable nature) with the hydrodynamic radius confirmed as between 200-400 nm with PDA <0.5. The resulting colloidal suspensions (5 mg / ml polymer and 0.176mg / ml IgG or AB) were added to human leukemia monocytic cells THP-1 at a final concentration on cells 1.25 mg / ml polymer and 0.044mg / ml IgG or AB.

[0127] THP-1 cells were treated with lipopolysaccharide (LPS) for 3hrs and then either electroporated with various suspensions of IgG control and AB samples or incubated with the same concentration of AB encapsulated within the polymersome. Post-electroporation (6 hr), cells were treated with nigericin for 1 hr. Supernatants were removed and IL-1 p was quantified using an ELISA kit. It was concluded that the polymersomal formulation performed to the same extend as electroporation, indicating the ability to encapsulate and delivery Abs.

[0128] Figure 12 shows the IL-1 quantified secretion from the supernatant of THP-1 cells subjected to either the AB only, the AB with electroporation to aid cell delivery, or the AB encapsulated within a nanoparticle (polymersome) without electroporation.

Claims

Claims1. A polymersome comprising: a) a hydrophobic monomer; and b) a hydrophilic monomer wherein the hydrophilic monomer is polyethylene glycol (PEG) methacrylate and the hydrophobic monomer is decyl methacrylate.

2. The polymersome according to claim 1, wherein the molar ratio of hydrophilic:hydrophobic monomer is about 5:3.

3. The polymersome according to claim 1 or claim 2 further comprising cholesteryl.

4. The polymersome according to any of claims 1-3 further comprising a quaternary amine monomer.

5. The polymersome according to claim 4, wherein the quaternary amine monomer is 2- hydroxy-dimethyl[(10-(2-methylprop-2-enoyloxy)decyl]ammonium.

6. The polymersome according to any of claims 1-5 further comprising a fluorescent component.

7. The polymersome according to claim 6, wherein the fluorescent component is bodipy.

8. The polymersome according to any of claims 1-7, wherein the polymersome has a diameter of 50 - 450 nm.

9. The polymersome according to any of claims 1-8 further comprising a composition for use as a medicament.

10. The polymersome according to any of claims 1-8 further comprising a composition for use in treating an ocular disorder and / or a pulmonary disorder.

11. The polymersome according to claim 10, wherein the ocular disorder is selected from any one or more of age-related macular degeneration (AMD), macular oedema, retinopathy, diabetic retinopathy, glaucoma, ocular melanoma, proliferative vitreoretinopathy and uvleal melanoma.

12. The polymersome according to claim 10, wherein the pulmonary disorder is selected form any one or more of chronic obstructive pulmonary disease (COPD), lung cancers, asthma,infections like pulmonary tuberculosis (TB), cystic fibrosis, pneumonia, and idiopathic pulmonary fibrosis (IPF).

13. The polymersome according to any of claims 9-11 wherein the composition comprises any one or more of monoclonal antibodies (mAb), oligonucleotides, siRNAs, miRNA, steroids, antibiotics and anti-inflammatories.

14. The polymersome according to any of claims 1-8 for enhancing uptake of a composition to a region of a subject.

15. The polymersome according to claim 14 further comprising a viscosity enhancer.

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