Biopolymers for encapsulation

Abstract

The present invention is inter alia directed to amphiphilic pH-sensitive polymers, wherein the polymer is soluble in an aqueous environment at a trigger physiological pH. Such polymers are suitable for encapsulation of a cargo, such as a biologically active constituent, to protect the cargo from undesirable interactions with co-formulated substances, such as in a parenteral formulation. The present invention is also directed to self-assembled particles, compositions, immunogenic compositions, and vaccines comprising such. Self-assembled particles, compositions, immunogenic compositions, and vaccines provided herein are suitable for use in medicine, in particular for the treatment or prevention of an infection, caused directly or indirectly by a pathogen.

Description

[0001] BIOPOLYMERS FOR ENCAPSULATION

[0002] TECHNICAL FIELD

[0003] The present invention is inter alia directed to amphiphilic pH-sensitive polymers suitable for encapsulation of a cargo, such as a biologically active constituent, to protect the cargo from undesirable interactions with co-formulated substances, such as in a parenteral formulation. The present invention is also directed to self-assembled particles and immunogenic compositions comprising such. Immunogenic compositions provided herein are suitable for use in medicine, in particular for the treatment or prevention of an infection, caused directly or indirectly by a pathogen.

[0004] BACKGROUND

[0005] Many medicinal compositions, such as drug or vaccine compositions, are combination products which contain two or more biologically active constituents e.g., pharmaceutical substances or antigens. Such compositions may exhibit a synergistic effect or may offer advantages such as increased compliance with a treatment regimen e.g., due to a reduced total number of administrations, especially in the case of paediatric immunisation schedules.

[0006] The respective biologically active constituents may have associated with them other constituents such as adjuvants, in the case of vaccines, or pharmaceutically acceptable excipients, often in an aqueous formulation. It is known than when co-formulated ( / .e., formulated together as a single composition such as a parenteral formulation), one biologically active constituent may interact with another biologically active constituent, or with an associated constituent, such as an adjuvant, an excipient or even water, present in the formulation. Such interaction may have a deleterious impact on the biological effect mediated by at least one of the interacting biologically active constituents (such impact being deleterious relative to the biological effect that such biologically active constituent would mediate if formulated alone i.e., as the some biologically active constituent.

[0007] In the case of vaccines, such deleterious interaction may manifest as a physical or biochemical incompatibility, such as an effect on the stability of the biologically active constituent, and / or as an in vivo phenomenon adversely impacting on the immune response elicited by the constituent (known as immunological interference). For example, in the paediatric combination vaccine INFANRIX HEXA, the lyophilized Hib (Haemophilus influenzae type B) antigen must be packaged separately from the aqueous formulation containing DTPa (diphteria toxoid, tetanus toxoid and acellular pertussis ant / gensj / aluminium hydroxide because: (a) the Hib polysaccharide polyribosylribitol (PRP) in the Hib-antigen conjugate is susceptible to degradation in an aqueous environment; and (b) the reaction of PRP with aluminium hydroxide causes flocculation, with consequent masking of PRP epitopes. The vaccine must be therefore reconstituted by medical personnel at the time of administration. A vaccine with all components in a single container would offer advantages such as simplified filling / packaging, transport / storage, and administration.

[0008] Therefore, there remains a need to provide solutions to the problem of how to combine physically, biochemically and / or immunologically incompatible constituents of medicinal compositions into one-part liquid, aqueous compositions which can be packaged and stored in single containers, while avoiding the deleterious consequences of such incompatibilities.

[0009] SUMMARY OF THE INVENTION

[0010] In a first aspect, the invention provides an amphiphilic pH-sensitive polymer, wherein the polymer is soluble in an aqueous environment at a trigger physiological pH.

[0011] The amphiphilic pH-sensitive polymer of the invention is suitable for encapsulation of a cargo, such as a biologically active constituent, to protect the cargo from undesirable interactions with co-formulated substances, such as in a parenteral formulation. The amphiphilic polymer is engineered to be sensitive f / .e., responsive) to pH in an aqueous environment so that it is soluble at a trigger physiological pH, such as at the injection site tissue of the subject.

[0012] In some embodiments, the amphiphilic pH-sensitive polymer of the invention is insoluble in an aqueous environment at a sub-physiological pH, so that it is insoluble at the pH of the final medicinal composition (e.g., during storage).

[0013] Also provided herein is a polymer comprising at least one repeat unit according to formula (A):

[0014] wherein at least one of Ri, R2, R3, R4, R5 and R6is a hydrophobic group, and

[0015] - when R1 is a hydrophobic group, Bi is a type of bond, p is 0 or 1 , and when p is 1 , Li is a linker and B2is a type of bond,

[0016] - when R2is a hydrophobic group, B3is a type of bond, q is 0 or 1 , and when q is 1 , l_2is a linker and B4is a type of bond,

[0017] - when R3is a hydrophobic group, B5is a type of bond, r is 0 or 1 , and when r is 1 , l_3is a linker and Be is a type of bond,

[0018] - when R4is a hydrophobic group, B7is a type of bond, s is 0 or 1 , and when s is 1 , l_4is a linker and B8is a type of bond,

[0019] - when R5is a hydrophobic group, B9is a type of bond, t is 0 or 1 , and when t is 1 , l_5is a linker and B10 is a type of bond, and / or

[0020] - when Re is a hydrophobic group, Bn is a type of bond, u is 0 or 1 , and when u is 1 , Le is a linker and BI2is a type of bond.

[0021] In a second aspect, the invention provides a self-assembled particle comprising at least one amphiphilic pH-sensitive polymer as defined herein. In some embodiments, the selfassembled particle further comprises a cargo, such as a biologically active constituent of a medicinal composition, being encapsulated within said at least one amphiphilic pH-sensitive polymer, as defined herein, in an aqueous environment having a sub-physiological pH.

[0022] In the context of a composition comprising such, the particle can protect the cargo contained therein from potentially deleterious interactions with co-formulated substances of the composition (e.g., a biologically active constituent or an associated constituent, such as an adjuvant) external to the particle, during storage. Further, such particle can release said cargo in response to being administered such that the cargo is then free to exert its effect within the body of the recipient subject. In a third aspect, the invention provides a composition, an immunogenic composition or a vaccine comprising at least one, suitably a plurality of, self-assembled particle as defined herein in an aqueous environment, wherein said composition, immunogenic composition or vaccine is of sub-physiological pH.

[0023] In a fourth aspect, the invention relates to self-assembled particles, compositions, immunogenic compositions, or vaccines as described herein, for use as a medicament, such as prophylactically or therapeutically.

[0024] In a fifth aspect, the invention provides self-assembled particles, compositions, immunogenic compositions, or vaccines as described herein, for use in the treatment or prevention of a disorder or a disease, such as an infection, caused directly or indirectly by a pathogen, or of a pathology associated with immunologically distinct host cells such as cancer.

[0025] In a sixth aspect, the invention relates to a method of treating or preventing a disorder or a disease, such as an infection, caused directly or indirectly by a pathogen, or of a pathology associated with immunologically distinct host cells such as cancer, wherein the method comprises applying or administering to a subject in need thereof the self-assembled particles, compositions, immunogenic compositions, or vaccines as described herein.

[0026] In a seventh aspect, the invention provides a method of eliciting an immune response, wherein the method comprises applying or administering to a subject in need thereof an effective amount of the self-assembled particles, compositions, immunogenic compositions or vaccines as described herein.

[0027] In an eighth aspect, the invention provides a method for preventing or reducing interaction between a cargo, such as a biologically active constituent, and co-formulated substances of an aqueous environment, such as in a parenteral formulation, comprising:

[0028] (i) forming said at least one self-assembled particle, as described herein, comprising said cargo; and

[0029] (ii) formulating said at least one self-assembled particle, as described herein, in said aqueous environment, comprising any necessary adjustment to render said environment of sub-physiological pH.

[0030] Also provided herein is a method for preventing or reducing interaction between a cargo, such as a biologically active constituent, and co-formulated substances of an aqueous environment, such as in a parenteral formulation, of sub-physiological pH, comprising:

[0031] (i) forming said at least one self-assembled particle, as described herein, comprising said cargo; and (ii) formulating said at least one self-assembled particle, as described herein, in said aqueous environment.

[0032] DESCRIPTION OF THE FIGURES

[0033] FIG. 1 1 H-NMR spectroscopy analysis of the synthesized conjugates. The 1 H-NMR spectra were recorded on a Bruker Avance III™ HD 500 MHz NMR spectrometer using a 5 mm TCI cryo probe at 298 K. Spectra were processed using MestreNova software.

[0034] FIG. 2 Qualitative analysis of the behaviour of HA-SA 30 % w / w conjugate in phosphate buffer at different pH.

[0035] FIG. 3 Qualitative analysis of the behaviour of HA-SA 10 % w / w conjugate in phosphate buffer at different pH.

[0036] FIG. 4 Qualitative analysis of the behaviour of HA-SA 5 % w / w conjugate in phosphate buffer at different pH.

[0037] FIG. 5 Qualitative analysis of the behaviour of HA-PEG-SA 30 % w / w conjugate in phosphate buffer at different pH.

[0038] FIG. 6 Qualitative analysis of the behaviour of HA-PEG-SA 10 % w / w conjugate in phosphate buffer at different pH.

[0039] FIG. 7 Transmissivity test to study pH dependent solubility of conjugate solutions (HA- SA 5 % w / w, HA-SA 10 % w / w, and HA-SA 30 % w / w) at different pH.

[0040] FIG. 8 Turbidity analysis to study pH dependent solubility of conjugate solutions (HA- SA 5 % w / w, HA-SA 10 % w / w, and HA-SA 30 % w / w) at different pH.

[0041] FIG. 9 TEM analysis to assess the coating of conjugates over the BSA (Set C - BSA + HA-SA 10 % w / w), at pH 5.5.

[0042] FIG. 10 TEM analysis to assess the coating of conjugates over the BSA (Set A - BSA alone), at pH 5.5.

[0043] FIG. 11 In vitro release of BSA at pH 5.5: BSA alone and BSA with HA-SA 10 % w / w.

[0044] FIG. 12 In vitro release of BSA at pH 7.4: BSA alone and BSA with HA-SA 10 % w / w.

[0045] FIG. 13 In vitro release of BSA from HA-SA 10 % w / w stored for 0, 3 and 7 days. FIG. 14A-B In vitro release of BSAfrom HA-SA 10 % w / w stored at 3 (A) and 7 (B) days in buffer.

[0046] FIG. 15 In vitro release of BSAfrom HA-SA 10 % w / w at concentration of 1 and 2 mg / ml in water.

[0047] DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention is concerned with the stable storage and delivery of medicinal compositions, such as drug or vaccine compositions, which contain, in a one-part aqueous liquid composition, substances ( / .e., constituents) which are incompatible such as mutually physically or biochemically reactive or, if the composition is an immunogenic composition or a vaccine, prone to interfere immunologically. This is achieved by sequestering at least one of the incompatible constituents within particles, such as micro- or nano- particles, in order than it is not exposed to the surrounding environment ( / .e., the aqueous composition) containing the constituent with which it is incompatible. The constituent sequestered within such particles is referred to herein as the "cargo".

[0049] The inventors overcame the drawbacks of the prior art by providing an amphiphilic pH- sensitive polymer which is soluble in an aqueous environment at a trigger physiological pH.

[0050] Amphiphilic polymers are copolymers including both hydrophilic and hydrophobic chains. This category of polymers has the property of displaying a self-assembling behaviour in selective solvents (e.g., in an aqueous environment). This behaviour is triggered by hydrophilic-hydrophobic interactions among the polymer chains, which form micellar structures in the nano- and / or micro- scale (e.g., self-assembled particles).

[0051] The polymer of the invention is engineered to be pH-sensitive. “pH-sensitive” which as referred to herein means being responsive to a pH trigger such that outside of a pre-determined range of pH (e.g., below a pre-determined threshold pH such as at sub-physiological pH) the polymer remains insoluble in an aqueous environment, whereas within the pre-determined range of pH (e.g., at physiological pH) the polymer become soluble in an aqueous environment. In the context of self-assembled particles comprising such, outside of a pre-determined range of pH (e.g., below a pre-determined threshold pH such as at sub-physiological pH) the cargo remains sequestered within the particles, whereas within the pre-determined range of pH (e.g., at physiological pH) the cargo is no longer sequestered and is accessible to the surrounding environment. By tuning the polymer to have an appropriate sensitivity to pH with respect to the local pH of the target administration site of the intended subject, the delivery (i.e., accessibility of the cargo following the release from the particles) occurs only after administration. pH-Sensitive polymers are a class of polyelectrolytes with ionizable groups in their backbones, side groups, or end groups. These polymers may undergo pH-sensitive conformational changes in three different ways: a) dissociation, b) destabilization (e.g., via collapse or swelling), and c) changes of partition coefficient (e.g., dissolution).

[0052] It has been found that the amphiphilic pH-sensitive polymers of the invention are suitable for encapsulation of a cargo, such as a biologically active constituent, to protect the cargo from undesirable interactions with co-formulated substances, such as in a parenteral formulation.

[0053] In particular, or in addition, the amphiphilic pH-sensitive polymers of the invention are responsive to a pH trigger such that outside of a pre-determined range of pH (e.g., below a pre-determined threshold pH such as at sub-physiological pH) the polymer remains insoluble in an aqueous environment, whereas within the pre-determined range of pH (e.g., at physiological pH) the polymer become soluble in an aqueous environment.

[0054] In particular, or in addition, the amphiphilic pH-sensitive polymers of the invention have an appropriate sensitivity to pH with respect to the local pH of the target administration site, such as at the injection site tissue, of the subject.

[0055] In particular, or in addition, the amphiphilic pH-sensitive polymers of the invention display a self-assembling behaviour in an aqueous environment, to provide self-assembled particles according to the invention, such as micro- and / or nano- particles.

[0056] In particular, or in addition, the amphiphilic pH-sensitive polymers of the invention allow to combine physically, biochemically and / or immunologically incompatible constituents of medicinal compositions, such as drug or vaccine compositions.

[0057] In particular, or in addition, the amphiphilic pH-sensitive polymers of the invention are suitable for use in combination products which contain one or more biologically active constituents e.g., pharmaceutical substances or antigens, and / or other associated constituents such as adjuvants, in the case of vaccines, or pharmaceutically acceptable excipients.

[0058] In particular, or in addition, the amphiphilic pH-sensitive polymers of the invention allow to provide one-part liquid, aqueous compositions which can be packaged and stored in single containers, while avoiding the deleterious consequences of potential incompatibilities.

[0059] Suitably, the amphiphilic pH-sensitive polymers of the invention have at least some of the following advantageous features:

[0060] • Biodegradable;

[0061] • FDA approved;

[0062] • Inert towards the cargo e.g., a biologically active constituent such as an antigen; • Insoluble at a pH below 6.8 e.g., 5.5;

[0063] • Soluble at a pH of 7.4; and / or

[0064] • Approved as injectable material.

[0065] Polymer:

[0066] Therefore, in a first aspect, the invention provides an amphiphilic pH-sensitive polymer, wherein the polymer is soluble in an aqueous environment at a trigger physiological pH.

[0067] In some embodiments, the polymer of the invention is insoluble in an aqueous environment at a sub-physiological pH, such that it is insoluble at the pH of the final medicinal composition (e.g., during storage).

[0068] As used herein, the meaning of “sub-physiological pH” and “physiological pH” is with respect to the local physiology of the intended recipient subject of the polymer (as formulated into an administrable composition) e.g., the pH of the tissue of the subject’s injection site. In some embodiments, “sub-physiological pH” and “physiological pH” respectively mean sub- physiological and physiological with respect to the pH of human tissue, in particular adult and / or infant muscle.

[0069] In some embodiments, said aqueous environment at sub-physiological pH comprises a buffer, such as a saline phosphate, phosphate, Tris, borate, succinate, histidine, citrate and / or maleate buffer.

[0070] In some embodiments, said physiological pH is at or above 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6 or 7.7, or comprises a range with these respective values as the lower limit and an upper limit selected from 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0.

[0071] In some embodiments, said sub-physiological pH is at or below 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1 , 6.0, 5.9, 5.8, 5.7, 5.6 or 5.5, or comprises a range with these respective values as the upper limit and a lower limit selected from 6.6, 6.5, 6.4, 6.3, 6.2, 6.1 , 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1 , 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.3, 4.2, 4.1 or 4.0.

[0072] In some embodiments, said sub-physiological pH and physiological pH differ from each other by at least 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .1 , 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9 or 2.0 pH units i.e., the physiological pH is at least 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 pH units higher than the sub- physiological pH.

[0073] In some embodiments, subjecting the polymer to a trigger physiological pH occurs at a temperature of around the body temperature of the recipient, in particular at around the temperature of injection site tissue of the recipient, such as at or around 37 °C in the case of a human.

[0074] In some embodiments, the polymer, when in the aqueous environment at sub-physiological pH, is in an at least partially protonated state and / or may have an approximately or exactly neutral charge and be insoluble.

[0075] In some embodiments, the polymer has a pKa below the threshold physiological pH. In some embodiments, the polymer has a pKa comprised between 3 and 4, optionally of 4.

[0076] In some embodiments, the polymer is intact and / or stable at said sub-physiological pH.

[0077] In some embodiments, on subjecting the polymer to said trigger physiological pH, the polymer is subjected to a change of partition coefficient (e.g., dissolution).

[0078] In some embodiments, on subjecting the polymer to said trigger physiological pH, the polymer is at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 % dissolved. In some embodiments, on subjecting the polymer to said trigger physiological pH, the polymer is at least 30 % dissolved.

[0079] In some embodiments, on subjecting the polymer to said trigger physiological pH, the polymer is subjected to substantially or completely dissociation.

[0080] In some embodiments, on subjecting the polymer to said trigger physiological pH, the polymer is at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 % dissociated. In some embodiments, on subjecting the polymer to said trigger physiological pH, the polymer is at least 30 % dissociated.

[0081] In some embodiments, on subjecting the polymer to said trigger physiological pH, the polymer is subjected to substantially or completely destabilization e.g., via collapse or swelling.

[0082] In some embodiments, on subjecting the polymer to said trigger physiological pH, the polymer is at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 % destabilized. In some embodiments, on subjecting the polymer to said trigger physiological pH, the polymer is at least 30 % destabilized.

[0083] In some embodiments, on subjecting the polymer to said trigger physiological pH, the polymer is substantially or completely, such as at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 %, optionally at least 30 %, dissolved and / or dissociated and / or destabilized within 24 hours or less, such as within 20, 16, 12, 10, 8, 6, 4, 2 or 1 hours or 45, 30, 15, 10 or 5 minutes. These values may represent the upper limit of a range which is bounded at the lower end by a value selected from 16, 12, 10, 8, 6, 4, 2 or 1 hours or 45, 30, 15, 10, 5 or 1 minutes. The solubility of a substance (e.g., a solid) is the amount of that substance being required to form a saturated solution in a given amount of solvent, at a specified temperature. Solubility may be measured in grams of solute per 100 ml of solvent.

[0084] In some embodiments, the polymer has a solubility comprised between 10 and 1000 mg per 100 ml at trigger physiological pH. In some embodiments, the polymer has a solubility of at least 10, 20, 30, 40, 50, 60, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mg per 100 ml at trigger physiological pH, or comprises a range with these respective values as the upper limit and a lower limit selected from 10, 20, 30, 40, 50, 60, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mg per 100 ml at trigger physiological pH. Polymers are synthetic or natural substances or materials that are constituted by repeat units derived from one or more species of monomers.

[0085] In some embodiments, the polymer is a biopolymer.

[0086] Biopolymers are natural polymers produced by the cells of living organisms. Like other polymers, biopolymers consist of repeating units (i.e. monomers) that are covalently bonded in chains to form larger molecules. There are three main classes of biopolymers, classified according to the monomers used and the structure of the biopolymer formed: polynucleotides, polypeptides, and polysaccharides.

[0087] In some embodiments, the biopolymer is selected from the group consisting of polynucleotides, polypeptides, and polysaccharides.

[0088] In some embodiments, the biopolymer is a polysaccharide.

[0089] In some embodiments, the polymer is biocompatible, suitably with the human body.

[0090] In some embodiments, the polymer is biodegradable, bioresorbable, bioabsorbable and / or excretable, suitably in or from the human body.

[0091] In some embodiments, the polymer is biodegradable.

[0092] By the term “biodegradable” is meant the ability of a biomaterial to decompose in a nontoxic matter in the biological body after its designed purpose.

[0093] In some embodiments, the polymer is biodegradable enzymatically.

[0094] In some embodiments, the polymer is injectable, suitably in the human body.

[0095] In some embodiments, the polymer is suitable for parenteral administration, such as intradermal, subcutaneous, or intramuscular administration. The polymer of the invention is an amphiphilic polymer. In some embodiments, the polymer displays a self-assembling behaviour in said aqueous environment at said sub-physiological pH.

[0096] In some embodiments, at least one repeat unit within the polymer comprises a hydrophilic scaffold functionalized with at least one hydrophobic group.

[0097] The terms “functionalized”, “derivatized” and “substituted” are used interchangeably herein.

[0098] In some embodiments, the hydrophilic scaffold is a repeat unit of a poly-anionic polymer.

[0099] In some embodiments, the functionalization is done on at least one carboxylic acid group and / or at least one sulphonamide group of at least one repeat unit of the poly-anionic polymer.

[0100] In some embodiments, the functionalization is done on at least one carboxylic acid group of at least one repeat unit of the poly-anionic polymer.

[0101] In some embodiments, the hydrophilic scaffold is a repeat unit of a glycosaminoglycan.

[0102] In some embodiments, the hydrophilic scaffold is a repeat unit of hyaluronic acid.

[0103] In some embodiments, the functionalization is done on the carboxylic acid group at least one repeat unit of the hyaluronic acid.

[0104] In some embodiments, the hydrophobic group comprises an alkyl chain comprising from 6 to 36 carbon atoms, optionally from 10 to 18 carbon atoms. Such alkyl chain may be of 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 carbon atoms. In particular, they are of 17 or 18 carbon atoms.

[0105] In some embodiments, the hydrophobic group is derived from a compound selected from the group consisting of poly lactic-co-glycolic acid (PLGA), polylactic acid (PLA), and lipids.

[0106] In some embodiments, the hydrophobic group is a lipophilic group.

[0107] In some embodiments, the lipophilic group is derived from a compound selected from the group consisting of phospholipids, triglycerides, triacylglycerols, sterols, and fatty acids.

[0108] In some embodiments, the lipophilic group is derived from a compound selected from distearoylphosphatidylcholine / 1 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine DOPE), 8-[(2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1 -octylnonyl ester / SM-102, [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) / ALC-0315, stearyl amine, and stearic acid. In some embodiments, the lipophilic group is derived from a phospholipid.

[0109] In some embodiments, the lipophilic group is derived from a fatty acid.

[0110] In some embodiments, the fatty acid is stearyl amine or stearic acid.

[0111] In some embodiments, the hydrophobic group is directly grafted to the hydrophilic scaffold.

[0112] In some embodiments, the hydrophobic group is derived from stearyl amine, and the hydrophilic scaffold is a repeat unit of hyaluronic acid.

[0113] In some embodiments, stearyl amine is grafted on said the carboxylic acid group of hyaluronic acid.

[0114] In some embodiments, the hydrophobic group is grafted to the hydrophilic scaffold via a linker.

[0115] In some embodiments, the linker is hydrophilic.

[0116] In some embodiments, the linker is a polyethylene glycol (PEG) linker.

[0117] In some embodiments, the hydrophobic group is derived from stearic acid, and the hydrophilic scaffold is a repeat unit of hyaluronic acid.

[0118] In some embodiments, stearic acid is grafted on the carboxylic acid group of hyaluronic acid via said PEG linker.

[0119] In some embodiments, the polymer comprises 1 to 100 % by mole of repeat units that are functionalized.

[0120] By the term “mole” (or mol) is meant the base unit of measurement in the International System of Units (SI) for amount of substance, a quantity proportional to the number of elementary entities of a substance. One mole is an aggregate of exactly 6.02214076 x 1023elementary entities, which can be atoms, molecules, ions, ion pairs, or other particles.

[0121] In some embodiments, the polymer comprises 1 to 50 % by mole of said functionalized repeat units, such as 5%, 10% or 30% by mole.

[0122] In some embodiments, the hydrophobic group is derived from stearic acid, the hydrophilic scaffold is a repeat unit of hyaluronic acid, and the polymer comprises 1 to 50 % by mole of said functionalized repeat units, such as 5%, 10% or 30% by mole. In some embodiments, the polymer comprises 1 to 30 % by mole of said functionalized repeat units. Optionally, the polymer comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 % by mole of said functionalized repeat units. In some embodiments, the polymer comprises 5 % by mole of said functionalized repeat units. In some embodiments, the polymer comprises 10 % by mole of said functionalized repeat units. In some embodiments, the polymer comprises 30 % by mole of said functionalized repeat units.

[0123] In some embodiments, the hydrophobic group is derived from stearyl amine, the hydrophilic scaffold is a repeat unit of hyaluronic acid, and the polymer comprises 1 to 50 % by mole of said functionalized repeat units, such as 5%, 10% or 30% by mole. In some embodiments, the polymer comprises 1 to 30 % by mole of said functionalized repeat units. Optionally, the polymer comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 % by mole of said functionalized repeat units. In some embodiments, the polymer comprises 5 % by mole of said functionalized repeat units. In some embodiments, the polymer comprises 10 % by mole of said functionalized repeat units. In some embodiments, the polymer comprises 30 % by mole of said functionalized repeat units.

[0124] In some embodiments, the hydrophilic scaffold functionalized with at least one hydrophobic group is according to formula (A): wherein at least one of Ri, R2, R3, R4, R5 and Re is a hydrophobic group, and

[0125] - when R1 is a hydrophobic group, Bi is a type of bond, p is 0 or 1 , and when p is 1 , Li is a linker and B2is a type of bond,

[0126] - when R2is a hydrophobic group, B3is a type of bond, q is 0 or 1 , and when q is 1 , l_2is a linker and B4 is a type of bond,

[0127] - when R3is a hydrophobic group, B5is a type of bond, r is 0 or 1 , and when r is 1 , l_3is a linker and B6is a type of bond,

[0128] - when R4is a hydrophobic group, B7is a type of bond, s is 0 or 1 , and when s is 1 , l_4is a linker and B3is a type of bond,

[0129] - when R5is a hydrophobic group, B9is a type of bond, t is 0 or 1 , and when t is 1 , l_5is a linker and B10 is a type of bond, and / or

[0130] - when R6is a hydrophobic group, Bn is a type of bond, u is 0 or 1 , and when u is 1 , l_6is a linker and B12 is a type of bond. In some embodiments, Ri may not be a hydrophobic group. In this case, p is 0 and -B1-R1 is -NH-(CO)-CH3. Additionally, or alternatively, R2may not be a hydrophobic group. In this case, q is 0 and -B3-R2 is -OH. Additionally, or alternatively, R3may also not be a hydrophobic group. In this case, r is 0 and -Bs-R3is -OH. Additionally, or alternatively, R4 may not a hydrophobic group. When that is the case, s is 0 and -B7-R4 is -OH. Additionally, or alternatively, R5may not be a hydrophobic group. When that is the case, t is 0 and -B9-R5 is - OH. Additionally, or alternatively, there are instances where R6is not a hydrophobic group. In these cases, u is 0 and -Bn-Re is -(CO)-OH or a salt thereof, such as -(CO)-ONa, or -(CO)- OK.

[0131] Saturated and unsaturated alkyl chains are components of various organic molecules, including lipids, and play significant roles in determining the physical and chemical properties of these compounds.

[0132] Saturated alkyl chains are carbon-carbon single bonds, resulting in a straight, linear structure without any double or triple bonds. Each carbon atom in a saturated alkyl chain is bonded to the maximum number of hydrogen atoms. The absence of double bonds allows for tight packing of the chains and confers rigidity.

[0133] Unsaturated alkyl chains contain one or more carbon-carbon double bonds, introducing kinks or bends in the molecular structure. The presence of double bonds reduces the number of hydrogen atoms bonded to the carbon chain, prevents tight packing, and imparts fluidity and flexibility.

[0134] In some embodiments, R1, R2, R3, R4, R5 and / or R6that are hydrophobic groups may be saturated alkyl chains, or unsaturated alkyl chains. Such alkyl chains may be from 6 to 36 carbon atoms, optionally from 10 to 18 carbon atoms. Such alkyl chain may be of 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 carbon atoms. In particular, they are of 17 or 18 carbon atoms.

[0135] Phospholipids represent a class of lipids integral to the structural and functional framework of cellular membranes. These molecules are characterized by their amphiphilic nature, comprising two hydrophobic fatty acid tails and a hydrophilic phosphate-containing head group. This unique molecular architecture facilitates the formation of lipid bilayers, which can play a role in delineating cellular boundaries and maintaining membrane integrity. Examples of phospholipids include but are not limited to phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) and sphingomyelin. In some embodiments, Ri, R2, R3, R4, R5 and / or R6that are hydrophobic groups may be phospholipids.

[0136] Sterols are a subclass of steroids. These compounds are integral to various biological processes and can be utilized as groups to derivatize polymers, thereby modifying their properties for specific applications. The incorporation of sterols into polymeric structures can enhance properties such as biocompatibility, mechanical strength, and thermal stability. Nonlimiting examples of sterols include cholesterol, ergosterol, stigmasterol, and beta-sitosterol.

[0137] In some embodiments, R1, R2, R3, R4, R5 and / or Re that are hydrophobic groups may be sterols. In further embodiments, R1, R2, R3, R4, R5 and / or R6that are hydrophobic groups may be polymers. In particular said polymers are at least 1000 Da. Additionally or alternatively, they may be less than 10000 Da. In some embodiments, they are from 1000 to 9000 Da, 2000 to 8000 Da, 3000 to 7000 Da, 4000 to 6000 Da, e.g., 5000 Da. Said polymers may be: polylactic acid (PLA) having the following repeat unit:

[0138] Poly(lactic-co-glycolic) acid (PLGA) having the following repeat unit: wherein x is the number of repeat units of lactic acid and y is the number is units of glycolic acid.

[0139] In one embodiment, R1 may be a hydrophobic group and Bi is an amide bond, such as an amide bond wherein the carbon atom of position 2 is directly linked to the nitrogen atom of said amide bond.

[0140] Alternatively, or additionally, R2may be a hydrophobic group and B3 an ester bond or a carbamate bond, such as ester or carbamate bonds wherein the oxygen not involved in the carbonyl of said bonds is directly linked to the carbon atom of position 4. Alternatively, or additionally, R3may be a hydrophobic group and B5an ester bond or a carbamate bond, such as ester or carbamate bonds wherein the oxygen not involved in the carbonyl of said bonds is directly linked to the carbon atom of position 6.

[0141] Additionally, or alternatively, R4 may be a hydrophobic group and B7 an ester bond or a carbamate bond, such as ester or carbamate bonds wherein the oxygen not involved in the carbonyl of said bonds is directly linked to the carbon atom of position 8.

[0142] Alternatively, or additionally, R5may be a hydrophobic group and B9an ester bond or a carbamate bond, such as ester or carbamate bonds wherein the oxygen not involved in the carbonyl of said bonds is directly linked to the carbon atom of position 9.

[0143] Alternatively, or additionally, R6may be a hydrophobic group and Bn an ester bond or an amide bond, such as ester or amide bonds wherein the carbonyl of said bonds is directly linked to the carbon atom of position 11 .

[0144] In embodiments where R1, R2, R3, R4, R5 and / or R6are hydrophobic groups, and where p, q, r, s, t and / or u are 0, Bi, B3, B5, B7, B9and / or Bn, respectively, are directly linked to R1, R2, R3, R4, Rs and / or R6. For example, when R1 is a hydrophobic group and p is 0, R1 is directly linked to Bi ; when R2is a hydrophobic group and q is 0, R2is directly linked to B3; when R3is a hydrophobic group and r is 0, R3is directly linked to B5; when R4is a hydrophobic group and s is 0, R4is directly linked to B7; when R5is a hydrophobic group and t is 0, R5is directly linked to B9; and when R6is a hydrophobic group and u is 0, R6is directly linked to Bn.

[0145] In the embodiments where Li, l_2, l_3, L4, Ls and / or l_6 are linkers, said linkers, may be from 72 to 3200 Da, such as from 200 to 2800 Da, 400 to 2600 Da, 600 to 2400 Da, 800 to 2200 Da, 1000 to 2000 Da, 1200 to 1800 Da, 1400 to 1600 Da.

[0146] Alternatively, or additionally, said linkers may comprise polyethylene glycol (PEG) repeat units. In particular, they may comprise the following structure, wherein n is the number of PEG repeat unit:

[0147] In some embodiments, n is from 20 to 60, such as from 25 to 55, 30 to 50, 35 to 45, 36,

[0148] 37, 38, 39, 40, 41 , 42, 43, or 44. In other embodiments, n is from 35 to 45, for example 36, 37,

[0149] 38, 39, 40, 41 , 42, 43 or 44, in particular n is 39 or n is 40. In exemplary embodiments, when Ri is a hydrophobic group, and p is 1 , Li is a linker as described above; when R2is a hydrophobic group, and q is 1 , l_2is a linker as described above; when R3is a hydrophobic group, and r is 1 , l_3is a linker as described above; when R4is a hydrophobic group, and s is 1 , l_4is a linker as described above; when Rs is a hydrophobic group, and t is 1 , l_5is a linker as described above; and / or when R6is a hydrophobic group, and u is 1 , l_6 is a linker as described above.

[0150] In embodiments where a linker (i.e., Li, l_2, l_3, l_4, l_5and / or l_6) is present, said linker is linked to the hydrophobic group (i.e., Ri, R2, R3, R4, Rs and / or Re, respectively), by an amide or an ester bond.

[0151] Therefore, in such embodiments B2, B4, B5, B6, B8, Bio and / or BI2are amide bonds or ester bonds. In embodiments where B2, B4, B6, B8, Bio and / or BI2are amide bonds, Ri, R2, R3, R4, Rs and / or Re, respectively, are directly linked to the carbonyl of said amide bond. For example, when B2is an amide bond, Ri is directly linked to the carbonyl of said amide bond; when B4is an amide bond, R2is directly linked to the carbonyl of said amide bond; when B6is an amide bond, R3is directly linked to the carbonyl of said amide bond; when B8is an amide bond, R4is directly linked to the carbonyl of said amide bond; when B is an amide bond, Rs is directly linked to the carbonyl of said amide bond; and when BI2is an amide bond, R6is directly linked to the carbonyl of said amide bond.

[0152] Furthermore, in embodiments where B2, B4, B6, B8, Bio and / or BI2are ester bonds, Ri, R2, R3, R4, RS and / or Re, respectively, are directly linked to the carbonyl of said ester bond. For example, when B2is an ester bond, Ri is directly linked to the carbonyl of said ester bond; when B4is an ester bond, R2is directly linked to the carbonyl of said ester bond; when B6is an ester bond, R3is directly linked to the carbonyl of said ester bond; when B8is an ester bond, R4is directly linked to the carbonyl of said ester bond; when Bio is an ester bond, Rs is directly linked to the carbonyl of said ester bond; and when BI2is an ester bond, R6is directly linked to the carbonyl of said ester bond.

[0153] In one embodiment, the stereochemistry of formula (A) is as depicted in structure (AA):

[0154]

[0155] In such embodiment, formula (A) has the same stereochemistry as the repeat unit of hyaluronic acid.

[0156] In some embodiments, R6is a hydrophobic group.

[0157] Additionally, R6 may be the only hydrophobic group, such that none of Ri, R2, R3, R4, or R5 are hydrophobic groups.

[0158] Additionally, or alternatively, Bn may be an amide bond or an ester bond, such as an amide bond or an ester bond wherein the carbonyl of said bonds is directly linked to the carbon atom of position 11 . In particular, Bn is an amide bond, such as an amide bond wherein the carbonyl of said bond is directly linked to the carbon atom of position 11 .

[0159] Additionally, the hydrophobic group may be a saturated or unsaturated alkyl chain. Additionally, such alkyl chain may have from 6 to 36 carbon atoms. In one embodiment, the alkyl chain has from 11 to 31 carbon atoms. In another embodiment, the alkyl chain has from 16 to 26 carbon atoms. For example, the alkyl chain may have 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 carbon atoms. In particular, the alkyl chain has 18 carbon atoms.

[0160] Additionally, or alternatively, u is 0. In such embodiment, there is no l_6or B12, and Bn is directly linked to Re.

[0161] Therefore, in one exemplary embodiment, the repeat unit according to formula (A) is as depicted in formula (A1): In other exemplary embodiments where R6is a hydrophobic group, R6may be an alkyl chain having 17 carbon atoms. Additionally, u may be 1 , and l_6may be a linker as defined supra. Additionally, R6may be the only hydrophobic group, such that none of Ri, R2, R3, R4, or Rs are hydrophobic groups.

[0162] Additionally, or alternatively, Bn may be an amide bond or an ester bond, such as an amide bond or an ester bond wherein the carbonyl of said bonds is directly linked to the carbon atom of position 11 . In particular, Bn is an amide bond, such as an amide bond wherein the carbonyl of said bond is directly linked to the carbon atom of position 11 .

[0163] Additionally, or alternatively, B12 may be an amide bond or an ester bond, such as an amide bond or an ester bond wherein R6is directly linked to the carbonyl of said bonds. In particular, B12 is an amide bond, such as an amide bond wherein R6is directly linked to the carbonyl of said bond.

[0164] Therefore, in one exemplary embodiment, the repeat unit according to formula (A) is as depicted in formula (A2): wherein the fragment about 2000 Da and / or wherein n is 39 or n is 40.

[0165] Additionally, or alternatively to previous embodiments, the polymer comprises from 2 to 45% by mole, such as 5, 10, 15, 20, 25, 30 or 35 % by mole, in particular 10 to 30 % by mole, of hydrophilic scaffold functionalized with at least one hydrophobic group according to formula

[0166] (A).

[0167] In some embodiments, the polymer further comprises repeat units according to formula

[0168] (B), wherein formula (B) corresponds to formula (A) wherein: - p is 0 and -B1-R1 is -NH-(CO)-CH3, q is 0 and -B3-R2 is -OH, r is 0 and -B5-R3 is -OH, s is 0 and -B7-R4 is -OH,

[0169] - t is 0 and -B9-R5 is -OH,

[0170] - u is 0 and -Bn-R6is -(CO)-OH, -(CO)-ONa, or -(CO)-OK.

[0171] In one embodiment, formula (B) is as follows: salt thereof.

[0172] In some embodiments, only a portion of the repeat units according to formula (B) are salts, in other embodiments, all of these repeat units are salts. In some other embodiments, none of these repeat units according to formula (B) are salts. In some embodiments, the salt is formed on the carboxylic acid of formula (B).

[0173] When repeat units according to formula (B) are salts, these may be sodium salts or potassium salts.

[0174] In one embodiment, the polymer comprises repeat units according to formula (A) and repeat units according to formula (B). In particular, in the polymer of the first aspect, the total content of repeat units according to formula (A) and repeat units according to formula (B) constitutes 100% (by mole) of the polymer.

[0175] In some embodiments, the polymer has a molecular weight comprised between 1 and 100 kDa, such as 10, 20, 30, 40, 50, 60, 70, 80, or 90 kDa. In some embodiments, the molecular weight of the polymer according to the first aspect is from 10 to 40 KDa, such as 15, 20, 25, 30 or 35 kDa, in particular about 20 kDa. Molecular weight may be measured by size exclusion chromatography.

[0176] In some embodiments, the polymer is a homo-, a hetero-, or co- polymer, such as an alternating or block copolymer.

[0177] In some embodiments, the repeat units are derived from one species of monomers. Also provided herein is a polymer comprising at least one repeat unit according to formula (A): wherein at least one of Ri, R2, R3, R4, R5 and R6is a hydrophobic group, and

[0178] - when R1 is a hydrophobic group, Bi is a type of bond, p is 0 or 1 , and when p is 1 , Li is a linker and B2is a type of bond,

[0179] - when R2is a hydrophobic group, B3 is a type of bond, q is 0 or 1 , and when q is 1 , l_2is a linker and B4is a type of bond,

[0180] - when R3is a hydrophobic group, B5is a type of bond, r is 0 or 1 , and when r is 1 , l_3is a linker and B6is a type of bond,

[0181] - when R4is a hydrophobic group, B7is a type of bond, s is 0 or 1 , and when s is 1 , l_4is a linker and B8is a type of bond,

[0182] - when R5is a hydrophobic group, B9is a type of bond, t is 0 or 1 , and when t is 1 , l_5is a linker and B10 is a type of bond, and / or

[0183] - when R6is a hydrophobic group, Bn is a type of bond, u is 0 or 1 , and when u is 1 , l_6is a linker and B12 is a type of bond.

[0184] In some embodiments, R1 may not be a hydrophobic group. In this case, p is 0 and -B1-R1 is -NH-(CO)-CH3. Additionally, or alternatively, R2may not be a hydrophobic group. In this case, q is 0 and -B3-R2is -OH. Additionally, or alternatively, R3 may also not be a hydrophobic group. In this case, r is 0 and -B5-R3 is -OH. Additionally, or alternatively, R4may not a hydrophobic group. When that is the case, s is 0 and -B7-R4is -OH. Additionally, or alternatively, R5may not be a hydrophobic group. When that is the case, t is 0 and -B9-R5 is - OH. Additionally, or alternatively, there are instances where Re is not a hydrophobic group. In these cases, u is 0 and -Bn-Re is -(CO)-OH or a salt thereof, such as -(CO)-ONa, or -(CO)- OK. In some embodiments, Ri, R2, R3, R4, R5 and / or R6that are hydrophobic groups may be saturated alkyl chains, or unsaturated alkyl chains. Such alkyl chains may be from 6 to 36 carbon atoms, optionally from 10 to 18 carbon atoms. Such alkyl chain may be of 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 carbon atoms. In particular, they are of 17 or 18 carbon atoms.

[0185] In some embodiments, R1, R2, R3, R4, R5 and / or R6that are hydrophobic groups may be phospholipids.

[0186] In some embodiments, R1, R2, R3, R4, R5 and / or Re that are hydrophobic groups may be sterols. In further embodiments, R1, R2, R3, R4, R5 and / or R6that are hydrophobic groups may be polymers. In particular said polymers are at least 1000 Da. Additionally or alternatively, they may be less than 10000 Da. In some embodiments, they are from 1000 to 9000 Da, 2000 to 8000 Da, 3000 to 7000 Da, 4000 to 6000 Da, e.g., 5000 Da. Said polymers may be: polylactic acid (PLA) having the following repeat unit:

[0187] Poly(lactic-co-glycolic) acid (PLGA) having the following repeat unit: wherein x is the number of repeat units of lactic acid and y is the number is units of glycolic acid.

[0188] In one embodiment, R1 may be a hydrophobic group and Bi is an amide bond, such as an amide bond wherein the carbon atom of position 2 is directly linked to the nitrogen atom of said amide bond.

[0189] Alternatively, or additionally, R2may be a hydrophobic group and B3 an ester bond or a carbamate bond, such as ester or carbamate bonds wherein the oxygen not involved in the carbonyl of said bonds is directly linked to the carbon atom of position 4. Alternatively, or additionally, R3may be a hydrophobic group and B5an ester bond or a carbamate bond, such as ester or carbamate bonds wherein the oxygen not involved in the carbonyl of said bonds is directly linked to the carbon atom of position 6.

[0190] Additionally, or alternatively, R4 may be a hydrophobic group and B7 an ester bond or a carbamate bond, such as ester or carbamate bonds wherein the oxygen not involved in the carbonyl of said bonds is directly linked to the carbon atom of position 8.

[0191] Alternatively, or additionally, R5may be a hydrophobic group and B9an ester bond or a carbamate bond, such as ester or carbamate bonds wherein the oxygen not involved in the carbonyl of said bonds is directly linked to the carbon atom of position 9.

[0192] Alternatively, or additionally, R6may be a hydrophobic group and Bn an ester bond or an amide bond, such as ester or amide bonds wherein the carbonyl of said bonds is directly linked to the carbon atom of position 11 .

[0193] In embodiments where R1, R2, R3, R4, R5 and / or R6are hydrophobic groups, and where p, q, r, s, t and / or u are 0, Bi, B3, B5, B7, B9and / or Bn, respectively, are directly linked to R1, R2, R3, R4, Rs and / or R6. For example, when R1 is a hydrophobic group and p is 0, R1 is directly linked to Bi ; when R2is a hydrophobic group and q is 0, R2is directly linked to B3; when R3is a hydrophobic group and r is 0, R3is directly linked to B5; when R4is a hydrophobic group and s is 0, R4is directly linked to B7; when R5is a hydrophobic group and t is 0, R5is directly linked to B9; and when R6is a hydrophobic group and u is 0, R6is directly linked to Bn.

[0194] In the embodiments where Li, l_2, l_3, L4, Ls and / or l_6 are linkers, said linkers, may be from 72 to 3200 Da, such as from 200 to 2800 Da, 400 to 2600 Da, 600 to 2400 Da, 800 to 2200 Da, 1000 to 2000 Da, 1200 to 1800 Da, 1400 to 1600 Da.

[0195] Alternatively, or additionally, said linkers may comprise polyethylene glycol (PEG) repeat units. In particular, they may comprise the following structure, wherein n is the number of PEG repeat unit:

[0196] In some embodiments, n is from 20 to 60, such as from 25 to 55, 30 to 50, 35 to 45, 36,

[0197] 37, 38, 39, 40, 41 , 42, 43, or 44. In other embodiments, n is from 35 to 45, for example 36, 37,

[0198] 38, 39, 40, 41 , 42, 43 or 44, in particular n is 39 or n is 40. In exemplary embodiments, when Ri is a hydrophobic group, and p is 1 , Li is a linker as described above; when R2is a hydrophobic group, and q is 1 , l_2is a linker as described above; when R3is a hydrophobic group, and r is 1 , l_3is a linker as described above; when R4is a hydrophobic group, and s is 1 , l_4is a linker as described above; when Rs is a hydrophobic group, and t is 1 , l_5is a linker as described above; and / or when R6is a hydrophobic group, and u is 1 , l_6 is a linker as described above.

[0199] In embodiments where a linker (i.e., Li, l_2, l_3, l_4, l_5and / or l_6) is present, said linker is linked to the hydrophobic group (i.e., Ri, R2, R3, R4, Rs and / or Re, respectively), by an amide or an ester bond.

[0200] Therefore, in such embodiments B2, B4, B5, B6, B8, Bio and / or BI2are amide bonds or ester bonds. In embodiments where B2, B4, B6, B8, Bio and / or BI2are amide bonds, Ri, R2, R3, R4, Rs and / or Re, respectively, are directly linked to the carbonyl of said amide bond. For example, when B2is an amide bond, Ri is directly linked to the carbonyl of said amide bond; when B4is an amide bond, R2is directly linked to the carbonyl of said amide bond; when B6is an amide bond, R3is directly linked to the carbonyl of said amide bond; when B8is an amide bond, R4is directly linked to the carbonyl of said amide bond; when B is an amide bond, Rs is directly linked to the carbonyl of said amide bond; and when BI2is an amide bond, R6is directly linked to the carbonyl of said amide bond.

[0201] Furthermore, in embodiments where B2, B4, B6, B8, Bio and / or BI2are ester bonds, Ri, R2, R3, R4, RS and / or Re, respectively, are directly linked to the carbonyl of said ester bond. For example, when B2is an ester bond, Ri is directly linked to the carbonyl of said ester bond; when B4is an ester bond, R2is directly linked to the carbonyl of said ester bond; when B6is an ester bond, R3is directly linked to the carbonyl of said ester bond; when B8is an ester bond, R4is directly linked to the carbonyl of said ester bond; when Bio is an ester bond, Rs is directly linked to the carbonyl of said ester bond; and when BI2is an ester bond, R6is directly linked to the carbonyl of said ester bond.

[0202] In one embodiment, the stereochemistry of formula (A) is as depicted in structure (AA):

[0203]

[0204] In such embodiment, formula (A) has the same stereochemistry as the repeat unit of hyaluronic acid.

[0205] In some embodiments, R6is a hydrophobic group.

[0206] Additionally, R6 may be the only hydrophobic group, such that none of Ri, R2, R3, R4, or R5 are hydrophobic groups.

[0207] Additionally, or alternatively, Bn may be an amide bond or an ester bond, such as an amide bond or an ester bond wherein the carbonyl of said bonds is directly linked to the carbon atom of position 11 . In particular, Bn is an amide bond, such as an amide bond wherein the carbonyl of said bond is directly linked to the carbon atom of position 11 .

[0208] Additionally, the hydrophobic group may be a saturated or unsaturated alkyl chain. Additionally, such alkyl chain may have from 6 to 36 carbon atoms. In one embodiment, the alkyl chain has from 11 to 31 carbon atoms. In another embodiment, the alkyl chain has from 16 to 26 carbon atoms. For example, the alkyl chain may have 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 carbon atoms. In particular, the alkyl chain has 18 carbon atoms.

[0209] Additionally, or alternatively, u is 0. In such embodiment, there is no l_6or B12, and Bn is directly linked to Re.

[0210] Therefore, in one exemplary embodiment, the repeat unit according to formula (A) is as depicted in formula (A1): In other exemplary embodiments where R6is a hydrophobic group, R6may be an alkyl chain having 17 carbon atoms. Additionally, u may be 1 , and l_6may be a linker as defined supra. Additionally, R6may be the only hydrophobic group, such that none of Ri, R2, R3, R4, or Rs are hydrophobic groups.

[0211] Additionally, or alternatively, Bn may be an amide bond or an ester bond, such as an amide bond or an ester bond wherein the carbonyl of said bonds is directly linked to the carbon atom of position 11 . In particular, Bn is an amide bond, such as an amide bond wherein the carbonyl of said bond is directly linked to the carbon atom of position 11 .

[0212] Additionally, or alternatively, B12 may be an amide bond or an ester bond, such as an amide bond or an ester bond wherein R6is directly linked to the carbonyl of said bonds. In particular, B12 is an amide bond, such as an amide bond wherein R6is directly linked to the carbonyl of said bond.

[0213] Therefore, in one exemplary embodiment, the repeat unit according to formula (A) is as depicted in formula (A2): wherein the fragment about 2000 Da and / or wherein n is 39 or n is 40.

[0214] Additionally, or alternatively to previous embodiments, the polymer comprises from 2 to 45% by mole, such as 5, 10, 15, 20, 25, 30 or 35 % by mole, in particular 10 to 30 % by mole, of hydrophilic scaffold functionalized with at least one hydrophobic group according to formula

[0215] (A).

[0216] In some embodiments, the polymer further comprises repeat units according to formula

[0217] (B), wherein formula (B) corresponds to formula (A) wherein: - p is 0 and -B1-R1 is -NH-(CO)-CH3, q is 0 and -B3-R2 is -OH, r is 0 and -B5-R3 is -OH, s is 0 and -B7-R4 is -OH,

[0218] - t is 0 and -B9-R5 is -OH,

[0219] - u is 0 and -Bn-R6is -(CO)-OH, -(CO)-ONa, or -(CO)-OK.

[0220] In one embodiment, formula (B) is as follows: salt thereof.

[0221] In some embodiments, only a portion of the repeat units according to formula (B) are salts, in other embodiments, all of these repeat units are salts. In some other embodiments, none of these repeat units according to formula (B) are salts. In some embodiments, the salt is formed on the carboxylic acid of formula (B).

[0222] When repeat units according to formula (B) are salts, these may be sodium salts or potassium salts.

[0223] In one embodiment, the polymer comprises repeat units according to formula (A) and repeat units according to formula (B). In particular, in the polymer of the first aspect, the total content of repeat units according to formula (A) and repeat units according to formula (B) constitutes 100% (by mole) of the polymer.

[0224] It has to be noted that specific features and embodiments that are described in the context of the first aspect of the invention, that is the polymers according to the invention, are likewise applicable to the second aspect (the self-assembled particles according to the invention), the third aspect (the compositions, immunogenic compositions and vaccines according to the invention), or further aspects including e.g., the self-assembled particles, compositions, immunogenic compositions and vaccines for use according to the invention, or methods of treatments.

[0225] Self-assembled particle:

[0226] In a second aspect, the invention provides a self-assembled particle comprising at least one amphiphilic pH-sensitive polymer as defined herein. The polymers of the invention provide a structural substrate ( / .e., a matrix such as a polymeric matrix) for forming particles and influences particle stability and the kinetics of their dissolution and / or dissociation and / or destabilization in response to a pH trigger. The particles of the invention therefore have tuneable cargo release profiles.

[0227] In some embodiments, said at least one amphiphilic pH-sensitive polymer forms a matrix e.g., a polymeric matrix, of said particle.

[0228] In some embodiments, said particle comprises a plurality of amphiphilic pH-sensitive polymers as defined herein.

[0229] In some embodiments, said particle comprises 1 to 100, optionally 1 to 50, optionally 1 to 20, optionally 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amphiphilic pH-sensitive polymers as defined herein.

[0230] In some embodiments, the particle further comprises a cargo, such as a biologically active constituent, being encapsulated within said at least one amphiphilic pH-sensitive polymer in an aqueous environment having a sub-physiological pH.

[0231] In some embodiments, the particle of the invention comprises a cargo, such as a biologically active constituent, within said matrix composed by at least one amphiphilic pH- sensitive polymer as defined herein, such as a plurality of amphiphilic pH-sensitive polymer as defined herein.

[0232] In some embodiments, the particle is triggered to release said cargo by being present in an aqueous environment having a physiological pH (e.g., a higher pH relative to the pH of the aqueous environment of sub-physiological pH in which said particle is present prior to being so triggered).

[0233] In some embodiments, the particle is induced to surrender its cargo by a change e.g., an increase, beyond a certain threshold pH e.g., at physiological pH, in the pH of the local environment.

[0234] For example, the particle being present as a component of a finally formulated parenteral composition which maintains a sub-physiological pH level during storage is triggered to release its cargo by the pH level e.g., physiological pH, encountered when the composition is injected into the muscle tissue of a human subject.

[0235] In the context of a composition comprising such, the particle can protect the cargo contained therein from potentially deleterious interactions with co-formulated substances of the composition (e.g., a biologically active constituent or an associated constituent, such as an adjuvant) external to the particle, during storage. Further, such particle can release said cargo in response to being administered such that the cargo is then free to exert its effect within the body of the recipient subject.

[0236] In some embodiments, the particle displays a self-assembling behaviour in said aqueous environment at said sub-physiological pH.

[0237] In some embodiments, the particle is engineered to be responsive to pH.

[0238] In some embodiments, said particle, when in the aqueous environment at sub-physiological pH, is in an at least partially protonated state and / or may have an approximately or exactly neutral charge and be insoluble.

[0239] In some embodiments, said particle has a pKa below the threshold physiological pH. In some embodiments, the particle has a pKa comprised between 3 and 4, optionally of 4.

[0240] In some embodiments, said particle is insoluble in an aqueous environment at said sub- physiological pH.

[0241] In some embodiments, said particle is intact and / or stable at said sub-physiological pH.

[0242] In some embodiments, said particle is self-assembled at said sub-physiological pH.

[0243] In some embodiments, the cargo is comprised within the particle at said sub-physiological pH.

[0244] In some embodiments, said particle encapsulates the cargo at said sub-physiological pH.

[0245] In some embodiments, the cargo is dispersed within the particle at said sub-physiological pH.

[0246] In some embodiments, said particle is associated and / or physically blended with the cargo at said sub-physiological pH.

[0247] In some embodiments, said particle is triggered to release said cargo, such as said biologically active constituent, by being present in said aqueous environment having a physiological pH.

[0248] In some embodiments, said particle is soluble in aqueous environment at said trigger physiological pH.

[0249] In some embodiments, on subjecting the particle to said trigger physiological pH, the particle is subjected to a change of partition coefficient (e.g., dissolution). In some embodiments, on subjecting the particle to said trigger physiological pH, the particle is at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 % dissolved. In some embodiments, on subjecting the particle to said trigger physiological pH, the particle is at least 30 % dissolved.

[0250] In some embodiments, on subjecting the particle to said trigger physiological pH, the particle is subjected to substantially or completely dissociation.

[0251] In some embodiments, on subjecting the particle to said trigger physiological pH, the particle is at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 % dissociated. In some embodiments, on subjecting the particle to said trigger physiological pH, the particle is at least 30 % dissociated.

[0252] In some embodiments, on subjecting the particle to said trigger physiological pH, the particle is subjected to substantially or completely destabilization e.g., via collapse or swelling.

[0253] In some embodiments, on subjecting the particle to said trigger physiological pH, the particle is at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 % destabilized. In some embodiments, on subjecting the particle to said trigger physiological pH, the particle is at least 30 % destabilized.

[0254] In some embodiments, on subjecting the particle to said trigger physiological pH, the particle is substantially or completely, such as at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 %, optionally at least 30 %, dissolved and / or dissociated and / or destabilized within 24 hours or less, such as within 20, 16, 12, 10, 8, 6, 4, 2 or 1 hours or 45, 30, 15, 10 or 5 minutes. These values may represent the upper limit of a range which is bounded at the lower end by a value selected from 16, 12, 10, 8, 6, 4, 2 or 1 hours or 45, 30, 15, 10, 5 or 1 minutes.

[0255] In some embodiments, on subjecting the particle to said trigger physiological pH, the particle is at least 80, 85, 90, 95, 99 or 100 % dissolved and / or dissociated and / or destabilized. The degree to which the particle is dissolved and / or dissociated and / or destabilized can be determined in vitro by microscopy such as optical or electronic microscopy.

[0256] In some embodiments, the particle has a solubility comprised between 10 and 1000 mg per 100 ml at trigger physiological pH. In some embodiments, the particle has a solubility of at least 10, 20, 30, 40, 50, 60, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mg per 100 ml at trigger physiological pH, or comprises a range with these respective values as the upper limit and a lower limit selected from 10, 20, 30, 40, 50, 60, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mg per 100 ml at trigger physiological pH. In some embodiments, the amount of cargo, such as said biologically active constituent, released from said particle when present in an aqueous environment for at least 6 months, optionally for at least 3 years, at a sub-physiological pH is no more than 30 wt%, such as no more than 25, 20, 15, 10 or 5 wt%, of the total amount of cargo, such as said biologically active constituent, and on subjecting said particle to said trigger physiological pH the amount of cargo, such as said biologically active constituent, released within 24 hours or less is no less than 50 wt%, such as no less than 55, 60, 65, 70, 75, 80, 85, 90 or 95 wt%, of the total amount of cargo, such as said biologically active constituent.

[0257] By "released” in the context of the above aspect is meant that cargo is detectable in the supernatant, rather than the pellet, of a particle-containing sample after separation of the particle e.g., by centrifugation. Herein, the aqueous part of a particle-containing composition which remains when the particles are separated from it is referred to as the "aqueous environment" or "storage buffer". This may or may not contain one or more biologically active constituents.) It is expressed as the proportion of cargo measured as being present in the supernatant relative to the total amount detected at the same timepoint i.e., the supernatant amount plus the amount detected as being associated with the particle-containing fraction e.g., centrifugation pellet. The amount of cargo released can be determined in vitro by several methods know in the art, depending on its nature. For example, a polysaccharide can be detected by colorimetric or chromatography methods, such as by HPAEC-PAD. However, it should be noted that more generally the concept of the particles "releasing" cargo as used herein is meant that the cargo is no longer "sequestered" by the particle, in the sense that the cargo is exposed or is accessible to the aqueous environment. Thus, except as mentioned above in the specific context of quantifying association of cargo with the particles, "released" is not intended necessarily to imply a physical dissociation or separation of the cargo from the particle; rather it means that the particle structure or integrity has been altered by the change in pH in such a way that cargo becomes exposed or accessible to the local aqueous environment.

[0258] Reference to the particle “comprising a cargo” as used herein is intended to encompass various ways in which such a cargo and the polymer forming the matrix of the particle can together form a particle. When “comprised within a matrix” in this sense, the cargo can be said to be sequestered, meaning it is largely or entirely inaccessible to the external environment e.g., the aqueous environment of the composition. In some embodiments, the cargo is encapsulated within the matrix. In preferred embodiments, the cargo is substantially homogeneously dispersed throughout or entangled with the matrix of the particle. In some embodiments, said aqueous environment at sub-physiological pH comprises a buffer, such as a saline phosphate, phosphate, Tris, borate, succinate, histidine, citrate and / or maleate buffer.

[0259] In some embodiments, said physiological pH is at or above 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6 or 7.7, or comprises a range with these respective values as the lower limit and an upper limit selected from 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0.

[0260] In some embodiments, said sub-physiological pH is at or below 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1 , 6.0, 5.9, 5.8, 5.7, 5.6 or 5.5, or comprises a range with these respective values as the upper limit and a lower limit selected from 6.6, 6.5, 6.4, 6.3, 6.2, 6.1 , 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1 , 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.3, 4.2, 4.1 or 4.0.

[0261] In some embodiments, said sub-physiological pH and physiological pH differ from each other by at least 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .1 , 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9 or 2.0 pH units i.e., the physiological pH is at least 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 pH units higher than the sub- physiological pH.

[0262] In some embodiments, subjecting the particle to a trigger physiological pH occurs at a temperature of around the body temperature of the recipient, in particular at around the temperature of injection site tissue of the recipient, such as at or around 37 °C in the case of a human.

[0263] In some embodiments, the amount of cargo, such as said biologically active constituent, released from said particle when present in an aqueous environment for at least 6 months, optionally for at least 3 years, at a sub-physiological pH is less than or equal to 30 wt% of total amount of cargo, such as no more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 wt%. These values may represent the upper limit of a range which is bounded at the lower end by a value selected from 29, 28, 27, 26, 25, 24 ,23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1. Such a level of release of cargo (conversely, such a level of sequestration within the particles) when present in an aqueous environment at a sub-physiological pH is, in some embodiments, achievable over a longer duration, such as at least 7, 8, 9, 10, 11 , 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36 months. These values may represent the lower limit of a range which is bounded at the upper end by a value selected from 8, 9, 10, 11 , 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 48 or 60 months.

[0264] The amount of cargo, such as said biologically active constituent, released from said particles within 24 hours or less of subjecting said particles to a trigger physiological pH is, in some embodiments, greater than or equal to 50 wt% of the total amount of cargo, such as no less than 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100 wt%. These values may represent the lower limit of a range which is bounded at the upper end by a value selected from 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100. Such a level of release of cargo in response to a trigger physiological pH is, in some embodiments, achievable over a shorter duration, such as within 20, 16, 12, 10, 8, 6, 4, 2 or 1 hours or 45, 30, 15, 10 or 5 minutes. These values may represent the upper limit of a range which is bounded at the lower end by a value selected from 16, 12, 10, 8, 6, 4, 2 or 1 hours or 45, 30, 15, 10, 5 or 1 minutes.

[0265] In some embodiments, the sub-physiological pH aqueous environment in which the particles are present for at least 6 months, optionally for at least 3 years, is maintained at between 2-8°C e.g., at about 4 °C or about 5 °C.

[0266] In some embodiments, the particles of the invention are thermostable. This means that while present in the sub-physiological pH aqueous environment at a temperature in the range 2-8°C ( / .e., not exceeding 2, 3, 5, 6, 7, 8 or, preferably, 4 °C or 5 °C), the particles may be subjected to a temperature excursion ( / .e., a temperature exceeding 2, 3, 4, 5, 6, 7 or 8 °C) not exceeding about 25°C or 37°C for up to 12 weeks, such as for a duration of between 1 day and 2, 4, 6, 8, 10 12 weeks.

[0267] In some embodiments, the particle is biocompatible, suitably with the human body.

[0268] In some embodiments, the particle is biodegradable, bioresorbable, bioabsorbable and / or excretable, suitably in or from the human body.

[0269] In some embodiments, the particle is biodegradable.

[0270] In some embodiments, the particle is biodegradable enzymatically.

[0271] In some embodiments, the particle is injectable, suitably in the human body.

[0272] In some embodiments, the particle is suitable for parenteral administration, such as intradermal, subcutaneous, or intramuscular administration.

[0273] In some embodiments, the particles are highly uniform with respect to shape, size and / or composition.

[0274] In some embodiments, the particles may be micro- or nano- particles.

[0275] In some embodiments, the particle is a nanoparticle.

[0276] In some embodiments, the nanoparticle may have largest dimension of less than about 1000 nm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 50 nm.

[0277] In some embodiments, the particle is a microparticle.

[0278] In some embodiments, the microparticle may have a largest dimension of less than about 1000 pm, less than about 900 pm, less than about 800 pm, less than about 700 pm, less than about 600 pm, less than about 500 pm, less than about 400 pm, less than about 300 pm, less than about 200 pm, less than about 100 pm, less than about 50 pm, less than about 10 pm, less than about 5 pm, or about 1 pm.

[0279] In some embodiments, the particle has a size low enough to be able to be injected with a needle. Less than 50 pm was suggested as broadest. In some embodiments, the particle has a largest dimension of less than about 50 pm, less than about 10 pm, less than about 5 pm, less than about 1 pm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 50 nm.

[0280] In some embodiments, the particle of the invention comprises, in addition to the matrix, a cargo. Such cargo is sequestered within the particles, permitting storage within an aqueous environment while preventing any undesirable interactions between the cargo and coformulated substances of the aqueous environment.

[0281] In some embodiments, the cargo comprises a biologically active cargo ( / .e., biologically active constituent).

[0282] By "biologically active" as used herein in connection with the cargo is meant that the cargo is not inert with respect to the biological (e.g., physiological, immunological, etc) functioning of the body of the recipient to which the particles are administered. In other words, such biologically active cargo is capable of interacting with the body of the recipient to mediate some manner of biological effect. The biological effect may be a therapeutic or prophylactic effect, and the cargo may therefore be a drug, which in connection with the particles of the invention is herein used in its broadest sense. Therefore, the cargo may be an active agent, a pharmaceutical agent, a therapeutic agent, or a vaccine agent.

[0283] In some embodiments, the particles may each comprise more than one biologically active cargo, such as one, two, three, four or more different cargoes.

[0284] In some embodiments, the particles may each comprise more than one biologically active cargo that are of the same type, such as two, three, four, or more drugs or two, three, four, or more therapeutic agents. In some embodiments, the particles may each comprise more than one biologically active cargo that are of different types, such as one, two, three, four or more drugs and one, two, three, or four or more vaccine agents.

[0285] The biologically active cargo may more particularly comprise an antigen, antibody, small molecule drug compound, immunoglobulin, protein, polysaccharide, protein-polysaccharide conjugate, nucleic acid or adjuvant (non-specific immunomodulatory agent). The biologically active cargo may in some embodiments be hydrolytically sensitive meaning that, subject to prevailing parameters such as pH, temperature, ionic strength etc, the cargo is susceptible to a material degree of hydrolytic degradation when in contact with an aqueous environment. For example, a "material" degree of hydrolytic degradation might be, in the context of an antigen cargo, a degree of degradation which causes a detectable reduction in immunogenicity or antigenicity. In some embodiments, the biologically active cargo has a low isoelectric point (pl), such as a pl of 4 or below, in particular 3 or 2 or below. In some embodiments, the biologically active cargo comprises phosphate groups, such as in phosphodiester bonds.

[0286] In particular embodiments, the biologically active cargo comprises an antigen. The term "antigen" is well-understood by those of skill in the art to mean an agent capable of eliciting an immune response in a human or animal body. Antigens are therefore the active agent in immunogenic compositions and vaccines. An antigen may comprise or consist of, for example, a protein or polypeptide, a saccharide such as an oligo- or polysaccharide, a conjugate of a protein and a saccharide or a nucleic acid. Antigens may be presented in various forms, such as purified or recombinant proteins, polysaccharides, conjugates of such proteins and polysaccharides, nucleic acid vectors for in vivo antigen production, inactivated whole bacteria or viruses, viral fragments, virus-like particles, live attenuated bacteria, replicating attenuated viruses or bacterial outer membrane complexes. Antigens, being cargo according to the invention, can be any type of antigen as described above, and may be antigens derived from or related to a pathogen (such as a bacteria, virus or other pathogen), a cancer / tumour, an allergic or autoimmune condition, a non-infectious disease condition, an addiction condition, or any other physiological condition potentially amenable to prophylactic or therapeutic intervention via immunisation.

[0287] In some embodiments, wherein the biologically active cargo is an antigen, said cargo comprises a saccharide such as an oligo- or polysaccharide. The expression "oligo / polysaccharide" will be used herein to mean an oligosaccharide or polysaccharide which has been isolated from a pathogen. In some such embodiments, the oligo / polysaccharide has a low isoelectric point (pl), such as a pl of 4 or below, in particular 3 or 2 or below. The oligo / polysaccharide may be used in its native form as isolated from the pathogen or may be processed. Such processing may be e.g., sizing of the native saccharides by e.g., microfluidisation.

[0288] In some embodiments, the oligo / polysaccharide is derived from a bacterial pathogen and in particular may be derived from bacterial capsular saccharide or lipooligosaccharide (LOS) or lipopolysaccharide (LPS). For example, the oligo / polysaccharide may be derived from a bacterial pathogen selected from the group consisting of: Haemophilus influenzae type b (Hib) Neisseria meningitidis (in particular serotypes A, C, W and / or Y); Streptococcus pneumoniae (in particular serotypes 1 , 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, I0A, HA, 12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F and / or 33F); Staphylococcus aureus, Bordetella pertussis, and / or Salmonella typhi.

[0289] In a particular embodiment, the saccharide in said saccharide-comprising antigen cargo is an oligo / polysaccharide conjugated to a carrier protein, i.e., said cargo is an oligo / polysaccharide-protein conjugate antigen. Such conjugates are well-known in the art as a means to confer upon the oligo / polysaccharide antigen the T-cell dependent character of the immune response elicited by the carrier protein. Hence, carrier proteins are selected for their ability to provide a source of T-helper cell epitopes. In a given oligo / polysaccharide-protein conjugate, the carrier protein may be derived from the same pathogen as the oligo / polysaccharide, or from a different pathogen. Carrier proteins suitable for use in the oligo / polysaccharide-protein conjugate antigen cargoes of the invention are well known in the art, and include tetanus toxoid, fragment C of tetanus toxoid, diphtheria toxoid, CRM 197 or another non-toxic mutant of diphtheria toxin, protein D of non-typeable Haemophilus influenzae, outer membrane protein complex (OMPC) of Neisseria meningitidis, pneumococcal PhtD, pneumococcal pneumolysin, exotoxin A of Pseudomonas aeruginosa (EPA), detoxified haemolysin of Staphylococcus aureus, detoxified adenylate cyclase of Bordetella sp, detoxified Escherichia coli heat labile enterotoxin, or cholera toxin subunit B (CTB) or detoxified cholera toxin.

[0290] As discussed above, the biologically active cargo of the particles of the invention may be hydrolytically sensitive. In the case of an oligo / polysaccharide-protein conjugate antigen, hydrolytic sensitivity can manifest as hydrolytic cleavage within the saccharide chain or between the saccharide and carrier protein, in either case resulting in the production of 'free' (unconjugated) saccharide i.e., saccharide that is not conjugated to protein, which is not desirable. As the sequestration of the oligo / polysaccharide-protein conjugate antigen within the particles of the invention serves to protect the conjugate antigen from possible hydrolytic interactions with a (sub-physiological pH) aqueous environment in which the particles may be present, loss of conjugate integrity during storage of the particles in an aqueous environment is minimised. Thus, in some embodiments wherein the biologically-active cargo is an oligo / polysaccharide-protein conjugate antigen, the amount of free (unconjugated) saccharide, derived from said oligo / polysaccharide conjugate, present collectively in the drug delivery particles and aqueous environment is no more than 30 or 25 or 20 or 15 or 10 wt% of the total amount of conjugated and free saccharide present collectively in the particles and aqueous environment during the at least 6 months, optionally the at least 3 years, in an aqueous environment at sub-physiological pH. These values may respectively represent the upper end of a range which is bounded at the lower end by a value selected from 25, 20, 15, 10 or 5 wt%. In some such embodiments, such a maximum level of free saccharide increase applies during a period of longer than 6 months, such as at least 7, 8, 9, 10, 11 , 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36 months. These values may represent the lower limit of a range which is bounded at the upper end by a value selected from 8, 9, 10, 11 , 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 48 or 60 months. In some such embodiments, the sub-physiological pH aqueous environment in which the particles are present for at least 6 months, optionally for at least 3 years, is maintained at between 2-8°C. In particular embodiments, the particles may be subjected to a single excursion to a temperature exceeding this range, however not exceeding about 37°C for no longer than about 2 weeks. Preferably, the excursion does not exceed about 25°C.

[0291] In preferred embodiments, the biologically active cargo comprises oligo / polysaccharide derived from the capsular saccharide of Haemophilus influenzae type b (H / b; polyribosylribitol phosphate or PRP), optionally in its full-length native form, conjugated to CRM 197 or, more preferably, tetanus toxoid. In other preferred embodiments, the oligo / polysaccharide is derived from capsular saccharide from Neisseria meningitidis, in particular serotype A. In these preferred embodiments, the conjugate antigens are preferably substantially homogeneously dispersed throughout the matrix of the particles.

[0292] Compositions, immunogenic compositions, and vaccines:

[0293] The self-assembled particles of the invention may be stored and delivered to a subject, being an animal, in particular a mammal, more particularly a human, in a parenterally acceptable composition.

[0294] Hence, in a third aspect, the invention provides a composition comprising at least one, suitably a plurality of, self-assembled particle as defined herein in an aqueous environment, wherein said composition is of sub-physiological pH.

[0295] Such a composition of the invention may be an immunogenic composition i.e., a composition capable of eliciting in a subject an immune response directed specifically to one or more antigenic components present in the composition. Such an immunogenic composition may be a vaccine. Put another way, the invention provides a vaccine comprising a composition (e.g., an immunogenic composition) of the invention as described herein.

[0296] Said sub-physiological pH of the composition or the aqueous environment thereof is determined relative to the local physiological pH of the particular tissue type / anatomical region (e.g., intramuscular, intravenous) of the particular subject type (e.g., animal, mammal, human, adult, infant) to which the composition is intended to be directly administered. In some embodiments, the composition / aqueous environment is adjusted to have a pH at or below 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1 or 6.0. The values may respectively define the upper end of a range which is defined at the lower end by a value selected from 6.6, 6.5, 6.4, 6.3, 6.2, 6.1 , 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1 , 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.3, 4.2, 4.1 or 4.0. The aqueous environment may contain one or more physiologically acceptable excipients and / or a buffer such as a saline, phosphate, Tris, borate, succinate, histidine, citrate or maleate buffer.

[0297] In some embodiments, the composition, the immunogenic composition, or the vaccine comprises a plurality of self-assembled particle as defined herein, in an aqueous environment of said sub-physiological pH.

[0298] The composition the immunogenic composition, or the vaccine may comprise more than one population of self-assembled particles as defined herein, each population containing different cargoes and comprising the same or different particle matrices. Thus, in some embodiments the composition comprises a first plurality and a second plurality of particles, wherein said second plurality of particles comprises a cargo other than the cargo of the first plurality of particles. In some embodiments, the composition may contain a plurality of populations of particles differing in physical characteristics such as matrix polymer, size, and shape; such populations may respectively comprise the same or different cargoes.

[0299] As a result of the cargo being sequestered within the matrix of the particles, accessibility of the cargo to the aqueous environment is impaired or substantially prevented. The cargo is therefore substantially prevented by the matrix from interacting with components of the aqueous environment, or such interaction is at least reduced relative to the situation in the absence of the particle matrix. In some embodiments, said interaction is prevented or reduced for at least 6 months, such as 7, 8, 9, 10, 11 , 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36 months (these values may represent the lower limit of a range which is bounded at the upper end by a value selected from 8, 9, 10, 11 , 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 48 or 60 months). In some such embodiments, the composition is maintained at about 4° C or at about 5 °C.

[0300] In some embodiments, wherein the composition is an immunogenic composition or a vaccine, the aqueous environment ( / .e., not including the particles present therein) comprises one or more antigens, and optionally associated constituents such as one or more adjuvants. Such an adjuvant may have a high pl, such as a pl of 8 or above, such as 9 or 10 or above, in particular 11 or above. Suitably, the adjuvant is aluminium hydroxide.

[0301] The one or more antigens comprised in the aqueous environment in some embodiments of the immunogenic composition or of the vaccine may, in some embodiments, be selected from: diphtheria toxoid, tetanus toxoid, acellular pertussis antigens (such as pertussis toxoid, filamentous haemagglutinin, pertactin), Hepatitis B Surface Antigen (HBsAg) and Inactivated Polio Vaccine (IPV), Haemophilus influenzae type b oligo / polysaccharide conjugate antigen, N. meningitidis serotype C oligo / polysaccharide conjugate antigen, N. meningitidis serotype A oligo / polysaccharide conjugate antigen, N. meningitidis serotype W oligo / polysaccharide conjugate antigen, N. meningitidis serotype Y oligo / polysaccharide conjugate antigen and N. meningitidis serotype B antigen. In particular, the following combinations of antigens may be comprised within the aqueous environment of immunogenic compositions or vaccines of the invention: i. diphtheria toxoid and tetanus toxoid; ii. diphtheria toxoid, tetanus toxoid, acellular pertussis antigens;

[0302] Hi. diphtheria toxoid, tetanus toxoid, acellular pertussis antigens and HBsAg; iv. diphtheria toxoid, tetanus toxoid, acellular pertussis antigens and IPV; v. diphtheria toxoid, tetanus toxoid, acellular pertussis antigens, HBsAg and IPV; vi. Neisseria meningitidis serotype C (MenC) capsular oligo / polysaccharide conjugated to carrier protein, Neisseria meningitidis serotype W (MenW) capsular oligo / polysaccharide conjugated to carrier protein and Neisseria meningitidis serotype Y (MenY) capsular oligo / polysaccharide conjugated to carrier protein; vii. Neisseria meningitidis serotype C (MenC) capsular oligo / polysaccharide conjugated to carrier protein, Neisseria meningitidis serotype W (MenW) capsular oligo / polysaccharide conjugated to carrier protein, Neisseria meningitidis serotype Y (MenY) capsular oligo / polysaccharide conjugated to carrier protein and antigen derived from Neisseria meningitidis serotype B (MenB).

[0303] Suitably, wherein the aqueous environment of the immunogenic composition or vaccine comprises one of combinations (i)-(v) above, the cargo is a Hib oligo / polysaccharide conjugate antigen. Also suitably, wherein the aqueous environment of the immunogenic composition comprises one of combinations (vi)-(vii) above, the cargo is a MenA oligo / polysaccharide conjugate antigen. In some such embodiments wherein diphtheria toxoid, tetanus toxoid, acellular pertussis antigens and / or HBsAg are present in the aqueous environment, the diphtheria toxoid, tetanus toxoid, acellular pertussis antigens are adsorbed onto aluminium hydroxide and the HBsAg is adsorbed onto aluminium phosphate. In some such embodiments, diphtheria toxoid is present at the amount per dose of 1-10 International Units (IU) (for example exactly or approximately 2 IU) or 10-40 IU (for example exactly or approximately 20 or 30 IU) or 1-10 Limit of flocculation (Lf) units (for example exactly or approximately 2 or 2.5 or 9 Lf) or 10-30 Lf (for example exactly or approximately 15 or 25 Lf), and tetanus toxoid is present at the amount per dose of 10-30 IU (for example exactly or approximately 20 IU) or 30-50 IU (for example exactly or approximately 40 IU) or 1-15 Lf (for example exactly or approximately 5 or I0 Lf).

[0304] In some embodiments, in addition to particle-associated Hib oligo / polysaccharide conjugate antigen, the immunogenic composition comprises, in its aqueous environment, diphtheria toxoid and tetanus toxoid at the respective exact or approximate amounts per dose: 30:40 IU; 25: 10 Lf; 20:40 IU; 15:5 Lf; 2:20 IU; 2.5:5 Lf; 2:5 Lf; 25: 10 Lf; 9:5 Lf. Acellular pertussis (Pa) antigens including pertussis toxoid (PT), filamentous haemagglutinin (FHA) and pertactin (PRN) may also be present, such that the aqueous environment comprises DTPa antigens in the following amounts:

[0305] 20-30 ug, for example exactly or approximately 25 ug of PT;

[0306] 20-30 ug, for example exactly or approximately 25 ug of FHA;

[0307] 1-10 ug, for example exactly or approximately 3 or 8 ug of PRN;

[0308] 10-30 Lf, for example exactly or approximately 15 or 25 Lf of D; and

[0309] 1-15 Lf, for example exactly or approximately 5 of 10 Lf of T; or

[0310] 2-10 ug, for example exactly or approximately 2.5 or 8 ug of PT;

[0311] 2-10 ug, for example exactly or approximately 5 or 8 ug of FHA;

[0312] 0.5-4 ug, for example 2-3ug such as exactly or approximately 2.5 or 3 ug of PRN;

[0313] 1-10 Lf, for example exactly or approximately 2 or 2.5 or 9 Lf of D; and

[0314] 1-15 Lf, for example exactly or approximately 5 of 10 Lf of T.

[0315] As mentioned above, interaction between the cargo and constituents of the aqueous environment is reduced or substantially prevented by the particle matrix. In this way, in embodiments of the immunogenic compositions or vaccines of the invention, the particle matrix prevents or reduces aggregation or flocculation and / or prevents or reduces immunological interference and / or prevents hydrolytic degradation of the cargo, relative to an equivalent composition wherein the cargo is not sequestered within particles and is accessible to the aqueous environment. In some embodiments, said interaction, and in particular said aggregation / flocculation and / or immunological interference and / or hydrolytic degradation, is prevented or reduced for at least 6 months, such as 7, 8, 9, 10, 11 , 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36 months (these values may represent the lower limit of a range which is bounded at the upper end by a value selected from 8, 9, 10, 11 , 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 48 or 60 months), optionally wherein said composition is maintained at about 4 °C, or at about 5 °C.

[0316] The phenomenon of aggregation or flocculation, which may be observed visually or by optical microscopy, occurs when certain cargoes interact with certain components of the aqueous environment, resulting in the formation of a network of particles. In the case of an immunogenic composition or vaccine containing an antigen cargo, such a network of particles may mask epitopes and interfere negatively with the elicited immune response. In some cases, the aggregation / flocculation and resulting interference may be the result of the cargo and aqueous environment component having respectively low and high (or vice versa) isoelectric points (pl), such that they are drawn to interact with each other. This is thought to be the reason for observed aggregation / flocculation / interference between the PRP saccharide of Hib conjugate vaccine (low pl) and the aluminium hydroxide adjuvant (high pl) used to adsorb other antigens in some Hib conjugate-containing combination vaccines. Thus, in some embodiments, said cargo has a low pl such as a pl of 4 or below, in particular 3 or 2 or below and / or the aqueous environment comprises a component having a high pl, such as a pl of 8 or above, such as 9 or 10 or 11. Such low pl cargo may comprise phosphate groups, e.g. in the context of phosphodiester bonds. However, the immunological interference reduced or prevented by the immunogenic compositions or vaccines provided herein is not necessarily associated with flocculation or aggregation. In embodiments wherein the aqueous environment comprises one or more antigens, preferably the particle matrix does not interfere with the immunogenicity of said one or more antigens.

[0317] In particular embodiments of the immunogenic compositions or vaccines of the invention, the matrix of the particles prevents or reduces aggregation or flocculation of the Hib-TT or Hib- CRM197 or MenACRM197 and / or prevents or reduces immunological interference on the Hib- TT or Hib-CRM197 or MenA-CRM197.

[0318] Hydrolytic degradation, such as cleavage, of hydrolytically sensitive cargoes is discussed above, and can occur through interaction of said cargo with water molecules in the aqueous environment. Therefore, preventing or minimising exposure of the cargo to the water molecules can reduce or prevent the occurrence of hydrolysis. Accordingly, the present immunogenic compositions or vaccines achieve this through sequestration of the antigen cargo within the particle. Some saccharide-based antigens, such as Hib and MenA, can be particularly prone to hydrolytic degradation, such as depolymerisation. In particular embodiments of the immunogenic compositions of the invention, the matrix of the particles prevents or reduces hydrolytic degradation of the Hib-TT or Hib-CRM197 or MenA-CRM197.

[0319] The immunogenic compositions or vaccines of the invention, in some embodiments, are suitable for parenteral administration. The person skilled in the art is aware of how to formulate therapeutic compositions for compatibility with a given parenteral route of administration e.g., intramuscular. In particular, the skilled person knows how to formulate such compositions to be of a particular pH, this being a key characteristic of the immunogenic compositions or vaccines of at least some embodiments of the invention wherein the aqueous environment in which the particles are administered (and optionally stored) is of sub-physiological pH.

[0320] The invention further provides a composition or immunogenic composition or vaccine of the invention, packaged in a therapeutically suitable container. The composition or immunogenic composition or vaccine may be presented in a vial from which the contents may be extracted when needed, for example using a needle and syringe. Alternatively, the composition or immunogenic composition or vaccine may be pre-filled in a parenteral administration device such as a syringe. Such a syringe may be a conventional singlechambered syringe or may be a dual-chambered syringe. The dual-chambered syringe may be designed to deliver the respective contents of the chambers sequentially, or simultaneously following extemporaneous mixing within the syringe.

[0321] Uses and administration:

[0322] In a fourth aspect, the invention relates to self-assembled particles, compositions, immunogenic compositions, or vaccines as described herein, for use as a medicament, such as prophylactically or therapeutically.

[0323] They may be administered to a subject in need thereof. Typically, the subject is an animal, such as a mammal, and is preferably a human subject. In some embodiments, the subject is an infant or a child or an adolescent or an adult or an elderly adult. The subject may be a pregnant female, optionally wherein the gestational infant is the subject in need. The subject may be an immunocompromised individual. In a particular embodiment, the plurality of particles and the immunogenic compositions and vaccines are for use in immunisation, such as paediatric immunisation.

[0324] Also described herein is a use of self-assembled particles, compositions, immunogenic compositions, or vaccines as described herein, as a medicament.

[0325] Also described herein is a use of self-assembled particles, compositions, immunogenic compositions, or vaccines as described herein in the manufacture of a medicament. In a fifth aspect, the invention provides self-assembled particles, compositions, immunogenic compositions, or vaccines as described herein, for use in the treatment or prevention of a disorder or a disease, such as an infection, caused directly or indirectly by a pathogen, or of a pathology associated with immunologically distinct host cells such as cancer.

[0326] Also described herein is a use of self-assembled particles, compositions, immunogenic compositions, or vaccines as described herein as treatment or prevention of a disorder or a disease, such as an infection, caused directly or indirectly by a pathogen, or of a pathology associated with immunologically distinct host cells such as cancer.

[0327] Also described herein is a use of self-assembled particles, compositions, immunogenic compositions, or vaccines as described herein in the manufacture of a medicament for the treatment or prevention of a disorder or a disease, such as an infection, caused directly or indirectly by a pathogen, or of a pathology associated with immunologically distinct host cells such as cancer.

[0328] In some embodiment, the use is for parenteral administration, suitably intramuscular administration.

[0329] In some embodiments, an immune response is elicited, suitably an adaptative immune response, more suitably a protective adaptative immune response. In some embodiments, the immune response is elicited against an infection- or pathology-causing pathogen or allergen, or immunologically distinct host cells responsible for a pathology such as cancer.

[0330] In a sixth aspect, the invention relates to a method of treating or preventing a disorder or a disease, such as an infection, caused directly or indirectly by a pathogen, or of a pathology associated with immunologically distinct host cells such as cancer, wherein the method comprises applying or administering to a subject in need thereof the self-assembled particles, compositions, immunogenic compositions, or vaccines as described herein.

[0331] In some embodiments, the composition, thereof the self-assembled particles, compositions, immunogenic compositions, or vaccines are administered at a therapeutically effective amount.

[0332] In some embodiments, the subject in need is a mammalian subject, suitably a human subject.

[0333] In a seventh aspect, the invention provides a method of eliciting an immune response, wherein the method comprises applying or administering to a subject in need thereof an effective amount of the self-assemble particles, compositions, immunogenic compositions or vaccines as described herein. In some embodiments, an immune response is elicited, suitably an adaptative immune response, more suitably a protective adaptative immune response. In some embodiments, the immune response is elicited against an infection- or pathology-causing pathogen or allergen, or immunologically distinct host cells responsible for a pathology such as cancer.

[0334] In some embodiments, the composition, thereof the self-assembled particles, compositions, immunogenic compositions, or vaccines are administered at a therapeutically effective amount.

[0335] In some embodiments, the subject in need is a mammalian subject, suitably a human subject.

[0336] The self-assembled particles, compositions, immunogenic compositions, or vaccines of the invention may be administered in a liquid form i.e., as a suspension containing the particles. Prior to administration, the particles may be stored in the finally formulated, administrable liquid composition, such as the composition, immunogenic composition; or vaccine of the invention in which the particles are in the aqueous environment as defined herein. However, the particles may be stored in lyophilised, such as freeze-dried, form, or powdered form, to be reconstituted into liquid form through mixing with the aqueous environment (as defined herein) extemporaneously with administration to a subject. In some embodiments, the particles may be stored in liquid medium other than said aqueous environment, such that mixing with said aqueous environment, to give the composition, immunogenic composition, or vaccine of the invention, takes place extemporaneously with administration. The self-assembled particles, compositions, immunogenic compositions, or vaccines of the invention may be presented in unit-dose or multi-dose sealed containers such as vials or may be pre-filled into administration devices such as syringes.

[0337] In particular embodiments of the above self-assembled particles, compositions, immunogenic compositions, or vaccines; use of such; or method of treatment or of eliciting an immune response, the pathogen is selected from the list consisting of: Haemophilus influenzae type b (Hib); Neisseria meningitidis (in particular serotypes A, C, W and / or Y); Streptococcus pneumoniae, Staphylococcus aureus, Bordetella sp; and Salmonella typhi. Suitably, the pathogen is Haemophilus influenzae type b (Hib) or Neisseria meningitidis serotype A (MenA).

[0338] In an eighth aspect, the invention provides a method for preventing or reducing interaction between a cargo, such as a biologically active constituent, and co-formulated substances of an aqueous environment, such as in a parenteral formulation, comprising:

[0339] (i) forming said at least one self-assembled particle as described herein, comprising said cargo; and (ii) formulating said at least one self-assembled particle as described herein, in said aqueous environment, comprising any necessary adjustment to render said environment of sub-physiological pH.

[0340] In step (ii) above, said adjustment may not be necessary if the aqueous environment is already at sub-physiological pH. Thus, also provided herein is a method for preventing or reducing interaction between a cargo, such as a biologically active constituent, and coformulated substances of an aqueous environment, such as in a parenteral formulation, of sub- physiological pH, comprising:

[0341] (i) forming said at least one self-assembled particle as described herein, comprising said cargo; and

[0342] (ii) formulating said at least one self-assembled particle as described herein, in said aqueous environment.

[0343] In some embodiments, the result of formulating according to the steps (ii) above is the production of a composition, immunogenic composition, or vaccine as defined herein.

[0344] As discussed above, such prevention or reduction of said interaction is advantageous in various circumstances. Accordingly, in some embodiments, the above methods are methods for storing a biologically active cargo in an aqueous environment (wherein the storage is effected by the prevention or reduction of interactions between the biologically active cargo and components of an aqueous environment). Similarly, in some such embodiments as well as in other embodiments, the above methods are for preventing or reducing interaction between said biologically active cargo and water molecules in said aqueous environment, or between said biologically active cargo and a constituent of the aqueous environment other than water. Such a constituent may be, for example, an adjuvant or antigen or biological or pharmaceutical active constituent, or a formulation excipient. In particular, said methods may be for preventing or reducing degradation, such as hydrolytic degradation ( / .e., through interaction with water molecules), of said cargo in said aqueous environment.

[0345] Wherein the above methods are for storing a biologically active cargo in an aqueous environment, said storing may be for at least 6 months, for example 7, 8, 9, 10, 11 , 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36 months. These values may represent the lower limit of a range which is bounded at the upper end by a value selected from 8, 9, 10, 11 , 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 48 or 60 months.

[0346] Wherein the above methods are for preventing or reducing interaction between said biologically active cargo and an adjuvant component of the aqueous environment, in some embodiments, said adjuvant is aluminium hydroxide. In some embodiments, the cargo contains phosphate groups such as in phosphodiester moieties and / or has a low pl. In some embodiments, the cargo is Hib-TT or Hib-CRM197 or MenA-CRM197.

[0347] The above methods may be for preventing or reducing interaction between said biologically active cargo and a second cargo. Such embodiments comprise, in addition to the above recited steps (i) and (ii), a step (iii): forming a second plurality of particles comprising said second cargo within a matrix, wherein said second plurality of particles is as defined herein for the plurality of particles but with the proviso that the cargo is not the same as said biologically- active cargo, i.e., the second plurality of particles is as defined herein for the plurality of particles with the exception of the cargo, in the sense that in such embodiments comprising two pluralities (populations) of particles, the two pluralities comprise different cargoes. In some alternative embodiments, the two pluralities may comprise the same cargo in a different polymer matrix.

[0348] Manufacture and formulation:

[0349] The self-assembled particles of the invention may comprise a cargo, such as a biologically active constituent (i.e., biologically active cargo) within a matrix, such as at least one amphiphilic pH-sensitive polymer as defined herein.

[0350] The cargo may associate with the particle matrix in various ways such that it is 'comprised within the matrix'. For example, the cargo may be encapsulated within the matrix, or the cargo may be dispersed within or physically blended with the matrix. The combination of cargo and matrix may be described as a physical association, such as a non-covalent association.

[0351] In particular embodiments, the cargo is substantially homogeneously dispersed throughout the matrix of the particle. This may be achieved by fabricating particles from a homogeneous mixture i.e., a solution) comprising the cargo and matrix polymer(s).

[0352] Thus, also described herein is a method for manufacturing at least one, suitably a plurality, of self-assembled particles, suitably self-assembled particles as described herein, comprising a cargo, such as a biologically active constituent (i.e., biologically active cargo) within a matrix, such as at least one amphiphilic pH-sensitive polymer as defined herein, wherein the method comprises the step of making a solution (i.e., an homogeneous mixture) comprising said cargo and matrix polymer(s), such as at least one amphiphilic pH-sensitive polymer as defined herein.

[0353] Also provided is a method for making at least one, suitably a plurality, of self-assembled particles, suitably self-assembled particles as described herein, comprising a cargo, such as a biologically active constituent (i.e., biologically active cargo) within a matrix, such as at least one amphiphilic pH-sensitive polymer as defined herein, wherein the method comprises the use of a solution comprising at least one amphiphilic pH-sensitive polymer as defined herein, and a cargo, wherein said cargo is optionally as defined herein.

[0354] In some embodiments of the above methods, said solution comprises said cargo at an amount not exceeding 30 wt%. For example, the solution may comprise the cargo at an amount of 0.1-5 wt%, such as 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1 .5, 2, 2.5 or 5 wt%.

[0355] The cargo may, for example, be selected from Hib-TT, Hib-CRM197 and MenA-CRM197. In particular embodiments, said solution comprises 0.2-1.2, more particularly 0.4-1 , wt% Hib- TT or Hib-CRM197 or MenA-CRM197.

[0356] The particles of the invention may be fabricated from such solutions through known techniques for fabricating micro- or nano- particles, for example coacervation, emulsification, double emulsification, microfluidic, impinging jet mixing or spray-drying.

[0357] In another aspect, the invention provides at least one, suitably a plurality, of self-assembled particles, suitably self-assembled particles as described herein, obtainable or obtained by the foregoing methods.

[0358] In a further aspect, is provided a method for making a composition, such as compositions, immunogenic compositions or vaccines as described herein, said method comprising making at least one, suitably a plurality, of self-assembled particles, suitably self-assembled particles as described herein, according to a method as defined herein, and formulating said particles in an aqueous environment.

[0359] In some embodiments, the composition is an immunogenic composition and said environment comprises an antigen and / or an adjuvant. Said antigen and / or adjuvant may, for example, be as defined herein in relation to the immunogenic compositions of the invention.

[0360] The invention further provides, in another aspect, a method for making a composition, such as compositions, immunogenic compositions or vaccines as described herein, comprising at least one, suitably a plurality, of self-assembled particles, suitably self-assembled particles as described herein, comprising a cargo, such as a biologically active constituent ( / .e., biologically active cargo) within a matrix, such as at least one amphiphilic pH-sensitive polymer as defined herein, the method comprising the steps of:

[0361] (i) introducing at least one, suitably a plurality, of self-assembled particles, made according to a method as defined herein, to an acidic aqueous environment such that the acidic environment protonates the matrix polymer making the polymer insoluble in said environment; (ii) raising the pH of the acidic aqueous environment to a sub-physiological pH which is acceptable for parenteral administration while retaining the insoluble state of the matrix polymer in said environment; and optionally

[0362] (iii) formulating said particles in an aqueous environment of sub-physiological pH.

[0363] In step (i) of this method, referred to as herein as "stabilisation", the particles are brought into contact with a "stabilising solution" at acidic pH, resulting in protonation of the matrix polymer making the polymer insoluble. For example, for particles comprising matrix polymer initially containing COOH groups predominantly in the ionised, negatively charged ( / .e. COO-) form, such protonation alters the balance in favour of the charge-neutral COOH form. The protonation of the matrix polymer causes the particles to become insoluble in the stabilising solution. In some embodiments, the stabilising solution has a pH in the range 1-5, such as about or exactly pH 3.5 or 4.5. The duration of the contact ( / .e., before step (ii) begins) may be up to 60, 50, 40, 30, 20, 10 or 5 minutes, or these values may respectively delineate the upper end of a time range which is bounded at the lower end by 1 minute.

[0364] In step (ii), known herein as "neutralisation", the environment containing the insoluble particles is adjusted to a pH which is compatible with parenteral administration. Such pH clearly must be below the threshold pH above which the particles would release their cargo, i.e. the pH must be sub-physiological. Both “sub-physiological” and “acceptable for parental administration” in this sense must be with respect to the particular target administration site of / route into the particular intended subject. This adjustment in pH, for example by continuous or stepwise addition of a solution of higher pH (for example by addition of a buffer which is less acidic than the stabilising solution), must be done at an appropriate rate which maintains the insoluble state of the matrix polymer. Thus, in some embodiments, step (ii) comprises increasing the pH of the aqueous environment towards a sub-physiological pH in a stepwise manner. In some embodiments, the pH of the aqueous environment is increased by 0.1-10 pH units per minute, such as 0.5, 1 , 2 or 5 pH units per minute, in particular 0.5 pH units per minute. By way of clarification, this means that in every minute elapsing between the first adjustment and the attainment of the finally adjusted sub-physiological and parenterally acceptable pH, the increase in solution pH is in the range of 0.1-10 pH units. The combined steps of stabilisation and neutralisation are termed herein as "transition".

[0365] In optional step (iii) the particles are formulated in an aqueous environment of sub- physiological pH, i.e. the particle-containing solution at sub-physiological pH is combined with other components to produce a composition, which composition must also be at sub- physiological pH in order that the particles retain the cargo during storage. Such "other components" present within the aqueous composition of step (iii) may include formulation excipients such as buffers, tonicity modifiers, preservatives, adjuvants, etc, as well as biologically active constituents such as drug compounds or antigens. In particular, the aqueous environment of step (iii) may be as defined herein for the compositions, immunogenic compositions or vaccines of the invention.

[0366] In another aspect the invention provides a composition, immunogenic composition or vaccine, obtainable or obtained by the foregoing methods.

[0367] The following Examples are provided to illustrate certain particular features and / or embodiments. These Examples should not be construed to limit the invention to the particular features or embodiments described.

[0368] EXAMPLES

[0369] The following polymers were synthesized:

[0370] 1. Lipid-X-Polymer: Hyaluronic Acid-PEG-Stearic Acid (HA-PEG-SA)

[0371] • 10 % w / w stearic acid

[0372] • 30 % w / w stearic acid

[0373] 2. Lipid-Polymer: Hyaluronic Acid-Stearyl Amine (HA-SA)

[0374] • 5 % w / w stearyl amine

[0375] • 10 % w / w stearyl amine

[0376] • 30 % w / w stearyl amine

[0377] In case of HA-SA, the acid group of HA is conjugated with the amine group of stearyl amine (Sam), whereas, in case of HA-PEG-SA, one amine group of bis amine PEG is conjugated with HA and the other amine group of bis amine PEG is conjugated with stearic acid (Sac).

[0378] Materials

[0379] • Hyaluronic Acid, MW of -20000 (HA) (from TCI)

[0380] • Dimethylsulfoxide (DMSO) (from Sigma)

[0381] • N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) (from Sigma)

[0382] • N-hydroxysuccinimide (NHS) (from Sigma)

[0383] • Triethylamine (TEA) (from Sigma)

[0384] • Stearylamine (Sam) (from TCI)

[0385] • Pyridine (from Sigma)

[0386] • Stearic acid (Sac) (from Sigma)

[0387] • t-Boc Amine PEG Amine (t-boc NH2-PEG-NH2, MW 2000) (from Broadpharm) • Bovine Serum Albumin (BSA) (from Sigma)

[0388] Reagents solubility studies

[0389] Solubility studies of the excipients (HA, PEG, Sam and Sac) were performed. The following observations were found:

[0390] • HA: Hydrophilic - soluble in water, insoluble in organic solvents including DMSO and ethanol

[0391] • PEG: Hydrophilic - soluble in water and DMSO

[0392] • Lipid (Sac / Sam): Hydrophobic - slightly soluble in chloroform, acetone, and ethanol. Insoluble in water

[0393] Considering the solubility of the raw materials used for the conjugation no common solvent was found. Thus, a combination of water (for HA and PEG) and ethanol (for lipids) was investigated. However, the solution obtained was turbid suggesting insolubility of lipids and polymers (HA and PEG). Thereafter, a basic solvent pyridine was added i.e., solvent mixture in a ratio of 1 :1 :1 of water, ethanol, pyridine. However, the raw materials were found to be not soluble. Thereafter, pyridine was replaced with Triethylamine (TEA) in the ratio of 1 :1 :1 of water, ethanol, TEA. Using TEA, the raw materials were found to be soluble and thus used for the chemical reaction.

[0394] Synthesis of HA-S A conjugate

[0395] HA-SA conjugate was synthesized utilizing free -COOH groups of HA which binds with - NH2group of Sam. The covalent modification of HA groups involved three sequential steps: (i) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) activation of -COOH of HA (ii) Covalent conjugation with free -NH2group of Sam with activated HA (iii) Dialysis of synthesized conjugate followed by lyophilization.

[0396] Briefly, HA (100 mg) dissolved in 1 :1 :1 ratio of Triethylamine: Water: Ethanol (5 ml) in round bottom flask and EDC (1 .5 moles in respect to Sam) and NHS (1 .5 moles in respect to Sam) were added and allowed to stir for 12 h at room temperature. Thereafter, 5, 10 and 30 mg Sam dissolved in 1 :1 :1 ratio of Triethylamine: Water: Ethanol (5 ml) was added, in case of synthesis of 5 % w / w HA-SA, 10 % w / w HA-SA and 30 % w / w HA-SA, respectively, and allowed to stir at 350 rpm for 24 h at room temperature. As a result, activated carboxylic group of HA was conjugated with free amine group of Sam. The prepared conjugates were purified using a dialysis membrane (cutoff value of MW 12kD). The samples were placed in dialysis bag and placed in two litres of milli Q water for 48 h. The milli Q water was replaced three times. The samples after dialysis were freeze dried and used for the solubility analysis. The conjugates prepared were also analysed using 1 H-NMR. Synthesis of HA-PEG-SA conjugate

[0397] HA-PEG-SA conjugate was synthesized utilizing -COOH groups from HA and Sac bounded through the -NH2groups of bis amine PEG. The covalent modification of these groups involved the following sequential steps: (i) EDC and NHS activation of -COOH of HA; EDC and NHS activation of -COOH of Sac (ii) Covalent conjugation with free - NH2group of t-boc-NH2-PEG- NH2with activated Sac (iii) Dialysis of synthesized conjugate (t-boc-NH-PEG-NH-Sac) followed by lyophilization (iv) Deprotection of the t-boc group done by dissolving the conjugated in 10 ml of undiluted Trifluoroacetic acid for 12 h (v) Covalent conjugation with free -NH2group of NH2-PEG-NH-Sac with activated HA (vi) Dialysis of synthesized conjugate (HA-NH-PEG-NH- Sac) followed by lyophilization.

[0398] Briefly, HA (100 mg) dissolved in 1 :1 :1 ratio of Triethylamine: Water: Ethanol (5 ml) in round bottom flask and EDC (1 .5 moles in respect to Sac) and NHS (1 .5 moles in respect to Sac) were added and allowed to stir for 12 h at room temperature to produce activated HA. Separately, the stearic acid was activated, Sac dissolved in 1 :1 :1 ratio of Triethylamine: Water: Ethanol (5 ml) in round bottom flask and EDC (1.5 moles in respect to Sac) and NHS (1.5 moles in respect to Sac) were added and allowed to stir for 12h at room temperature to produce activated Sac.

[0399] Thereafter, t-boc bis amine PEG (10 or 30 mg of activated Sac) was dissolved in 1 : 1 : 1 ratio of Triethylamine: Water: Ethanol (5 ml) and was added to the activated 10 mg or 30 mg of Sac (1 :1 mole ratio of Sac and t-boc bis amine PEG). The solution was stirred for 24 h at 350 rpm in round bottom flask. The solution was dialysed for 48 h and lyophilized. The lyophilized sample was dissolved in 10 ml of TFA for deprotection and kept for 12 h at RT. Thereafter, the solution was dialyzed again using dialysis bag of 1 kD molecular weight cutoff and lyophilized resulting in synthesis of NH2-PEG-NH-Sac conjugate.

[0400] The lyophilized conjugate (NH2-PEG-NH-Sac) (10 and 30 mg) was dissolved in the 1 :1 :1 ratio of Triethylamine: Water: Ethanol (5 ml) and was added to the 100 mg of activated HA (dissolved in 5 ml of 1 :1 :1 ratio of Triethylamine: Water: Ethanol) separately. The solution was stirred at 350 rpm for 24 h at room temperature. As a result, activated carboxylic group of HA was conjugated with free amine group of NH2-PEG-NH-Sac. The prepared conjugate was purified using a dialysis membrane (cutoff value of MW 12 kD). The sample was placed in dialysis bag and placed in two litres of milli Q water for 48 h. The milli Q water was replaced three times. The sample after dialysis was freeze dried and used for the solubility analysis. The conjugates prepared were also analysed using 1 H-NMR.

[0401] A purification step, via precipitation method, was further developed to improve the polymer purity. Briefly, the pH of the polymer solution obtained after the dialysis was adjusted to 5.5 using 0.1 N HCI. The solution was then centrifuged, and the precipitate was collected and dried using a lyophilizer. The pegylated conjugates (10 and 30 % w / w HA-PEG-SA) were centrifuged at 25000 rpm for 30 mins, whereas the non-pegylated conjugates (5, 10 and 30 % w / w HA- SA) were centrifuged at 5000 rpm for 5 min. This purification step was integrated in the synthesis steps. Furthermore, all the conjugates (5, 10 and 30 % w / w HA-SA, and 10 and 30 % w / w HA-PEG-SA) were synthesized confirming reproducibility of the conjugate synthesis.

[0402] Qualitative analysis of the chemical derivatization by 1H-NMR analysis

[0403] For the confirmation of chemical conjugation, the conjugates were analysed using 1 H-NMR spectroscopy. Briefly, approximatively 20 mg of the samples were dissolved in 1 ml of a mixture ratio of 1 :1 of deuterated water to deuterated methanol and analysed using NMR. The 1 H- NMR spectra were recorded on a Bruker Avance III™ HD 500 MHz NMR spectrometer using a 5 mm TCI cryo probe at 298 K. Spectra were processed using MestreNova software. As an outcome of optimization part, the acquisition parameters used were spectral width 7997.6 Hz, relaxation delay 3.0 s, number of scans 64, and pulse width 45°. As shown in FIG. 1, all the synthesized conjugates contain an amide bond at ~10.2 ppm, which is not present in case of any reagent used for the synthesis of conjugates (HA, PEG), thus, confirming the synthesis of conjugates.

[0404] HA-SA conjugates

[0405] As shown in FIG. 2-4, the conjugates show a pH-dependent solubility (more soluble at physiological and basic pH). With an increase in the amount of Sam conjugated, the hydrophobicity of HA was found to increase (30 % Sam conjugated HA was found to be floating on the surface (at 10 mg / ml)). Whereas 10 and 5 % Sam conjugated HA was settled in the bottom, suggesting higher wettability of 10 and 5 % Sam conjugated HA. The floating of the 30% Sam conjugated HA polymer was found to be in case of pH 6.5 only, suggesting higher wettability or solubility on increasing the pH. The floating of the developed conjugate was found to reduce on diluting the sample to 5 mg / ml. To check the reproducibility, the 10 % w / w HA-SA was resynthesized and evaluated for the solubility studies. The resynthesized 10 % w / w HA- SA was found to follow the solubility trend of the previous conjugate (data not shown), suggesting reproducibility of the conjugate synthesis.

[0406] HA-PEG-SA conjugates

[0407] As shown in FIG. 5-6, the conjugates with PEG show pH dependent solubility (more soluble at physiological and basic pH). In line with the HA-SA conjugates, 30 % w / w HA-PEG-SA was found to float on the surface of the buffer of pH 5.5 and 6.5, suggesting lower solubility as compared to the pH 7.4 and 9.0. Furthermore, less particles were observed in case of 10 % w / w HA-PEG-SA as compared to 30 % w / w HA-PEG-SA at pH 5.5 and 6.5, suggesting higher solubility of 10 % w / w HA-PEG-SA.

[0408] Particle size, polydispersity index (PDI) and transmittance

[0409] The effect of SA and SA-PEG on the HA were evaluated via characterization of conjugate solutions using Litesizer and analyzed for the particle size, PDI, and transmittance. Briefly, 100 pl of purified conjugates (purified after the synthesis using a dialysis membrane cutoff value of MW 12 kD) were diluted with 900 pl deionized water and characterized for the particle size, PDI and transmittance. Results are depicted in Table 1. The particle size and PDI values of HA-SA conjugates were higher as compared to HA-PEG-SA conjugates. Probably, HA-SA conjugates, due to high hydrophobicity, tends to aggregate and form particles in micron range. Whereas, due to the presence of PEG, HA-PEG-SA conjugates are hydrophilic in nature and more soluble, thus the particle size and PDI values were found to be < 1 pm and < 0.3 pm, respectively. Furthermore, due to higher water solubility and hydrophilicity, HA-PEG-SA showed higher transmittance as compared to HA-SA conjugates.

[0410] Table 1 : Mean particle size, PDI and transmittance of the conjugates pH dependent solubility study of the conjugates

[0411] The pH dependent solubility of the conjugates was evaluated through gravimetric, transmittivity, and turbidity studies.

[0412] Gravimetric analysis

[0413] For the pH solubility analysis of conjugates HA-SA 5 % w / w, HA-SA 10 % w / w, and HA-SA 30 % w / w, the samples were dissolved or suspended in phosphate buffer (PBS) of pH 6.0 and 7.4 at a concentration of 2 mg / ml (10 mg in 5 ml). The solution was then sonicated for 10 min and centrifuged at 5000 rpm for 10 min (to precipitate the undissolved fraction). Thereafter, the supernatant and precipitate were separated via decantation. For the determination of solubility of conjugates, only the undissolved fraction (precipitate of conjugates) was evaluated. As depicted in Table 2, the precipitate in case of HA-SA 30 % w / w at pH 7.4 and 6.0 were found to be comparable, suggesting no or very low solubility of HA-SA 30 % w / w. Whereas an increase in the precipitation was found at pH 6.0 as compared to the pH 7.4, in case of HA-SA 10 % w / w. Interestingly, significant difference in the solubility was observed in case of HA-SA 5 % w / w i.e., no precipitation was observed at pH 7.4 as compared to pH 6.0. Thus, HA-SA 5% w / w and HA-SA 10 % w / w conjugates demonstrate pH dependent solubility.

[0414] Table 2: Weights of lyophilized precipitate fraction

[0415] Transmissivity test

[0416] The transmissivity of the conjugate (HA-SA 5 % w / w, HA-SA 10 % w / w, and HA-SA 30 % w / w) solutions (2 mg / ml) was evaluated as an indication of solubility. The conjugates were suspended in phosphate buffer at pH 5.5, 6.0, 6.5, 7.0, 7.4, and pH 8 and were sonicated for 10 min. After sonication, the samples were centrifuged at 5000 rpm for 10 min (to precipitate the undissolved fraction) and measured via Litesizer. Due to the hydrophobic chains, an increase in the conjugate solubility should result in a higher transmissivity (after precipitation of undissolved aggregates due to centrifugation step), and vice-versa.

[0417] As demonstrated in FIG. 7, no change in the transmissivity was observed in case of HA- SA 5 % w / w and HA-SA 30 % w / w samples with an increase in the pH. In case of HA-SA 5 % w / w, no or marginal change in transmissivity even at lower pH could be due to the lower weight fraction of hydrophobic fraction (Stearyl amine) resulting in lower level of transmissivity. Thus, higher concentration of HA-SA 5 % w / w should be required to attain similar level of turbidity compared to HA-SA 10 % w / w. Whereas, no change in transmissivity could be due to the negligible solubility of HA-SA 30 % w / w owing to its higher hydrophobic fraction and low solubility (HA-SA 30 % w / w due to higher hydrophobic fraction tends to aggregate, thus precipitate during centrifugation). HA-SA 10 % w / w showed very high transmissivity at lower pH (up to 6.5) due to very low solubility. Whereas, at higher pH from 7.0 pH onwards, higher fraction of the conjugates is soluble and thus the solution gets turbid resulting in lower transmissivity. Turbidity analysis

[0418] The turbidity of the conjugate (HA-SA 5 % w / w, HA-SA 10 % w / w, and HA-SA 30 % w / w) solutions (0.5 mg / ml) was also evaluated.

[0419] The turbidity analysis (European Pharmacopeia 9.2.1) was evaluated via UV-Vis. UV- 2700 (Shimadzu) in Wavelength range 595-605 nm (the maxima at 600 nm is used for data evaluation). The absorbance is measured against a Formazin standard and expressed as Nephelometric Turbidity Unit (NTU) i.e., higher turbidity should result in higher NTU values.

[0420] The conjugates were dissolved in water at pH 5.5, 6.0, 6.5, 7.0 and 7.4 (the pH was adjusted using NaOH and HCI) and after 10 min sonication the samples were centrifugation at 1000 rpm for 5 min and analysed for turbidity. An increase in the conjugate solubility should result in an increase of the turbidity and lower transmissivity, and vice-versa i.e., the turbidity is inversely proportional to the transmissivity.

[0421] The obtained data are in line with the results obtained from the transmissivity test. FIG. 8 demonstrates no significant change in turbidity in case of HA-SA 30 % w / w sample. Whereas, both 10 % w / w and 5 % w / w HA-SA samples showed increase in turbidity with increase in pH, suggesting pH dependent solubility. The increase in turbidity was significantly higher in case of HA-SA 10 % w / w as compared to HA-SA 5 % w / w (probably due to the higher solubility of HA-SA 5 % w / w in water, for the low number of hydrophobic chains).

[0422] Interaction studies between polymers and a model protein

[0423] The interaction of the developed polymers and a model protein (Bovine Serum Albumin; BSA) was evaluated to study the capacity of the developed polymers to encapsulate the protein, thus protect BSA from the environment and control the release according to the pH of the environment. The following set of samples were prepared and investigated at different pH conditions.

[0424] • Set A- BSA: add 750 pl of BSA stock (0.05 mg / ml, final concentration in 15 ml in water) + 14250 pl of water

[0425] • Set B - BSA + HA-PEG-SA 10 % w / w: add 750 pl of BSA stock (0.05 mg / mL, final concentration in 15 mL in water) + 2000 pl of polymer solution (HA-PEG-SA 10 % w / w) (0.1 mg / ml, final polymer concentration in 15 ml in water) + 12250 pl of water

[0426] • Set C - BSA + HA-SA 10 % w / w: add 750 pl of BSA stock (0.05 mg / ml, final concentration in 15 ml in water) + 2000 pl of polymer solution (HA-SA 10 % w / w) (0.1 mg / ml, final polymer concentration in 15 ml in water) + 12250 pl of water

[0427] • Set D - HA-PEG-SA 10 % w / w: add 2000 pl of polymer solution (HA-PEG-SA 10 % w / w) (0.1 mg / ml in water, final concentration in 15 ml) + 13000 pl of water • Set E - HA-SA 10 % w / w: add 2000 l of polymer solution (HA-SA 10 % w / w) (0.1 mg / ml in water, final concentration in 15 ml) + 13000 pl of water

[0428] Transmittance, particle size and PDI

[0429] The changes in particle size, transmissivity, and the PDI values of different sets (Set A to Set E) as a function of increase in pH (5.5 to 9.0) were evaluated. With an increase in pH, the particle size and the PDI of the samples should decrease owing to an increase in solubility of conjugates. At lower pH, the conjugate should not be soluble and result in encapsulation of BSA leading to higher particle size and PDI. Whereas, due to interaction of BSA and the conjugate, the samples could form nano-assemblies or nanoparticles, thus leading to higher transmissivity at all pH values.

[0430] The particle size, PDI and transmissivity were evaluated using Litesizer.

[0431] As anticipated, no significant difference in transmissivity was found in all the samples at different pH values (data not shown).

[0432] Interestingly, the particle size of the samples was found to be higher at low pH for Set D and Set E, suggesting pH dependent solubility of HA-PEG-SA 10 % w / w and HA-SA 10 % w / w conjugates. The particle size of Set C (HA-SA 10 % w / w + BSA) was also found to decrease with increase in pH, suggesting possible solubilisation of HA-SA 10 % w / w conjugate and thus releasing the BSA at higher pH (7.4 and 9.0) as compared to pH 5.5. (see Table 3).

[0433] Table 3: Effect of pH on the particle size and PDI values of the samples TEM analysis

[0434] To assess the coating of conjugates over the BSA, Set C (BSA + HA-SA 10 % w / w) at pH 5.5 was evaluated by TEM analysis. FIG. 9 shows the presence of two regions, i.e., inner black core and outer peripheral translucent part. The translucent part was found to be made up to fibrous part, which suggests presence of conjugate over the surface of BSA. Furthermore, the BSA alone sample (Set A) showed dark black core only i.e., without any fibrous structure over the surface of the BSA (FIG. 10).

[0435] In vitro release study

[0436] In vitro BSA release study in the presence of HA-SA 10% w / w:

[0437] The in vitro release of BSA from HA-SA 10 % w / w conjugate was evaluated in order to verify the interaction of developed conjugates with the protein molecules and the pH- dependent release. Briefly, 1 ml of the following samples was filled in the dialysis bag:

[0438] • BSA (250 pg) used as reference

[0439] • BSA (250 pg) + HA-SA 10 % w / w (500 pg)

[0440] The polymer and BSA were dissolved in water at pH 9.0 and sonicated for 10 min. Thereafter, the pH was adjusted to the pH of 5.5 (to mimic the pH formulation) and 7.4 (to mimic the physiological conditions) using 0.1 N HCI. After pH adjustment, 1 ml of each sample was put in dialysis bag (molecular weight cutoff of 100 kDa) then kept in the 25 ml glass vial containing 20 ml of media (water at pH 5.5 or 7.4) under magnetic stirring (final concentration of BSA 12 pg / ml, 250 pg in 21 ml). At predetermined time intervals (5 min, 15 min, 30 min, 1 h, 3 h, 6 h and 71 h), 1 ml of the medium outside of the dialysis membrane was collected and evaluated via Size exclusion chromatography for the quantification of BSA (LOD: 0.54 pg / ml, LOQ: 1.81 pg / ml, Linearity: 0.02 mg / ml - 10 mg / ml).

[0441] As shown in FIG. 11 , the release profile of BSA was retarded in presence of developed polymer (HA-SA 10 % w / w) at pH 5.5. In the presence of HA-SA 10 % w / w sample, the BSA release was observed after a lag phase of 60 min at pH 5.5 compared to BSA alone at the same pH. Thus, the developed conjugate (HA-SA 10 % w / w) was able to encapsulate BSA and retarded its release at pH 5.5. After 60 min, the release of BSA can be due to dilution of the sample (BSA and conjugate) with the external media. Dilution (sink condition) with the external media could result in solubilization of the conjugate resulting in BSA release after 60 min. Whereas, no marked difference in the BSA release profile was observed between free BSA and BSA in presence of HA-SA 10 % w / w at pH 7.4 (see FIG. 12). Thus, the developed conjugate HA-SA 10 % w / w demonstrated potential in encapsulating the protein molecule at pH 5.5 and release it at pH 7.4. In vitro BSA release study from HA-SA 10% w / w after a storage period:

[0442] In order to verify the protein release from HA-SA 10 % w / w after storage conditions, the delivery systems were stored for 3 and 7 days at 5 °C. The BSA released was evaluated by UPLC method To attain the final concentration within the linearity range (1 pg / ml - 45 pg / ml) of the UPLC method, the concentration of the sample in 1 ml of the media in dialysis bag was increased to 4 times i.e., the amount of BSA and HA-SA 10 % w / w was increased to 1 mg and 2 mg, respectively. Briefly, 1 ml of the following samples was filled in the dialysis bag:

[0443] • BSA (1 mg)

[0444] • BSA (1 mg) + HA-SA 10 % w / w (2 mg)

[0445] The polymer and BSA were dissolved in water of pH 9.0 and sonicated for 10 min. Thereafter, the pH was adjusted to the pH of 6.0 (to mimic formulation pH) and 7.4 (to mimic physiological medium) using 0.1 N HCI. Thereafter, the samples were stored in 25 ml falcon for 3 and 7 days at 5 °C. After storage, 1 ml of each sample (taken from the 25 ml flacon) was put in dialysis bag (molecular weight cut-off of 100 kDa). The dialysis bag was then kept in the 25 ml glass vial containing 20 ml of media (PBS buffer of pH 7.4 or 6.0) under magnetic stirring (final maximum concentration: 47.6 pg / ml of BSA in 21 ml on complete BSA release). At predetermined time intervals (5 min, 15 min, 30 min, 1 h, 3 h and 6 h), 1 ml of the medium outside of the dialysis membrane was collected from the same vial and evaluated via UPLC for the quantification of BSA.

[0446] As demonstrated in FIG. 13, even after storing, HA-SA 10 % w / w reduces the BSA release rate at pH 6.0 compared to pH 7.4. Interestingly, with increase in storage time (from 0 to 7 days), the amount of BSA released considering the same pH, is lower, possibly due to a stronger interaction between polymer and BSA increasing the contact time or to degradation of BSA upon storage.

[0447] In vitro BSA release study from HA-SA 10 % w / w after solubilization in buffer:

[0448] The BSA control release capacity of the HA-SA 10 % w / w polymer in buffer (6.0 and 7.4) was evaluated and compared with the water at different pH. Briefly, instead of sample preparation using pH shift method (from 9.0 to pH 7.4 and 6.0), the conjugates were directly dissolved in buffer of pH 7.4 and 6.0. The following samples were prepared:

[0449] • BSA (1 mg / ml) + HA-SA 10 % w / w (2 mg / ml) o PBS buffer of pH 7.4 o PBS buffer of pH 6.0 After suspending, the samples were stored in 25 ml falcon for 3 and 7 days at 5 °C. After storage, 1 ml of each sample (taken from the 25 ml flacon) was put in dialysis bag (molecular weight cut-off of 100 kDa). The dialysis bags were kept in the 25 ml glass vial containing 20 ml of media (PBS buffer of pH 7.4 or 6.0) under magnetic stirring (final maximum concentration: 47.6 pg / ml of BSA in 21 ml on complete BSA release).

[0450] At predetermined time intervals (5 min, 15 min, 30 min, 1 h, 3 h and 6 h), 1 ml of the medium outside of the dialysis membrane was collected and evaluated via UPLC for the quantification of BSA and compared with the release profile mentioned in FIG. 13.

[0451] FIG. 14 demonstrates that, in line with the release profile after solubilization in water, the release of BSA was retarded in the presence of HA-SA 10 % w / w at pH 6.0 as compared to 7.4. However, the release was significantly higher in the presence of buffer as compared to the samples prepared using pH shift i.e., from 9 pH to 7.4 and 6.0, probably due to minor polymer solubilization or less BSA degradation.

[0452] In vitro BSA release study (after 7 days storage) - 2 mg vs. 1 mg polymer:

[0453] The effect of polymer concentration was also evaluated. The release of BSA was evaluated in presence of 1 mg and 2 mg of the HA-SA 10 % w / w conjugate. Briefly, 1 ml of the following samples was filled in the dialysis bag.

[0454] • BSA (1 mg) + HA-SA 10 % w / w (2 mg) o pH 7.4 o pH 6.0

[0455] • BSA (1 mg) + HA-SA 10 % w / w (1 mg) o pH 6.0

[0456] The polymer and BSA were dissolved in water of pH 9.0 and sonicated for 10 min. Thereafter, the pH was adjusted to the pH of 6.0 and 7.4 using 0.1 N HCI.

[0457] After pH adjustment, the samples were stored in 25 ml falcon for 7 days at 5 °C. After storage, 1 ml of each sample (taken from the 25 ml flacon) was put in dialysis bag (molecular weight cut-off of 100 kDa) then kept in the 25 ml glass vial containing 20 ml of media (PBS buffer of pH 7.4 or 6.0) under magnetic stirring (final concentration: 47.6 pg / ml of BSA in 21 ml). At predetermined time intervals (5 min, 15 min, 30 min, 1 h, 3 h and 6 h), 1 ml of the medium outside of the dialysis membrane was collected and evaluated via UPLC for the quantification of BSA.

[0458] FIG. 15 shows BSA release retardation at pH 6.0 in presence of 1 and 2 mg HA-SA 10 % w / w. The BSA release retardation was found to be significantly higher in presence of 1 mg of HA- SA 10 % w / w polymer as compared to higher concentration (2 mg / ml). This may be due to better HA-SA 10 % w / w solubility at 1 mg / ml resulting in higher efficiency of BSA encapsulation (compared to HA-SA 10 % w / w at 2 mg / ml) or to higher amount of free BSA (due to lower ratio polymer / BSA and low encapsulation efficacy) that degraded in the release medium.

[0459] Conclusion

[0460] HA conjugates with different derivatization degree of stearic acid (Sac) or stearyl amine (Sam) were synthesized (100 mg of polymer was synthesized in one batch).

[0461] The developed conjugates showed pH-dependent solubility with an increased solubility in physiological pH as compared to acidic pH.

[0462] In vitro release studies of BSA from HA derivatives were performed. Slower BSA release rate was observed in case of BSA + HA-SA 10 % w / w at pH 5.5 as compared to the free BSA at pH 5.5 and to BSA + HA-SA 10 % w / w at pH 7.4. After storing (5 °C; up to 7 days), HA-SA 10 % w / w showed a slower BSA release rate at pH 6.0 compared to pH 7.4.

Claims

CLAIMS1. An amphiphilic pH-sensitive polymer, wherein the polymer is soluble in an aqueous environment at a trigger physiological pH.

2. The polymer of claim 1 , wherein the polymer is insoluble in an aqueous environment at a sub-physiological pH.

3. The polymer of any claim 1 or 2, wherein said physiological pH is at or above 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6 or 7.7.

4. The polymer of any claim 2 or 3, wherein said sub-physiological pH is below 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1 , 6.0, 5.9, 5.8, 5.7, 5.6 or 5.5.

5. The polymer of any claim 2 to 4, wherein said sub-physiological pH and physiological pH differ from each other by at least 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .1 , 1 .2, 1 .3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 pH units.

6. The polymer of any claim 1 to 5, wherein on subjecting the polymer to said trigger physiological pH, the polymer is substantially or completely dissolved and / or dissociated and / or destabilized within 24 hours or less.

7. The polymer of any claim 1 to 6, wherein the polymer has a solubility comprised between 10 and 1000 mg per 100 ml at said trigger physiological pH.

8. The polymer of any claim 1 to 7, wherein the polymer is a biopolymer.

9. The polymer of any claim 1 to 8, wherein the polymer is biocompatible.

10. The polymer of any claim 1 to 9, wherein the biopolymer is injectable.

11. The polymer of any claim 1 to 10, wherein at least one repeat unit within the polymer comprises a hydrophilic scaffold functionalized with at least one hydrophobic group.

12. The polymer of claim 11 , wherein the hydrophilic scaffold is a repeat unit of glycosaminoglycan.

13. The polymer of claim 11 or 12, wherein the hydrophilic scaffold is a repeat unit of hyaluronic acid.

14. The polymer of claim 13, wherein the functionalization is done on at least the carboxylic acid group of the hyaluronic acid repeat unit.

15. The polymer of any claim 11 to 14, wherein said at least one hydrophobic group is a lipophilic group.

16. The polymer of claim 15, wherein the lipophilic group is derived from a fatty acid.

17. The polymer of claim 16, wherein the fatty acid is stearyl amine or stearic acid .

18. The polymer of any claim 11 to 17, wherein said at least one hydrophobic group is directly grafted to the hydrophilic scaffold.

19. The polymer of claim 18, wherein said at least one hydrophobic group is derived from stearyl amine and the hydrophilic scaffold is hyaluronic acid.

20. The polymer of any claim 11 to 17, wherein said at least one hydrophobic group is grafted to the hydrophilic scaffold via a linker.21 . The polymer of claim 20, wherein the linker is a polyethylene glycol (PEG) linker.

22. The polymer of claim 20 or 21 , wherein said at least one hydrophobic group is derived from stearic acid, and the hydrophilic scaffold is hyaluronic acid.

23. The polymer of any claim 11 to 22, wherein the polymer comprises 1 to 50 % by mole of repeat units that are functionalized.

24. The polymer of claim 11 , wherein the hydrophilic scaffold functionalized with at least one hydrophobic group is according to formula (A):wherein at least one of Ri, R2, R3, R4, R5 and R6is a hydrophobic group, and- when R1 is a hydrophobic group, Bi is a type of bond, p is 0 or 1 , and when p is 1 , Li is a linker and B2is a type of bond,- when R2is a hydrophobic group, B3is a type of bond, q is 0 or 1 , and when q is 1 , l_2is a linker and B4 is a type of bond,- when R3is a hydrophobic group, B5is a type of bond, r is 0 or 1 , and when r is 1 , l_3is a linker and B6is a type of bond,- when R4is a hydrophobic group, B7is a type of bond, s is 0 or 1 , and when s is 1 , l_4 is a linker and B3is a type of bond,- when R5is a hydrophobic group, B9is a type of bond, t is 0 or 1 , and when t is 1 , l_5is a linker and B10 is a type of bond, and / or- when R6is a hydrophobic group, Bn is a type of bond, u is 0 or 1 , and when u is 1 , l_6 is a linker and B12 is a type of bond.

25. The polymer of claim 24, wherein when:R1 is not a hydrophobic group, p is 0 and -B1-R1 is -NH-(CO)-CH3,R2 is not a hydrophobic group, q is 0 and -B3-R2is -OH,R3 is not a hydrophobic group, r is 0 and -B5-R3is -OH,R4 is not a hydrophobic group, s is 0 and -B7-R4 is -OH,R5 is not a hydrophobic group, t is 0 and -B9-R5 is -OH,R6 is not a hydrophobic group, u is 0 and -Bn-Re is -(CO)-OH, -(CO)-ONa, or - (CO)-OK.

26. The polymer of any claim 24 or 25, wherein those of R1, R2, R3, R4, Rs and R6that are hydrophobic groups are each and independently selected from the group comprising such as consisting of: saturated or unsaturated alkyl chains, phospholipids, sterols, polymers such as polymers from 1000 to 10000 Da, such as PLA or PLGAfrom x to 10000 Da.

27. The polymer of any claim 24 to 26, wherein:R1 is a hydrophobic group and Bi is an amide bond, such as an amide bond wherein the carbon atom of position 2 is directly linked to the nitrogen atom of said amide bond,R2is a hydrophobic group and B3 is an ester bond or a carbamate bond, such as ester or carbamate bonds wherein the oxygen not involved in the carbonyl of said bonds is directly linked to the carbon atom of position 4,R3is a hydrophobic group and B5is an ester bond or a carbamate bond, such as ester or carbamate bonds wherein the oxygen not involved in the carbonyl of said bonds is directly linked to the carbon atom of position 6,R4is a hydrophobic group and B7is an ester bond or a carbamate bond, such as ester or carbamate bonds wherein the oxygen not involved in the carbonyl of said bonds is directly linked to the carbon atom of position 8,R5is a hydrophobic group and B9is an ester bond or a carbamate bond, such as ester or carbamate bonds wherein the oxygen not involved in the carbonyl of said bonds is directly linked to the carbon atom of position 9, and / orRe is a hydrophobic group, Bn is an ester bond or an amide bond, such as ester or amide bonds wherein the carbonyl of said bonds is directly linked to the carbon atom of position 11 .

28. The polymer of any claim 24 to 27, wherein when:R1 is a hydrophobic group and p is 0, R1 is directly linked to Bi ;R2 is a hydrophobic group and q is 0, R2is directly linked to B3;R3 is a hydrophobic group and r is 0, R3is directly linked to B5;R4 is a hydrophobic group and s is 0, R4is directly linked to B7;R5 is a hydrophobic group and t is 0, Rs is directly linked to B9; and / or R6 is a hydrophobic group and u is 0, R6is directly linked to Bn.

29. The polymer of any claim 24 to 27, wherein when p, q, r, s, t and / or u are 1 , Li, L2, L3, L4, l_5 and / or Le, respectively, are linkers, such as linkers from 72 to 3200 Da, such as from 200 to 2800 Da, 400 to 2600 Da, 600 to 2400 Da, 800 to 2200 Da, 1000 to 2000 Da, 1200 to 1800 Da, 1400 to 1600 Da.

30. The polymer of claim 29, wherein Li, L2, L3, L4, l_5and / or l_6comprise PEG repeat units.31 . The polymer of claim 29 or 30, wherein Li, L2, L3, L4, Ls and / or Le comprise such as arewherein n is the number of repeat units.

32. The polymer of claim 31 , wherein n is from 20 to 60, such as from 25 to 55, 30 to 50, 35 to 45, 36, 37, 38, 39, 40, 41 , 42, 43, or 44.

33. The polymer of claim 32, wherein n is from 35 to 45, for example 36, 37, 38, 39, 40, 41 , 42, 43 or 44, in particular 39 or 40.

34. The polymer of any claim 29 to 33, wherein B2, B4, B6, B8, Bio and / or B12, respectively, are amide or ester bonds.

35. The polymer of claim 34, wherein when B2, B4, B6, B8, Bio and / or BI2, are amide bonds, Ri, R2, R3, R4, R5and / or R6, respectively, are directly linked to the carbonyl of said amide bond.

36. The polymer of claim 34, wherein when B2, B4, B6, B8, Bio and / or BI2, are ester bonds, Ri, R2, R3, R4, R5and / or R6, respectively, are directly linked to the carbonyl of said ester bond.

37. The polymer of any claim 24 to 36, wherein the stereochemistry of formula (A) is as depicted in structure (AA):

38. The polymer of any claim 24 to 37, wherein R6is a hydrophobic group.

39. The polymer of any claim 24 to 38, wherein none of Ri , R2, R3, R4or Rs are hydrophobic groups.

40. The polymer of any claim 24 to 39, wherein the hydrophobic group is a saturated or unsaturated alkyl chain.41 . The polymer of claim 40, wherein the alkyl chain has from 6 to 36 carbon atoms.

42. The polymer of claim 41 , wherein the alkyl chain has from 11 to 31 carbon atoms.

43. The polymer of claim 42, wherein the alkyl chain has from 16 to 26 carbon atoms.

44. The polymer of claim 43, wherein the alkyl chain has 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 carbon atoms.

45. The polymer of claim 44, wherein the alkyl chain has 18 carbon atoms.

46. The polymer of any claim 38 to 45, wherein Bn is an amide bond, such as an amide bond wherein the carbonyl of said bond is directly linked to the carbon atom of position 11 .

47. The polymer of any claim 38 to 46, wherein u is 0.

48. The polymer of any claim 38 to 47, wherein the hydrophilic scaffold functionalized with at least one hydrophobic group has the following formula:

49. The polymer of any claim 38 to 44 and 46, wherein the alkyl chain has 17 carbon atoms.

50. The polymer of any claim 38 to 44, 46 and 49, wherein u is 1 .51 . The polymer of any claim 38 to 44, 46, 49 and 50, wherein l_6is as defined in any of claim 29 to 33.

52. The polymer of any claim 38 to 44, 46 and 49 to 51 , wherein B12 is an amide bond, wherein R6is directly linked to the carbonyl of said amide bond.

53. The polymer of any claim 38 to 44, 46 and 49 to 52, wherein the hydrophilic scaffold functionalized with at least one hydrophobic group has the following formula:about 2000Da and / or wherein n is 39 or n is 40.

54. The polymer of any claim 24 to 53, comprising from 2 to 45% by mole, such as 5, 10, 15, 20, 25, 30 or 35 % by mole, in particular 10 to 30 % by mole, of hydrophilic scaffold functionalized with at least one hydrophobic group according to formula (A).

55. The polymer of any claim 24 to 54, further comprising repeat units according to formula (B), and wherein formula (B) corresponds to formula (A) wherein: p is 0 and -B1-R1 is -NH-(CO)-CH3, q is 0 and -B3-R2 is -OH, r is 0 and -B5-R3 is -OH, s is 0 and -B7-R4 is -OH,- t is 0 and -B9-R5 is -OH,- u is 0 and -Bn-R6is -(CO)-OH, -(CO)-ONa, or -(CO)-OK.

56. The polymer of claim 55, wherein formula (B) is as follows:salt thereof.

57. The polymer of claim 56, wherein formula (B) is a sodium or potassium salt.

58. The polymer of any claim 24 to 57, wherein the total content of repeat units according to formula (A) and repeat units according to formula (B) constitutes 100% by weight of the polymer.

59. The polymer of any claim 1 to 58, having a molecular weight between 1 to 100 kDa, such as 10, 20, 30, 40, 50, 60, 70, 80, or 90 kDa.

60. The polymer of claim 59, wherein the molecular weight is from 10 to 40 KDa, such as 15, 20, 25, 30 or 35 kDa, in particular about 20 kDa.61 . The polymer of any claim 11 to 60, wherein the repeat units are derived from one species of monomers.

62. A self-assembled particle comprising at least one amphiphilic pH-sensitive polymer of any claim 1 to 61.

63. The self-assembled particle of claim 62, further comprising a cargo, such as a biologically active constituent, being encapsulated within the amphiphilic pH-sensitive polymer as defined in any claim 1 to 61 , in an aqueous environment having a sub-physiological pH.

64. The self-assembled particle of claim 63, wherein said particle is triggered to release said cargo, such as said biologically active constituent, by being present in said aqueous environment having a physiological pH.

65. The self-assembled particle of claim 63 or 64, wherein said cargo is an antigen.

66. The self-assembled particle from claim 65, wherein said cargo comprises a saccharide such as an oligo- or poly- saccharide derived from a bacterial pathogen selected from the group consisting of Haemophilus influenzae type b (Hib) Neisseria meningitidis (in particular serotypes A, C, W and / or Y); Streptococcus pneumoniae (in particular serotypes 1 , 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, I0A, HA, 12F, 14, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F and / or 33F); Staphylococcus aureus, Bordetella pertussis, and / or Salmonella typhi.

67. A composition, an immunogenic composition, or a vaccine comprising at least one self-assembled particle as defined in any claim 62 to 66 in an aqueous environment, wherein said composition is of sub-physiological pH.

68. A self-assembled particle of any claim 62 to 66, a composition, an immunogenic composition, or a vaccine of claim 67 for use as a medicament.

69. The composition, immunogenic composition or vaccine for use according to claim 68, wherein the cargo is released at physiological pH.

70. A self-assembled particle of any claim 62 to 66, a composition, an immunogenic composition, or a vaccine of claim 67 for use in the treatment or prevention of a disorder or a disease, such as an infection, caused directly or indirectly by a pathogen, or of a pathology associated with immunologically distinct host cells such as cancer.71 . A method of treating or preventing a disorder or a disease, such as an infection, caused directly or indirectly by a pathogen, or of a pathology associated with immunologically distinct host cells such as cancer, wherein the method comprises applying or administering to a subject in need thereof the self-assembled particle of any claim 62 to 66, the composition, the immunogenic composition, or the vaccine of claim 67.

72. A method of eliciting an immune response, wherein the method comprises applying or administering to a subject in need thereof an effective amount of the self-assembled particle of any claim 62 to 66, the composition, the immunogenic composition, or the vaccine of claim 67.

73. A method for preventing or reducing interaction between a cargo, such as a biologically active constituent, and co-formulated substances of an aqueous environment, such as in a parenteral formulation, comprising:(i) forming said at least one self-assembled particle of any claim 62 to 66, comprising said cargo; and(ii) formulating said at least one self-assembled particle in said aqueous environment, comprising any necessary adjustment to render said environment of sub- physiological pH.

74. A method for preventing or reducing interaction between a cargo, such as a biologically active constituent, and co-formulated substances of an aqueous environment, such as in a parenteral formulation, of sub-physiological pH, comprising:(iii) forming said at least one self-assembled particle of any claim 62 to 66, comprising said cargo; and(iv) formulating said at least one self-assembled particle in said aqueous environment.

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