Bioactive synthetic polymeric nanoparticles, related composition, polymers, macromolecules and methods thereof

Abstract

There is provided a polymeric nanoparticle comprising: a hydrophobic synthetic polymer core; and a hydrophilic bioactive shell, wherein the hydrophobic synthetic polymer core and hydrophilic bioactive shell are parts of a bioactive synthetic polymer with a poly(norbornene-dicarboximide) backbone having one or more repeating units selected from the group consisting of general formula (1), (2) and (3). Also provided are a composition comprising bioactive synthetic polymeric nanoparticles, bioactive synthetic polymers, macromolecules for preparing said bioactive synthetic polymers, related methods and uses.

Description

[0001] BIOACTIVE SYNTHETIC POLYMERIC NANOPARTICLES, RELATED COMPOSITION, POLYMERS, MACROMOLECULES AND METHODS THEREOF

[0002] TECHNICAL FIELD

[0003] The present disclosure relates broadly to bioactive synthetic polymeric nanoparticles and a method of preparing said bioactive synthetic polymeric nanoparticles. The present disclosure also relates to a composition comprising said bioactive synthetic polymeric nanoparticles, and related methods and uses. The present disclosure also relates to bioactive synthetic polymers, bioactive synthetic macromolecules, and related methods.

[0004] BACKGROUND

[0005] A better understanding of the biology and physiology of living things over the years has led to an appreciation of the potential of using alternative materials to enhance or replace existing functions in biological systems. Particularly, there is interest in designing nanoparticles that are capable of displaying biological functions while being thermally stable.

[0006] However, identifying a suitable material that meets both the thermal and biological requirements to function desirably in or with biological systems is often challenging.

[0007] This is because bioactive molecules (e.g. low molecular weight / short chain hyaluronic acid (HA) and arginyl-glycyl-aspartic acid (RGD) etc) that have the desired biological attributes are often unstable in physiological conditions and thermally unstable to withstand material processing. For example, many of such bioactive molecules are very hygroscopic, tend to be absorbed rapidly in human body and undergo rapid metabolism under physiological conditions. These limitations result in low biochemical functionality for cell attachment and proliferation. Sustained bioavailability of these bio molecules are required for effective therapeutic functions from the bio molecules.

[0008] Bio molecules can be stabilised through chemically linking with synthetic polymers. Combining these different materials with the hope that the resultant material obtained can achieve both the desired biological and thermal properties is also challenging. This is because bioactive molecules are often incompatible with synthetic polymers since the former is hydrophilic whereas the latter is hydrophobic. The inherent differences in their hydrophilicity likewise make chemically synthesizing a bioactive polymer from these materials extremely difficult, especially when the molecular weights of these materials are relatively high. This is in addition to the various complex chemical hurdles (e.g. potentially high intramolecular reactivity, unwanted chemical leaching of by-products etc) that need to be overcome when attempting to chemically combine these two chemically different types of materials together.

[0009] Studies have been performed to chemically combine these two different materials together. However, current methods have several limitations and are far from desirable.

[0010] For example, even though materials chemically combining bioactive molecules with synthetic polymers have been synthesized, the amount of bioactive molecules successfully incorporated into such materials is relatively low due to issues arising from their incompatibility and inherent differences in their hydrophilicity. With such relatively low amounts of bioactive molecules incorporated (e.g., a maximum of 10 wt%) in these materials, they lack a sufficient level of hydrophilicity and fail to provide good control over its stability for preparation into nanoparticles. Furthermore, such materials are typically made up of randomly connected polymer blocks which makes it impossible to control many aspects of nanoparticle preparation such as particle size distribution which is highly important for useful biomedical applications.

[0011] In view of the above, there is a need to address or at least ameliorate the above-mentioned problems. In particular, there is a need to provide bioactive synthetic polymeric nanoparticles and / or a composition comprising bioactive synthetic polymeric nanoparticles that address or at least ameliorate the above- mentioned problems. SUMMARY

[0012] In one aspect, there is provided a polymeric nanoparticle comprising:

[0013] (i) a hydrophobic synthetic polymer core; and

[0014] (ii) a hydrophilic bioactive shell, wherein the hydrophobic synthetic polymer core and hydrophilic bioactive shell are parts of a bioactive synthetic polymer with a poly(norbornene- dicarboximide) backbone having one or more repeating units selected from the group consisting of general formula (1), (2) and (3): and wherein

[0015] R1and R4are each independently selected from a single bond, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl or optionally substituted alkylcarbonylalkyl;

[0016] R2and R6are each optionally substituted alkyl;

[0017] R3, R5and R7are each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl;

[0018] L1and L2are each heteroalkylene;

[0019] X1and X2each independently comprise a bioactive moiety selected from the group consisting of proteins, peptides, oligopeptides, carbohydrates, oligosaccharides, sugar, collagen, hyaluronic acid, therapeutic / drug molecules and derivatives thereof;

[0020] Y1and Y2each independently comprise a synthetic polymer selected from the group consisting of poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid-co- lactic acid) (PGLA), poly(glycolic acid) (PGA), polystyrene (PS), poly (lactic acid) (PLA), poly(caprolactone) (PCL), polyacrylates, poly(meth)acrylates, polyesters, derivatives thereof and parts thereof; and

[0021] Z1, Z2and Z3are each independently selected from CRaRb, O, NRa, SiRaRb, PRaor S, wherein Raand Rbare each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl and optionally substituted alkynyl.

[0022] In one embodiment, the hydrophilic bioactive shell is at least 1 wt% of the nanoparticle.

[0023] In one embodiment, the hydrophobic synthetic polymer core is at least 10 wt% of the nanoparticle.

[0024] In one embodiment, the nanoparticle has an average size falling in the range of from 10 nm to 1 ,000 nm. In one embodiment, the repeating units represented by general formula (1); (2); and / or (3) are sequentially distributed within the bioactive synthetic copolymer.

[0025] In one embodiment, L1and L2are each polyethylene glycol (PEG).

[0026] In one embodiment, R1and R4are each independently selected from Ci- C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C1-C20 alkoxyalkyl, C2-C20 alkylcarbonyl or C3-C20 alkylcarbonylalkyl; R2and R6are each independently selected from straight or branched C1-C20 alkyl; and R3, R5and R7are each independently selected from H, C1-C20 alkyl, C2-C20 alkenyl or C2-C20 alkynyl.

[0027] In one aspect, there is provided a method of preparing a plurality of polymeric nanoparticles as claimed in any one of claims 1 to 7, the method comprising:

[0028] (a-i) mixing one or more bioactive synthetic polymer(s) with a polyfnorbornene- dicarboximide) backbone having one or more repeating units selected from the group consisting of general formula (1), (2) and (3) in a suitable organic solvent to obtain a solution;

[0029] (a-ii) precipitating polymeric nanoparticles from the solution into a non-solvent to obtain a dispersion of polymeric nanoparticles; and

[0030] (a-iii) removing the organic solvent and non-solvent from the dispersion to obtain polymeric nanoparticles.

[0031] In one embodiment, volume ratio of the organic solvent to the non-solvent is from 1 :1 to 1 :10.

[0032] In one embodiment, the step (a-i) comprises adding from 0.1 wt% to 50 wt% of the polymers.

[0033] In one embodiment, the step (a-ii) comprises adding the solution obtained from (a-i) into a non-solvent at a rate of from 0.1 mL / s to 5 mL / s. In one embodiment, the organic solvent is selected from tetrahydrofuran (THF), benzene, toluene, acetonitrile (ACN), dichloromethane (DCM), dimethyl sulfoxide (DMSO), acetone, methyl ethyl ketone (MEK), ethyl vinyl ether, methanol, ethanol, butanol and combinations thereof.

[0034] In one embodiment, the non-solvent comprises an aqueous medium.

[0035] In one embodiment, the method further comprises, prior to the step (a-iii) of removing organic solvent and non-solvent / aqueous medium, a step of sonicating the dispersion of nanoparticles under one or more of the following conditions:

[0036] (i) at a temperature that is no more than 35°C;

[0037] (ii) over a time duration of from 1 minute to 24 hours; and

[0038] (iii) at a stirring rate / speed of from 100 rpm to 5,000 rpm.

[0039] In one aspect, there is provided a polymeric nanoparticle composition comprising: a dispersion of a plurality of polymeric nanoparticles as disclosed herein.

[0040] In one embodiment, the polymeric nanoparticle composition further comprises:

[0041] (i) a buffer solution;

[0042] (ii) a thickening agent; and

[0043] (iii) optionally a stabilizer.

[0044] In one embodiment, the thickening agent is selected from the group consisting of hydroxyethyl cellulose (HEC), xanthan gum, hydroxypropyl cellulose and combinations thereof.

[0045] In one embodiment, the stabilizer is selected from the group consisting of propylene glycol, glycerol, glycerin and combinations thereof. In one embodiment, the composition is a topical skin formulation.

[0046] In one embodiment, the topical skin formulation is in the form of a liquid, gel or cream formulation.

[0047] In another aspect, there is provided a composition as disclosed herein for use in stimulating skin and / or tissue regeneration.

[0048] In another aspect, there is provided a composition as disclosed herein for use in the treatment of a skin condition.

[0049] In another aspect, there is provided use of a composition as disclosed herein in the manufacture of a medicament for stimulating skin and / or tissue regeneration.

[0050] In another aspect, there is provided use of a composition as disclosed herein in the manufacture of a medicament for treatment of a skin condition.

[0051] In another aspect, there is provided a method of stimulating skin and / or tissue regeneration in a subject in need thereof, the method comprising applying the composition as disclosed herein to a body part of the subject in need thereof.

[0052] In another aspect, there is provided a method of treating a skin condition, the method comprising applying the composition as disclosed herein to a body part of a subject in need thereof.

[0053] In one embodiment, the skin condition is selected from the group consisting of skin wounds, skin inflammation, psoriasis, rashes, aging skin eczema, atopic dermatitis / eczema, contact dermatitis, allergic dermatitis, dyshidrotic eczema, neurodermatitis, nummular eczema, seborrheic dermatitis, stasis dermatitis, and combinations thereof. In another aspect, there is provided a bioactive synthetic macromonomer represented by general formula (6) for preparing a bioactive synthetic polymer: wherein R4is selected from a single bond, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl or optionally substituted alkylcarbonylalkyl;

[0054] R5and R7are each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl;

[0055] R6is optionally substituted alkyl;

[0056] L2is heteroalkylene;

[0057] X2comprises a bioactive moiety selected from the group consisting of proteins, peptides, oligopeptides, carbohydrates, oligosaccharides, sugar, collagen, hyaluronic acid, therapeutic / drug molecules and derivatives thereof;

[0058] Y2comprises a synthetic polymer selected from the group consisting of poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid-co-lactic acid) (PGLA), polyfglycolic acid) (PGA), polystyrene (PS), poly(lactic acid) (PLA), poly(caprolactone) (PCL), polyacrylates, polyesters, derivatives thereof and parts thereof; and

[0059] Z3is selected from CRaRb, O, NRa, SiRaRb, PRaor S, wherein Raand Rbare each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl and optionally substituted alkynyl.

[0060] In another aspect, there is provided a method of preparing a bioactive synthetic macromonomer represented by general formula (6) as disclosed herein, the method comprising:

[0061] (e-i) reacting a synthetic macromonomer represented by general formula (7) with a diamine represented by general formula (8) to obtain an amine represented by general formula (9): wherein

[0062] R9is selected from H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl.

[0063] (e-ii) reacting said amine represented by general formula (9) with an acidcontaining bioactive moiety (e.g., X-C(=O)OH) to obtain the bioactive synthetic macromonomer represented by general formula (6). DEFINITIONS

[0064] The term "polymer" as used herein refer to a chemical compound comprising repeating units and is created through a process of polymerization. It will be appreciated that the term “polymerization” is not limited to homopolymerization but also encompasses copolymerization. The units composing the polymer are typically derived from monomers and / or macromonomers. A polymer or copolymer typically comprises repetition of a number of constitutional units. The term “polymer” encompasses the term “homopolymer”, where the polymer is derived from a single type of monomer or homomonomer (or contains a single type of repeating unit). The term “polymer” also encompasses the term “copolymer”, where the polymer is derived from two or more different types of monomers or comonomers (or contains two or more different types of repeating units).

[0065] The terms “monomer” or “macromonomer” as used herein refer to a chemical entity that may be covalently linked to one or more of such entities to form a polymer.

[0066] The term “bioactive” as used herein broadly refers to the property of having a biological effect, preferably a desirable or positive biological effect on a living organism, tissue, or cell.

[0067] The term “biocompatible” as used herein broadly refers to a property of being compatible with biological systems or parts of the biological systems without substantially or significantly eliciting an adverse physiological response such as a toxic reaction, an immune reaction, an injury or the like. Such biological systems or parts include blood, cells, tissues, organs or the like.

[0068] The term "bond" refers to a linkage between atoms in a compound or molecule. The bond may be a single bond, a double bond, or a triple bond. In the definitions of a number of substituents below, it is stated that “the group may be a terminal group or a bridging group”. This is intended to signify that the use of the term is intended to encompass the situation where the group is a terminal group / moiety as well as the situation where the group is a linker between two other portions of the molecule. Using the term “alkyl” having 1 carbon atom as an example, it will be appreciated that when existing as a terminal group, the term “alkyl” having 1 carbon atom may mean -CHs and when existing as a bridging group, the term “alkyl" having 1 carbon atom may mean -CH2- or the like.

[0069] The term "alkyl" as a group or part of a group refers to a straight or branched aliphatic hydrocarbon group having 1 to 20 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. Examples of suitable straight and branched alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, isopropyl, n- butyl, isobutyl, sec-butyl, t-butyl, hexyl, amyl, 1 ,2-dimethylpropyl, 1 ,1 - dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1 -methylpentyl, 2- methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1 ,2- dimethylbutyl, 1 ,3-dimethylbutyl, 1 ,2,2-trimethylpropyl, 1 ,1 ,2-trimethylpropyl, 2- ethylpentyl, 3-ethylpentyl, heptyl, 1 -methylhexyl, 2,2-dimethylpentyl, 3,3- dimethylpentyl, 4,4-dimethylpentyl, 1 ,2-dimethylpentyl, 1 ,3-dimethylpentyl, 1 ,4- dimethylpentyl, 1 ,2,3-trimethylbutyl, 1 ,1 ,2-trimethylbutyl, 1 ,1 ,3-trimethylbutyl, 5- methylheptyl, 1 -methylheptyl, octyl, nonyl, decyl and the like. The group may be a terminal group or a bridging group.

[0070] The term "alkenyl" as a group or part of a group denotes an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and which may be straight or branched having 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 6 carbon atoms, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms in the chain. The group may contain a plurality of double bonds and the orientation about each double bond is independently E or Z. Exemplary alkenyl groups include, but are not limited to, ethenyl, vinyl, allyl, 1 - methylvinyl, 1 -propenyl, 2-propenyl, 2-methyl-1 -propenyl, 2-methyl-1 -propenyl, 1 -butenyl, 2-butenyl, 3-butentyl, 1 ,3-butadienyl, 1 -pentenyl, 2-pententyl, 3- pentenyl, 4-pentenyl, 1 ,3-pentadienyl, 2,4-pentadienyl, 1 ,4-pentadienyl, 3- methyl-2-butenyl, 1 -hexenyl, 2-hexenyl, 3-hexenyl, 1 ,3-hexadienyl, 1 ,4- hexadienyl, 2-methylpentenyl, 1 -heptenyl, 2-heptentyl, 3-heptenyl, 1 -octenyl, 2- octenyl, 3-octenyl, 1 -nonenyl, 2-nonenyl, 3-nonenyl, 1 -decenyl, 2-decenyl, 3- decenyl and the like. The group may be a terminal group or a bridging group.

[0071] The term "alkynyl" as a group or part of a group denotes an aliphatic hydrocarbon group containing at least one carbon-carbon triple bond and which may be straight or branched having 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 6 carbon atoms, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms in the chain. The group may contain a plurality of triple bonds. Exemplary alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1 - butynyl, 2-butynyl, 3-butynyl, 1 -pentynyl, 2-pentynyl, 3-methyl-1 -butynyl, 4- pentynyl, 1 -hexynyl, 2-hexynyl, 5-hexynyl, 1 -heptynyl, 2-heptynyl, 6-heptynyl, 1 - octynyl, 2-octynyl, 7-octynyl, 1 -nonynyl, 2-nonynyl, 8-nonynyl, 1 -decynyl, 2- decynyl, 9-decynyl and the like. The group may be a terminal group or a bridging group.

[0072] The term "heteroalkylene" as used herein refers to alkylene having one or more -CH2- replaced with a heteroatom selected from O, NR, Si, P or S, where R is hydrogen or alkyl as defined herein. The term "heteroalkylene" can be linear, branched or cyclic and containing up to 500 carbon atoms.

[0073] The term "alkoxy" as used herein refers to straight chain or branched alkyloxy groups. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, tertbutoxy, and the like.

[0074] The term "alkoxyalkyl" as used herein is intended to broadly refer to a group containing -R-O-R’, where R and R’ are alkyl as defined herein. The group may be a terminal group or a bridging group. The term "alkylcarbonyl" as used herein is intended to broadly refer to a group containing -R-C(=O)-, where R is alkyl as defined herein. The group may be a terminal group or a bridging group.

[0075] The term "alkylcarbonylalkyl" as used herein is intended to broadly refer to a group containing -R-C(=O)-R’, where R and R’ are alkyl as defined herein. The group may be a terminal group or a bridging group.

[0076] The term "carboxylalkyl" as used herein is intended to broadly refer to a group containing -C(=O)-O-R, where R is alkyl as defined herein. The group may be a terminal group or a bridging group.

[0077] The term "oxycarbonylalkyl" as used herein is intended to broadly refer to a group containing -O-C(=O)-R, where R is alkyl as defined herein. The group may be a terminal group or a bridging group.

[0078] The term "alkylcarboxylalkyl" as used herein is intended to broadly refer to a group containing -R-C(=O)-O-R’, where R and R’ are alkyl as defined herein. The group may be a terminal group or a bridging group.

[0079] The term "alkoxycarbonylalkyl" as used herein is intended to broadly refer to a group containing -R-O-C(=O)-R’, where R and R’ are alkyl as defined herein. The group may be a terminal group or a bridging group.

[0080] The term "oxy" as used herein is intended to broadly refer to a group containing -O-.

[0081] The term "carbonyl" as used herein is intended to broadly refer to a group containing -C(=O)-.

[0082] The term "oxycarbonyl" as used herein is intended to broadly refer to a group containing -O-C(=O)-. The term "carboxyl" as used herein is intended to broadly refer to a group containing -C(=O)-O-R, where R is hydrogen or an organic group.

[0083] The term "halogen" represents chlorine, fluorine, bromine or iodine. The term "halo" represents chloro, fluoro, bromo or iodo.

[0084] The term "amine group" or the like is intended to broadly refer to a group containing -N 2, where R is independently a hydrogen or an organic group. The group may be a terminal group or a bridging group.

[0085] The term "amide group" or the like is intended to broadly refer to a group containing -C(=O)NR2, where R is independently a hydrogen or an organic group. The group may be a terminal group or a bridging group.

[0086] The term “optionally substituted,” when used to describe a chemical structure or moiety, refers to the chemical structure or moiety wherein one or more of its hydrogen atoms is optionally substituted with a chemical moiety or functional group such as alcohol, alkoxy, alkanoyloxy, alkoxycarbonyl, alkenyl, alkyl (e.g., methyl, ethyl, propyl, t-butyl), alkynyl, alkylcarbonyloxy (-OC(O)alkyl), amide (-C(O)NH-alkyl- or -alkylNHC(O)alkyl), amine (such as alkylamino, arylamino, arylalkylamino), aryl, aryloxy, azo, carbamoyl (-NHC(O)O-alkyl- or -OC(O)NH-alkyl), carbamyl (e.g., CONH2, as well as CONH-alkyl, CONH-aryl, and CONH-arylalkyl), carboxyl, carboxylic acid, cyano, ester, ether (e.g., methoxy, ethoxy), halo, haloalkyl (e.g., -CCh, -CF3, -C(CF3)3), heteroalkyl, isocyanate, isothiocyanate, nitrile, nitro, phosphodiester, sulfide, sulfonamido (e.g., SO2NH2), sulfone, sulfonyl (including alkylsulfonyl, arylsulfonyl and arylalkylsulfonyl), sulfoxide, thiol (e.g., sulfhydryl, thioether) or urea (-NHCONH-alkyl-).

[0087] The term "micro" as used herein is to be interpreted broadly to include dimensions from about 1 micron to about 1000 microns. The term "nano" as used herein is to be interpreted broadly to include dimensions less than about 1000 nm, less than about 500 nm, less than about 100 nm or less than about 50 nm.

[0088] The terms "coupled" or "connected" as used in this description are intended to cover both directly connected or connected through one or more intermediate means, unless otherwise stated.

[0089] The term "associated with", used herein when referring to two elements refers to a broad relationship between the two elements. The relationship includes, but is not limited to a physical, a chemical or a biological relationship. For example, when element A is associated with element B, elements A and B may be directly or indirectly attached to each other or element A may contain element B or vice versa.

[0090] The term "adjacent" used herein when referring to two elements refers to one element being in close proximity to another element and may be but is not limited to the elements contacting each other or may further include the elements being separated by one or more further elements disposed therebetween or in the space between.

[0091] The term "and / or", e.g., "X and / or Y" is understood to mean either "X and Y" or "X or Y" and should be taken to provide explicit support for both meanings or for either meaning.

[0092] Further, in the description herein, the word “substantially” whenever used is understood to include, but not restricted to, "entirely" or “completely” and the like. In addition, terms such as "comprising", "comprise", and the like whenever used, are intended to be non-restricting descriptive language in that they broadly include elements / components recited after such terms, in addition to other components not explicitly recited. For example, when “comprising” is used, reference to a “one” feature is also intended to be a reference to “at least one” of that feature. Terms such as “consisting”, “consist”, and the like, may in the appropriate context, be considered as a subset of terms such as "comprising", "comprise", and the like. Therefore, in embodiments disclosed herein using the terms such as "comprising", "comprise", and the like, it will be appreciated that these embodiments provide teaching for corresponding embodiments using terms such as “consisting”, “consist”, and the like. Further, terms such as "about", "approximately" and the like whenever used, typically means a reasonable variation, for example a variation of + / - 5% of the disclosed value, or a variance of 4% of the disclosed value, or a variance of 3% of the disclosed value, a variance of 2% of the disclosed value or a variance of 1% of the disclosed value.

[0093] Furthermore, in the description herein, certain values may be disclosed in a range. The values showing the end points of a range are intended to illustrate a preferred range. Whenever a range has been described, it is intended that the range covers and teaches all possible sub-ranges as well as individual numerical values within that range. That is, the end points of a range should not be interpreted as inflexible limitations. For example, a description of a range of 1% to 5% is intended to have specifically disclosed sub-ranges 1% to 2%, 1 % to 3%, 1 % to 4%, 2% to 3% etc., as well as individually, values within that range such as 1%, 2%, 3%, 4% and 5%. It is to be appreciated that the individual numerical values within the range also include integers, fractions and decimals. Furthermore, whenever a range has been described, it is also intended that the range covers and teaches values of up to 2 additional decimal places or significant figures (where appropriate) from the shown numerical end points. For example, a description of a range of 1% to 5% is intended to have specifically disclosed the ranges 1.00% to 5.00% and also 1.0% to 5.0% and all their intermediate values (such as 1.01 %, 1.02% ... 4.98%, 4.99%, 5.00% and 1.1 %, 1 .2% ... 4.8%, 4.9%, 5.0% etc.,) spanning the ranges. The intention of the above specific disclosure is applicable to any depth / breadth of a range.

[0094] Additionally, when describing some embodiments, the disclosure may have disclosed a method and / or process as a particular sequence of steps. However, unless otherwise required, it will be appreciated that the method or process should not be limited to the particular sequence of steps disclosed. Other sequences of steps may be possible. The particular order of the steps disclosed herein should not be construed as undue limitations. Unless otherwise required, a method and / or process disclosed herein should not be limited to the steps being carried out in the order written. The sequence of steps may be varied and still remain within the scope of the disclosure.

[0095] Furthermore, it will be appreciated that while the present disclosure provides embodiments having one or more of the features / characteristics discussed herein, one or more of these features / characteristics may also be disclaimed in other alternative embodiments and the present disclosure provides support for such disclaimers and these associated alternative embodiments.

[0096] DESCRIPTION OF EMBODIMENTS

[0097] Exemplary, non-limiting embodiments of bioactive synthetic polymeric nanoparticles, a method of preparing said bioactive synthetic polymeric nanoparticles, a composition comprising said bioactive synthetic polymeric nanoparticles, bioactive synthetic polymers, macromolecules for preparing said bioactive synthetic polymers, related methods and related uses are disclosed hereinafter.

[0098] BIOACTIVE SYNTHETIC POLYMERIC NANOPARTICLE

[0099] There is provided a polymeric nanoparticle comprising (i) a hydrophobic synthetic polymer core; and (ii) a hydrophilic bioactive shell. Accordingly, in various embodiments therefore, the term “polymeric nanoparticle” may comprise and / or may be used interchangeably with the term “bioactive synthetic polymeric nanoparticle”. In various embodiments, the polymeric nanoparticle comprises a core-shell configuration in which an (outer / exterior) shell encapsulates an (inner / interior) core. In various embodiments, the shell comprises a hydrophilic shell and the core comprises a hydrophobic core. In various embodiments therefore, the nanoparticle is amphiphilic. Accordingly, in various embodiments therefore, the term “polymeric nanoparticle’’ may comprise and / or may be used interchangeably with the terms “amphiphilic polymeric nanoparticle” or “amphipathic polymeric nanoparticle”.

[0100] Advantageously, the structure of the nanoparticle allows for embodiments of the nanoparticle to be used as an encapsulation / loading agent and / or delivery vehicle / system. In various embodiments, the nanoparticle is designed / configured to allow loading / encapsulation of one or more types of molecules or cargoes. In various embodiments, the nanoparticle is also designed / configured to allow the loaded / encapsulated cargo to be released from said nanoparticle and / or subsequently delivered to a desired target (e.g., cell, cytosol, tissue or organ). The molecules / cargoes to be loaded / encapsulated onto / into / within the compound may include but is not limited to a therapeutic agent, a prophylactic agent or the like. In various embodiments, the molecules / cargoes to be loaded / encapsulated comprise therapeutics. For example, the molecules / cargoes to be loaded / encapsulated may be drug molecules or therapeutics (e.g., for infection management) selected from anti-cancer drugs, antibiotic, antimicrobial agents, anti-bacterial agents, anti-fungal agents, anti-viral agents, anti-parasitic agents or the like or combinations thereof. Advantageously, the nanoparticle is suitable for use in encapsulating and / or delivering one or more therapeutic agent and / or prophylactic agent to a desired target (e.g., subject, cell, cytosol, tissue or organ). Accordingly, in various embodiments, there is also provided a carrier, nanocarrier or delivery system / vehicle comprising said nanoparticle, which may be provided in the form of injectable or topical formulation. Advantageously, the structure and / or amphiphilic property of the nanoparticle (due to presence of a hydrophobic synthetic polymer core and hydrophilic bioactive shell) allows embodiments of the nanoparticle to be suitable for use in promoting / stimulating skin regeneration / regrowth / repair, tissue regeneration / regrowth / repair and treating / relieving a skin condition such as skin wounds, burns, chronic and / or slow healing wounds such as pressure sores, venous ulcers, diabetic ulcers, foot ulcers, bed sores, skin inflammation, psoriasis, rashes, aging skin, atopic dermatitis / eczema, contact dermatitis, allergic dermatitis, dyshidrotic eczema, neurodermatitis, nummular eczema, seborrheic dermatitis, stasis dermatitis, the like, and combinations thereof. Accordingly, in various embodiments, there is also provided a composition comprising said nanoparticle, which may be provided in the form of a liquid, gel or cream formulation. In various embodiments, the nanoparticle is suitable for use in applications such as wound care, skin care, consumer care, beauty, anti-aging, aesthetic products, pet care, the like, and combinations thereof.

[0101] In various embodiments, the hydrophilic bioactive shell is / takes up at least about 1 .0 wt%, at least about 2.0 wt%, at least about 5.0 wt%, at least about 10.0 wt%, at least about 15.0 wt%, at least about 20.0 wt%, at least about 25.0 wt%, at least about 30.0 wt%, at least about 35.0 wt%, at least about 40.0 wt%, at least about 45.0 wt%, at least about 50.0 wt%, at least about 55.0 wt%, at least about 60.0 wt%, at least about 65.0 wt%, at least about 70.0 wt%, at least about 75.0 wt%, at least about 80.0 wt%, at least about 85.0 wt%, or at least about 90.0 wt% of the nanoparticle.

[0102] In various embodiments, the hydrophobic synthetic polymer core is / takes up at least about 99.0 wt%, at least about 98.0 wt%, at least about 95.0 wt%, at least about 90.0 wt%, at least about 85.0 wt%, at least about 80.0 wt%, at least about 75.0 wt%, at least about 70.0 wt%, at least about 65.0 wt%, at least about 60.0 wt%, at least about 55.0 wt%, at least about 50.0 wt%, at least about 45.0 wt%, at least about 40.0 wt%, at least about 35.0 wt%, at least about 30.0 wt%, at least about 25.0 wt%, or at least about 20.0 wt%, at least about 15.0 wt%, or at least about 10.0 wt% of the nanoparticle.

[0103] In various embodiments, the weight / mass / volume ratio of the hydrophilic bioactive shell to hydrophobic synthetic polymer core present in the nanoparticle is from about 1 .0 - 90.0 : 10.0 - 99.0, about 1 .0 - 50.0 : 50.0 - 99.0, or about 10.0 - 25.0 : 75.0 - 90.0. In various embodiments, the nanoparticle has an average size / diameter falling in the range of from about 10.0 nm to about 1 ,000.0 nm, from about 20.0 nm to about 950.0 nm, from about 25.0 nm to about 900.0 nm, from about 50.0 nm to about 850.0 nm, from about 75.0 nm to about 800.0 nm, from about 90.0 nm to about 750.0 nm, from about 100.0 nm to about 700.0 nm, from about 150.0 nm to about 650.0 nm, from about 200.0 nm to about 600.0 nm, from about 250.0 nm to about 550.0 nm, from about 300.0 nm to about 500.0 nm, from about 350.0 nm to about 450.0 nm, or about 400.0 nm. Advantageously, in various embodiments, the size of the nanoparticle is designed to controllable / adjustable as desired in order to suit a particular application.

[0104] In various embodiments, the nanoparticle is formed / fabricated via selfassembly of a bioactive synthetic polymer. That is, in various embodiments, polymer chains in the bioactive synthetic polymer undergo self assembly, spontaneously organizing / arranging the polymer into a core-shell structure, and thereby forming a nanoparticle.

[0105] In various embodiments, the hydrophobic synthetic polymer core and hydrophilic bioactive shell are parts of a bioactive synthetic polymer. The bioactive synthetic polymer may comprise a copolymer, homopolymer or combinations thereof. In various embodiments, the term “bioactive synthetic polymer” encompasses the terms “bioactive synthetic copolymer” and “bioactive synthetic homopolymer”. Accordingly, the term “bioactive synthetic polymeric nanoparticles” also encompasses the terms “bioactive synthetic copolymeric nanoparticles” and “bioactive synthetic homopolymeric nanoparticles”.

[0106] In various embodiments, the bioactive synthetic polymer comprises a brush, bottlebrush, block, comb, graft polymer structure, or combinations thereof. For example, the bioactive synthetic polymer may be a bioactive synthetic brush block copolymer or a bioactive synthetic brush homopolymer. In various embodiments, the bioactive synthetic polymer comprises a block polymer. The block polymer may be a di-block or a tri-block polymer. For example, the block polymer may have or is made up of two different polymer block (e.g., di-block polymer) or three different polymer blocks (e.g., tri-block polymer).

[0107] In various embodiments, the repeating units / polymer blocks are sequentially arranged / distributed within the bioactive synthetic polymer in an ordered manner, e.g., in a fixed / pre-fixed order. In various embodiments, the repeating units are orderly arranged / distributed within the bioactive synthetic polymer. It will be appreciated that the repeating units are not randomly distributed / arranged within the polymer. Advantageously, such a sequentially / orderly arrangement of the repeating units in the polymer allows the polymer to be fabricated into nanoparticles with substantial uniformity. Advantageously, as compared to randomly connected repeating units / polymer blocks, such a sequentially / orderly arrangement of the repeating units in the polymer allows better control of the particle size during fabrication into nanoparticles. Advantageously, in various embodiments, the nanoparticles have a narrow particle size distribution and / or the particle size of the nanoparticles are substantially uniform / homogenous.

[0108] In various embodiments, the hydrophobic synthetic polymer core and hydrophilic bioactive shell are parts of a bioactive synthetic polymer with a poly(norbornene-dicarboximide) backbone having one or more repeating units selected from the group consisting of general formula (1 ), general formula (2) and general formula (3)):

[0109]

[0110] (1) (2) (3) wherein

[0111] R1and R4are each independently selected from a single bond, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl or optionally substituted alkylcarbonylalkyl;

[0112] R2and R6are each optionally substituted alkyl;

[0113] R3, R5and R7are each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; L1and L2are each heteroalkylene;

[0114] X1and X2each comprises a bioactive moiety selected from the group consisting of proteins, peptides, oligopeptides, carbohydrates, oligosaccharides, sugar, collagen, hyaluronic acid, therapeutic / drug molecules and derivatives thereof;

[0115] Y1and Y2each comprises a synthetic polymer selected from the group consisting of poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid-co-lactic acid) (PGLA), polyglycolide or poly(glycolic acid) (PGA), polystyrene (PS), polylactide or poly(lactic acid) (PLA), poly(caprolactone) (PCL), polyacrylates, poly(meth)acrylates, polyesters, derivatives thereof and parts thereof; and Z1, Z2and Z3are each independently selected from CRaRb, O, NRa, SiRaRb, PRaor S, wherein Raand Rbare each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl and optionally substituted alkynyl.

[0116] In various embodiments, there is provided a plurality of different polymeric nanoparticles formed / prepared from one or more polymers selected from the group consisting of:

[0117] (A) a bioactive synthetic copolymer having one or more repeating units represented by general formula (1); one or more repeating units represented by general formula (2); and one or more repeating units represented by general formula (3);

[0118] (B) a bioactive synthetic copolymer having one or more repeating units represented by general formula (1); and one or more repeating units represented by general formula (2);

[0119] (C) a bioactive synthetic copolymer having one or more repeating units represented by general formula (1); and one or more repeating units represented by general formula (3);

[0120] (D) a bioactive synthetic copolymer having one or more repeating units represented by general formula (2); and one or more repeating units represented by general formula (3); and

[0121] (E) a bioactive synthetic homopolymer having one or more repeating units represented by general formula (3).

[0122] In various embodiments, the hydrophobic synthetic polymer core is part of the bioactive synthetic polymer (e.g. hydrophobic part) and the hydrophilic bioactive shell is part of the bioactive synthetic polymer (e.g. hydrophilic part). For example, the hydrophobic synthetic polymer core and the hydrophilic bioactive shell of the nanoparticle may be formed from the bioactive synthetic polymer. In one embodiment, they are formed from the same bioactive synthetic polymer. Advantageously, in various embodiments, the bioactive synthetic polymer is designed to contain sequentially / orderly connected repeating units / polymer blocks and a high concentration of hydrophilic polymer blocks (e.g., at least about 20 wt%, at least about 25 wt%, at least about 30 wt%, at least about 35 wt%, at least about 40 wt%, at least about 45 wt%, or at least about 50 wt%), which allows the polymer to be stabilized in water and therefore fabricated into uniform particles with controllable particle size.

[0123] In various embodiments, in the process of preparing / forming / fabricating nanoparticles (e.g., by reprecipitation in water,) the hydrophobic block / part of the polymer (e.g., repeating units represented by general formula (1) and / or (3)) is rearranged as core, while the hydrophilic block / part of the polymer (e.g., repeating units represented by general formula (2) and / or (3)) forms a shell around the core to encapsulate the core, thus organizing / arranging to form a core-shell structure, e.g., upon exposure to water.

[0124] In various embodiments, the core of the nanoparticle comprises the moiety Y1and / or Y2. In various embodiments, the shell of the nanoparticle comprises the moiety L1and / or L2; and X1and / or X2. In various embodiments, the shell comprises an inner shell and an outer shell. The inner shell may comprise the moiety L1and / or L2, e.g., PEG. The inner shell may comprise X1and / or X2, e.g., HA or RGD.

[0125] In various embodiments, Y1and Y2each comprises a synthetic polymer selected from the group consisting of poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid-co-lactic acid) (PGLA), polyglycolide or poly(glycolic acid) (PGA), polystyrene (PS), polylactide or poly(lactic acid) (PLA), poly(caprolactone) (PCL), polyacrylates, poly(meth)acrylates, polyesters, derivatives thereof and parts thereof.

[0126] In various embodiments, the term “poly(lactic-co-glycolic acid)” may comprise and / or may be used interchangeably with the terms “poly(lactide-co- glycolide)”, “poly(D-lactide-co-glycolide)”, ‘poly(L-lactide-co-glycolide)”, “poly(D,L-lactide-co-glycolide)” or the like. In various embodiments, the term “poly(glycolic acid-co-lactic acid)” may comprise and / or may be used interchangeably with the terms “poly(glycolide-co-lactide)”, “poly(glycolide-co-D- lactide)”, ‘poly(glycolide-co-L-lactide)”, “poly(glycolide-co-D-L-lactide)” or the like.

[0127] In various embodiments, Y1and / or Y2is a polyacrylate comprising one or more monomers selected from the group consisting of methyl acrylate, ethyl acrylate, n-propyl acrylate, isopropyl acrylate, n-butyl acrylate, isobutyl acrylate, tert-butyl acrylate, hexyl acrylate, cyclohexyl acrylate, 2-ethylhexyl acrylate, benzyl acrylate and phenyl acrylate. Y1and / or Y2may be poly(methyl acrylate), polyfethyl acrylate), poly (butyl acrylate) or poly (2-ethylhexyl acrylate). In various embodiments, Y1and / or Y2is a poly(meth)acrylate comprising one or more monomers selected from the group consisting of methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, isopropyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, tert-butyl methacrylate, hexyl methacrylate, cyclohexyl methacrylate, 2-ethylhexyl methacrylate, benzyl methacrylate and phenyl methacrylate. Y1and / or Y2may be poly(methyl methacrylate) (PMMA), polyfethyl methacrylate), poly(butyl methacrylate) or poly (2-ethylhexyl acrylate).

[0128] In various embodiments, Y1and / or Y2is substantially devoid of polyalkylene glycol such as polyethylene glycol.

[0129] In various embodiments, the nanoparticles are in a freeze dried or lyophilized form (e.g. for storage) or have been reconstituted in an appropriate solution (e.g. to form a dispersion or suspension).

[0130] In various embodiments, the repeating unit(s) represented by general formula (2), (3), moieties X1and / or X2possess bioactivity, biocompatibility and / or biodegradability. In various embodiments, the repeating unit(s) represented by general formula (1), (3), moieties Y1and / or Y2possess good mechanical strength / hardness. Advantageously, the presence of the repeating units in the bioactive synthetic copolymer / homopolymer imparts both bioactivity and mechanical strength to the copolymer / homopolymer, leading to a mechanically strong bioactive copolymer / homopolymer. In various embodiments, the copolymer / homopolymer may also be biocompatible and / or biodegradable. Accordingly, in various embodiments, the copolymer / homopolymer is capable of being classified as a biomaterial.

[0131] In various embodiments, L1and / or L2is a polymeric linker that links the bioactive moiety X1and / or X2to the poly(norbornene) backbone. Advantageously, L1and / or L2is designed to be adjustable and / or customizable based on the size of the bioactive moiety X1and / or X2and the size of the synthetic polymer present in Y1and / or Y2. The molecular weight and / or length of the polymeric linker L1and / or L2may be customized to suit the molecular weight and / or length of the bioactive moiety X1and / or X2and synthetic polymer chosen for Y1and / or Y2, depending on the application the copolymer / homopolymer is to be used for.

[0132] In various embodiments, the molecular weight and / or length of the polymeric linker L1is selected such that the overall molecular size of the repeating unit represented by general formula (1) is similar / comparable to the molecular size of the repeating unit represented by general formula (2). For example, if PCL having a molecular weight of 4,000 is selected as the choice of synthetic polymer for Y1and peptide having a molecular weight of from about 400 to about 500 is selected as the choice of bioactive moiety X1, then L1may be designed to comprise a molecular weight of about 3,400. It will be appreciated that in various embodiments, it is the length of L1that gets adjusted to match the molecular weight of general formula (1) to molecular weight of general formula (2).

[0133] In various embodiments, the molecular weight of general formula (1) is comparable / substantially similar with / to the molecular weight of general formula (2). In various embodiments, the molecular weight of general formula (1) does not differ from the molecular weight of general formula (2) by more than 30% of the molecular weight of general formula (2) or vice versa. For example, the molecular weight of general formula (1) may be at most about 30% more or at most 30% less than the molecular weight of general formula (2) or vice versa. The molecular weight of general formula (1) may not differ from the molecular weight of general formula (2) by more than about 30%, more than about 25%, more than about 20%, more than about 15%, more about 10%, more than about 5%, more than about 4%, more than about 3%, more than about 2%, or more than about 1% of the molecular weight of general formula (2) or vice versa. In various embodiments, the molecular weight of general formula (1 ) does not differ from the molecular weight of general formula (2) by more than about 20% of the molecular weight of general formula (2) or vice versa. For example, the molecular weight of general formula (1) may be at most about 20% more or at most 20% less than the molecular weight of general formula (2) or vice versa. Advantageously, as the bioactive moiety bearing repeating unit has a molecular size / weight / length that is similar to that of the synthetic polymer bearing repeating unit, the length of the bioactive moiety X1is extended, thereby allowing X1to be “visible”, available for binding to cells or accessible to its targeted physiological site for desired bioactivity, i.e. not buried in a sea / matrix of synthetic polymers.

[0134] In various embodiments, the molecular weight of general formula (1) is from about 100 to about 15,000, from about 200 to about 14,000, from about 300 to about 13,000, from about 400 to about 12,000, from about 500 to about 11 ,000, from about 1 ,000 to about 10,000, from about 1 ,500 to about 9,500, from about 2,000 to about 9,000, from about 2,500 to about 8,500, from about 3,000 to about 8,000, from about 3,500 to about 7,500, from about 4,000 to about 7,000, from about 4,500 to about 6,500, from about 5,000 to about 6,000 or about 5,500.

[0135] In various embodiments, the molecular weight of general formula (2) is about 15,000, about 14,000, about 13,000 or at least about 12,000. In various embodiments, the molecular weight of general formula (2) is from about 100 to about 15,000, from about 200 to about 14,000, from about 300 to about 13,000, from about 400 to about 12,000, from about 500 to about 11 ,000, from about 1 ,000 to about 10,000, from about 1 ,500 to about 9,500, from about 2,000 to about 9,000, from about 2,500 to about 8,500, from about 3,000 to about 8,000, from about 3,500 to about 7,500, from about 4,000 to about 7,000, from about 4,500 to about 6,500, from about 5,000 to about 6,000 or about 5,500. In various embodiments, when X1comprises longer peptides that contain more than 10 amino acids and the molecular weight of L1is about 6,000, then the molecular weight of general formula (2) is greater than about 7,000.

[0136] In various embodiments, the total molecular weight of general formula (1 ) and general formula (2) is kept to about 300,000, no more than about 300,000, no more than about 200,000, no more than about 100,000, no more than about 90,000, no more than about 80,000, no more than about 70,000, no more than about 60,000, no more than about 50,000, no more than about 45,000, no more than about 40,000, no more than about 35,000, no more than about 30,000, no more than about 25,000, no more than about 20,000, or no more than about 15,000 to facilitate copolymerization.

[0137] In various embodiments, the molecular weight of general formula (3) is from about 100 to about 15,000, from about 200 to about 14,000, from about 300 to about 13,000, from about 400 to about 12,000, from about 500 to about 11 ,000, from about 1 ,000 to about 10,000, from about 1 ,500 to about 9,500, from about 2,000 to about 9,000, from about 2,500 to about 8,500, from about 3,000 to about 8,000, from about 3,500 to about 7,500, from about 4,000 to about 7,000, from about 4,500 to about 6,500, from about 5,000 to about 6,000 or about 5,500.

[0138] In various embodiments, the number of repeating units represented by general formula (1 ) in the copolymer is from about 10 to about 1 ,000, from about 15 to about 950, from about 20 to about 900, from about 25 to about 850, from about 30 to about 800, from about 35 to about 750, from about 40 to about 700, from about 45 to about 650, from about 50 to about 600, from about 55 to about 550, from about 60 to about 500, from about 65 to about 450, from about 70 to about 400, from about 75 to about 350, from about 80 to about 300, from about 85 to about 250, from about 90 to about 200, from about 95 to about 150, or about 100.

[0139] In various embodiments, the number of repeating units represented by general formula (2) in the copolymer is from about 10 to about 1 ,000, from about 15 to about 950, from about 20 to about 900, from about 25 to about 850, from about 30 to about 800, from about 35 to about 750, from about 40 to about 700, from about 45 to about 650, from about 50 to about 600, from about 55 to about 550, from about 60 to about 500, from about 65 to about 450, from about 70 to about 400, from about 75 to about 350, from about 80 to about 300, from about 85 to about 250, from about 90 to about 200, from about 95 to about 150, or about 100.

[0140] In various embodiments, the number of repeating units represented by general formula (3) in the copolymer / homopolymer is from about 10 to about 1 ,000, from about 15 to about 950, from about 20 to about 900, from about 25 to about 850, from about 30 to about 800, from about 35 to about 750, from about 40 to about 700, from about 45 to about 650, from about 50 to about 600, from about 55 to about 550, from about 60 to about 500, from about 65 to about 450, from about 70 to about 400, from about 75 to about 350, from about 80 to about 300, from about 85 to about 250, from about 90 to about 200, from about 95 to about 150, or about 100.

[0141] In various embodiments, the ratio of the number of repeating units represented by general formula (2) to the number of repeating units represented by general formula (1) in the bioactive synthetic copolymer is from about 1 :1 to about 1 :100, from about 1 :2 to about 1 :99, from about 1 :3 to about 1 :98, from about 1 :4 to about 1 :97, from about 1 :5 to about 1 :96, from about 1 :6 to about 1 :95, from about 1 :7 to about 1 :90, from about 1 :8 to about 1 :85, from about 1 :9 to about 1 :80, from about 1 :10 to about 1 :75, from about 1 :15 to about 1 :70, from about 1 :20 to about 1 :65, from about 1 :25 to about 1 :60, from about 1 :30 to about 1 :55, from about 1 :35 to about 1 :50, or from about 1 :40 to about 1 :45. In various embodiments, the ratio of the number of repeating units represented by general formula (2) to the number of repeating units represented by general formula (1 ) in the bioactive synthetic copolymer is about 1 :10, about 1 :15, about 1 :20, about 1 :25, about 1 :30, about 1 :35, about 1 :40, about 1 :45 or about 1 :50.

[0142] In various embodiments, L1and / or L2is hydrophilic. As L1and / or L2is adjustable, the hydrophilicity of the repeating unit represented by general formula (2) and / or (3) and also the overall hydrophilicity of the bioactive synthetic copolymer / homopolymer may be adjusted as desired. Advantageously, the presence of L1and / or L2increases the hydrophilicity of the repeating unit represented by general formula (2) and / or (3) and also the overall hydrophilicity of the bioactive synthetic copolymer. It will be appreciated by a person skilled in the art that, bioactive moieties and synthetic polymers are typically mutually incompatible as the individual bioactive moiety is generally hydrophilic while synthetic polymer is generally hydrophobic. Advantageously, L1and / or L2in repeating unit represented by general formula (2) and / or (3) is also used to extend the chain length of the bioactive moiety X1and / or X2attached at the end of L1and / or L2.

[0143] In various embodiments, L1and / or L2is a heteroalkylene having at least 20 carbon atoms, at least 30 carbon atoms, at least 40 carbon atoms, at least 50 carbon atoms, at least 60 carbon atoms, at least 70 carbon atoms, at least 80 carbon atoms, at least 90 carbon atoms, at least 100 carbon atoms, at least 150 carbon atoms, at least 200 carbon atoms, at least 250 carbon atoms or at least 300 carbon atoms. In various embodiments, L1and / or L2is C20-C300 heteroalkylene or a heteroalkylene having from 20 carbon atoms to 300 carbon atoms.

[0144] In various embodiments, L1and / or L2has a number average molecular weight of between about 500 and about 7,000. L1and / or L2may have a number average molecular weight of about 600, about 700, about 800, about 900, about 1 ,000, about 1 ,500, about 2,000, about 2,500, about 3,000, about 3,500, about 4,000, about 4,500, about 5,000, about 5,500, about 6,000, about 6,500 or about 7,000. In various embodiments, when X1and / or X2comprises a small bioactive moiety, the molecular weight of L1and / or L2may be adjusted to about 7,000 so that the total molecular weight of general formula (1) and general formula (2) is kept to no more than about 10,000. In various embodiments, the number average molecular weight of L1and / or L2is from about 1 ,000 to about 6,000.

[0145] In various embodiments, the heteroatom in L1and / or L2is O. In various embodiments, L1and / or L2is polyalkylene glycol. In various embodiments, L1and / or L2is poly(C2-C4 alkylene glycol). L1and / or L2may be selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol (PPG), polytetramethylene glycol (PTMG), polybutylene glycol (PBG) and the like. Advantageously, the use of a polyalkylene glycol such as PEG can increase hydrophilicity of the macromonomer and the resultant copolymer / homopolymer. In various embodiments, the polyalkylene glycol such as PEG are used as spacers, linkers or linking groups in the overall polymers, instead of as terminal groups.

[0146] In various embodiments, L1and / or L2is polyalkylene glycol having at least about 10 repeating units, at least about 15 repeating units, at least about 20 repeating units, at least about 21 repeating units, at least about 22 repeating units, at least about 23 repeating units, at least about 24 repeating units, at least about 25 repeating units, at least about 30 repeating units, at least about 40 repeating units, at least about 50 repeating units, at least about 60 repeating units, at least about 70 repeating units, at least about 80 repeating units, at least about 90 repeating units, at least about 100 repeating units, at least about 150 repeating units, at least about 200 repeating units, or at least about 250 repeating units. In various embodiments, L1and / or L2comprises from about 10 monomers / repeating units to about 250 monomers / repeating units. Unlike conventional polymers which uses a short PEG chain, embodiments of the bioactive synthetic copolymer / homopolymer disclosed herein incorporate a long polyalkylene glycol chain of at least 21 repeating units at L1and / or L2. In various embodiments, L1and / or L2is selected from the group consisting of PEGsoo, PEGeoo, PEG700, PEGsoo, PEG900, PEG1000, PEG1100, PEG1200, PEG1300, PEG1400, PEG1500, PEG2000, PEG2500, PEG3000, PEG3500, PEG4000, PEG4500, PEG5000, PEG5500, PEGeooo, PEG6600 and mixtures thereof.

[0147] In various embodiments, R1and R4are each independently selected from C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C1-C20 alkoxyalkyl, C2-C20 alkylcarbonyl or C3-C20 alkylcarbonylalkyl. The C1-C20 alkyl substituents may be straight or branched substituents selected from methyl, ethyl, n-propyl, 2-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, hexyl, amyl, 1 ,2-dimethylpropyl, 1 ,1 - dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1 -methylpentyl, 2- methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1 ,2- dimethylbutyl, 1 ,3-dimethylbutyl, 1 ,2,2-trimethylpropyl, 1 ,1 ,2-trimethylpropyl, 2- ethylpentyl, 3-ethylpentyl, heptyl, 1 -methylhexyl, 2,2-dimethylpentyl, 3,3- dimethylpentyl, 4,4-dimethylpentyl, 1 ,2-dimethylpentyl, 1 ,3-dimethylpentyl, 1 ,4- dimethylpentyl, 1 ,2,3-trimethylbutyl, 1 ,1 ,2-trimethylbutyl, 1 ,1 ,3-trimethylbutyl, 5- methylheptyl, 1 -methylheptyl, octyl, nonyl, decyl or the like.

[0148] In various embodiments, R2and R6are each independently selected from straight or branched C1-C20 alkyl. The C1-C20 alkyl substituents may be straight or branched substituents selected from methyl, ethyl, n-propyl, 2-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, hexyl, amyl, 1 ,2-dimethylpropyl, 1 ,1 - dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1 -methylpentyl, 2- methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1 ,2- dimethylbutyl, 1 ,3-dimethylbutyl, 1 ,2,2-trimethylpropyl, 1 ,1 ,2-trimethylpropyl, 2- ethylpentyl, 3-ethylpentyl, heptyl, 1 -methylhexyl, 2,2-dimethylpentyl, 3,3- dimethylpentyl, 4,4-dimethylpentyl, 1 ,2-dimethylpentyl, 1 ,3-dimethylpentyl, 1 ,4- dimethylpentyl, 1 ,2,3-trimethylbutyl, 1 ,1 ,2-trimethylbutyl, 1 ,1 ,3-trimethylbutyl, 5- methylheptyl, 1 -methylheptyl, octyl, nonyl, decyl or the like. R2and R6may be straight or branched C1-C4 alkyl substituents. In various embodiments, the length of R2and / or R6is the same as the length of a repeating unit in L1and / or L2. For example, if L1and / or L2is poly(butylene glycol), then R2and R6is butyl. In another example, if L1and / or L2is polyethylene glycol), then R2and R6is ethyl. It will be appreciated that in various embodiments, R2and R6is carefully designed to match L1and / or L2.

[0149] In various embodiments, R3, R5and R7are each independently selected from H, C1-C20 alkyl, C2-C20 alkenyl or C2-C20 alkynyl.

[0150] In various embodiments, Z1, Z2and / or Z3are each independently selected from CRaRb, O, NRb, SiRaRb, PRaor S, wherein Raand Rbare each independently selected from the group consisting of H, C1-C20 alkyl, C1-C20 alkenyl and C1-C20 alkynyl. In various embodiments, Z1is CH2. In various embodiments, Z2is CH2. In various embodiments, Z3is CH2. In various embodiments, Z1, Z2and / or Z3are each independently selected from CH2, O, NH, SiRaRb, PRaor S. The poly(norbornene) backbone may be selected from the group consisting of poly(norbornene-imide), poly(norbornene-dicarboximide), poly(norbornene) backbone is poly(5-norbornene-2,3-dicarboximide), poly(7- oxanorbornene), poly(oxanorbornene-imide), poly(oxanorbornene- dicarboximide) and the like.

[0151] In various embodiments, X1and / or X2is coupled to the poly(norbornene dicarboximide) backbone through a carboxylic acid functionality in the following arrangement: -R2-L1-NR3-C(=O)-X1or -l_2-R6-NR7-C(=O)-X2.

[0152] Advantageously, by linking X1and / or X2through a carboxylic acid functionality, amine terminal group(s) in X1and / or X2is / are free up for delivering its bioactivity, therefore ensuring the bioavailability of X1and / or X2. It will be appreciated that as amine group(s) confer bioactivity, exhausting up amine groups in bioactive moieties for polymer binding may be undesirable.

[0153] In various embodiments, X1and / or X2is coupled to the poly(norbornene dicarboximide) backbone via peptide / amide linkage, i.e. -NR3-C(=O)- or -NR7- C(=O)-. Advantageously, the bioactive synthetic copolymer / homopolymer disclosed herein is considerably stronger and / or stable than conventional polymers that contain ester linkages. Without being bound by theory, it is believed that amide linkages are stronger than ester linkages because ester linkages are more prone to hydrolysis, which may release bioactive moieties into the bloodstream, leading to a premature metabolism of bioactive moieties.

[0154] In various embodiments, one or more of H atoms in alkyl, alkenyl, alkynyl, alkoxyalkyl, alkylcarbonyl and alkylcarbonylalkyl is / are optionally replaced by hydroxy, hydroxyalkyl, halogen, haloalkyl, cyano, cyanoalkyl and nitro.

[0155] In various embodiments, X1and / or X2comprises a bioactive moiety selected from proteins, peptides, carbohydrates, therapeutic / drug molecules and derivatives thereof. In various embodiments, proteins, peptides, carbohydrates or therapeutic / drug molecules derivatives thereof include proteins, peptides, carbohydrates or therapeutic / drug molecules that are or have been optionally modified to contain one carboxylic acid terminal group. In some embodiments, the bioactive moiety contains only one carboxylic acid terminal group.

[0156] In various embodiments, the bioactive synthetic polymer is designed / configured to contain bioactive moiety X1and / or X2having one or more carboxylic acid group(s). X1and / or X2may comprise one carboxylic acid group or more than one carboxylic acid groups, depending on the choice of bioactive moiety used.

[0157] In various embodiments, the bioactive moiety X1and / or X2comprises more than one carboxylic acid groups. In various embodiments, the bioactive moiety comprises multiple carboxylic acid groups. In such embodiments, the bioactive moiety comprises oligosaccharide(s). That is, in various embodiments, when the choice of bioactive moiety used is oligosaccharide(s), the bioactive moiety comprises multiple carboxylic acid groups. For example, a bioactive moiety comprising oligosaccharides (e.g., hyaluronic acid and / or heparin sulphate) may have more than about 1 , more than about 5, more than about 10, more than about 15, more than about 20, more than about 25, more than about 30, more than about 35, or about 40 carboxylic acid groups. In various embodiments, the bioactive moiety X1and / or X2comprises from 2 to 40 carboxylic acid groups, from 2 to 20 carboxylic acid groups or from 10 to 14 carboxylic acid groups. In various embodiments, X1and / or X2comprises 40 carboxylic acid groups, 30 carboxylic acid groups, 25 carboxylic acid groups, 20 carboxylic acid groups, 15 carboxylic acid groups, 14 carboxylic acid groups, 13 carboxylic acid groups, 12 carboxylic acid groups, 11 carboxylic acid groups, 10 carboxylic acid groups, 9 carboxylic acid groups, 8 carboxylic acid groups, 7 carboxylic acid groups, 6 carboxylic acid groups, 5 carboxylic acid groups, 4 carboxylic acid groups or 3 carboxylic acid groups or 2 carboxylic acid groups. In various embodiments, a disaccharide unit (or repeating unit) comprises one carboxylic acid group.

[0158] In various embodiments, the bioactive moiety X1and / or X2comprises no more than one carboxylic acid group. In various embodiments, the bioactive moiety comprises one or a single carboxylic acid group. In various embodiments, the bioactive moiety comprises a monocarboxylic acid. Advantageously, the use of a bioactive moiety having a monocarboxylic acid terminal group avoids the possibility of an undesirable crosslinking which may otherwise occur if there is more than one carboxylic acid. In various embodiments therefore, the bioactive moiety X1and / or X2is substantially devoid of more than one carboxylic acid terminal group, for e.g., a dicarboxylic acid or tricarboxylic acid. It will be appreciated that, in various embodiments, unless the bioactive moiety comprises oligosaccharide(s), the bioactive moiety X1and / or X2comprises no more than one carboxylic acid group.

[0159] In various embodiments, X1and / or X2comprises protein or peptide. X1and / or X2may be a peptide sequence, laminin-derived peptide, integrin binding peptide, cell-penetrating peptide, collagen mimics or collagen fragments. In various embodiments, X1and / or X2comprises from 2 to 50 amino acid residues, from 2 to 40 amino acid residues or from 2 to 20 amino acid residues in any sequence. In various embodiments, X1and / or X2comprises oligopeptides having from 2 to 50 amino acids, from 3 to 40 amino acids, or from 3 to 20 amino acids in any sequence. In various embodiments, X1and / or X2comprises 50 amino acid residues, 40 amino acid residues, 30 amino acid residues, 25 amino acid residues, 20 amino acid residues, 15 amino acid residues, 10 amino acid residues, 9 amino acid residues, 8 amino acid residues, 7 amino acid residues, 6 amino acid residues, 5 amino acid residues, 4 amino acid residues or 3 amino acid residues in any sequence. The amino acid residues may be selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, asparagine, glutamine, glycine, serine, threonine, serine, asparagine, glutamine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid and glutamic acid. In various embodiments, X1and / or X2is a peptide sequence comprising 3 to 20 natural amino acids. X1and / or X2may be integrin binding peptide selected from the group consisting of arginine-glycine- aspartic acid (RGD), SRGDS and RGDS; laminin-derived peptide A5G81 (AGQWHRVSVRWGC); osteopontin derived peptides SVVYGLR; and cell- penetrating / antimicrobial peptide selected from (IRIK)a or (IKKI)s. In various embodiments, X1and / or X2is a collagen sequence comprising 3 to 20 units of glycine (G), proline (P) and hydroxyproline (Hyp) in any sequence or permutation. X1and / or X2may be collagen fragment having a (PHypG)n type sequence, (PGHyp)n type sequence, (HypGP)n type sequence, (HypPG)n type sequence, (GHypP)n type sequence, (GPHyp)n type sequence or collagen mimic DGEA.

[0160] In various embodiments, X1and / or X2comprises carbohydrate or sugar. In various embodiments, X1and / or X2comprises monosaccharide, disaccharide, oligosaccharide or polysaccharide. In various embodiments, X1and / or X2comprises from 2 to 50 saccharide units, from 2 to 40 saccharide units, from 2 to 20 saccharide units or from 10 to 14 saccharide units. In various embodiments, X1and / or X2comprises 50 saccharide units, 40 saccharide units, 30 saccharide units, 25 saccharide units, 20 saccharide units, 15 saccharide units, 14 saccharide units, 13 saccharide units, 12 saccharide units, 11 saccharide units, 10 saccharide units, 9 saccharide units, 8 saccharide units, 7 saccharide units, 6 saccharide units, 5 saccharide units, 4 saccharide units or 3 saccharide units or 2 saccharide units. X1and / or X2may be heparin sulfate (HS) or glycosaminoglycans (GAGs). In various embodiments, X1and / or X2comprises oligosaccharides having from 1 to 40 disaccharide units, from 5 to 30 disaccharide units, or from 10 to 20 disaccharide units. In various embodiments, X1and / or X2is heparin sulfate / oligosaccharide selected from the group consisting of DP8, DP10, DP12, DP14 and DP16. In various embodiments, X1and / or X2is hyaluronic acid which is the simplest form of glycosaminoglycan (GAG).

[0161] In various embodiments, X1and / or X2is chemically coupled to the rest of general formula (2) and / or (3) via its hydroxy group. For example, when X1and / or X2is carbohydrate / saccharide, oxidation and / or reductive amination reactions may be performed on the carbohydrate’s hydroxy for linking X1and / or X2to general formula (2) and / or (3). -CH2OH on the saccharide may be oxidised to - C(=O)H, which subsequently undergoes reductive amination using the -NH2 terminal on L1and / or L2to create a peptide linkage.

[0162] In various embodiments, X1and / or X2comprises a carbohydrate / saccharide that contained or has been modified to contain one carboxylic acid terminal group. Modification by one or more chemical reaction(s) such as oxidation may be performed on the carbohydrate / saccharide to create a carboxylic acid group. In various embodiments, modification is performed on a hydroxyl group that is originally present in the carbohydrate / saccharide. In various embodiments, -CH2OH on the carbohydrate / saccharide is oxidized completely to -C(=O)OH, which subsequently reacts with a -NH2 terminal on L1and / or L2to create a peptide linkage that links the carbohydrate / saccharide to the rest of general formula (2) and / or (3): X1-C(=O)-NH-L1- or X2-C(=O)-NH-L2-. It will be appreciated, however, that no modification to the carbohydrate / saccharide may be required / necessary if a carboxylic acid is naturally present in the carbohydrate / saccharide.

[0163] In various embodiments, X1and / or X2comprises therapeutic / drug molecule. In various embodiments, X1and / or X2comprises antibiotic, antimicrobial, antibacterial, blood thinning agents or anti-inflammatory agents. X1and / or X2may be penicillin, amoxicillin, amphotericin, ciprofloxacin (CIF), vancomycin, atorvastatin, aspirin or aminoglycoside-based molecules selected from streptomycin, ribostamycin or gentamycin. It will be appreciated that X1and / or X2may be any therapeutic or drug molecule that contains a carboxylic acid group.

[0164] In various embodiments, X1and / or X2is chemically coupled to the rest of general formula (2) and / or (3) via one of its chemical moiety selected from the group consisting of -COOH, -CH2OH, -CH2NH2 and =CHNH2. For example, -CH2NH2 or =CHNH2 on the drug molecule may be coupled to a small dicarboxylic acid before reacting with a -NH2 terminal on L1and / or L2to create a peptide linkage that links the drug molecule to the rest of general formula (I): X- C(=O)-NH-L-

[0165] In various embodiments, X1and / or X2comprises a therapeutic / drug molecule that contained or has been modified to contain one carboxylic acid terminal group. Modification by one or more chemical reaction(s) such as oxidation may be performed on the therapeutic / drug molecule to create a carboxylic acid group. In various embodiments, modification is performed on a hydroxyl group that is originally present in the therapeutic / drug molecule. For example, in various embodiments when X1and / or X2is ribostamycin or gentamycin, -CH2OH on the drug molecule is oxidized completely to -C(=O)OH, which subsequently reacts with a -NH2 terminal on L1and / or L2to create a peptide linkage that links the drug molecule to the rest of general formula (2) and / or (3): X1-C(=O)-NH-L1- or X2-C(=O)-NH-L2-. It will be appreciated, however, that no modification to the therapeutic / drug molecule may be required / necessary if a carboxylic acid is already present in the therapeutic / drug molecule.

[0166] In various embodiments, the bioactive moiety is or has been modified to contain one carboxylic acid terminal group. For example, if a carboxylic acid terminal group is absent in a carbohydrate or therapeutic / drug molecule, the carbohydrate or therapeutic / drug molecule may be modified to add a carboxylic acid at one of the carbohydrate or therapeutic / drug molecule terminals. The modification may comprise oxidation reaction(s) to convert a hydroxy group in the carbohydrate to carboxylic acid.

[0167] In various embodiments, the repeating unit represented by general formula (2) is in an amount of from about 1 molar % to about 100 molar %, from about 2 molar % to about 99 from about 3 molar % to about 98 molar %, from about 4 molar % to about 97 molar %, from about 5 molar % to about 96 molar %, from about 10 molar % to about 95 molar %, from about 15 molar % to about 90 molar %, from about 20 molar % to molar %, about 85 molar %, from about 25 molar % to about 80 molar %, from about 30 molar % to about 75 molar %, from about 35 molar % to about 70 molar %, from about 40 molar % to about 65 molar %, from about 45 molar % to about 60 molar %, or from about 50 molar % to about 55 molar % relative to the copolymer. In various embodiments, the repeating unit represented by general formula (2) is in an amount of from about 1 molar % to about 10 molar % relative to the copolymer. In various embodiments, the bioactive moiety is about 2 molar %, about 3 molar %, about 4 molar %, about 5 molar %, about 6 molar %, about 7 molar %, about 8 molar %, about 9 molar % or about 10 molar % of the bioactive synthetic copolymer.

[0168] In various embodiments, the total molecular weight of general formula (1) is kept to no more than about 15,000 or no more than about 10,000. It will be appreciated that copolymerisation may become inefficient when the total molecular weight of general formula (1) and (2) is too high. In various embodiments, when the bioactive synthetic copolymer is used for applications which require fast biodegradation, the molecular weight of general formula (II) is kept low by adjusting the value of n and / or m. In various embodiments, the bioactive synthetic copolymer / homopolymer has a number average molecular weight (Mn) of from about 1 ,000 to about 500,000, from about 1 ,250 to about 400,000, from about 1 ,500 to about 300,000, from about 2,000 to about 250,000, from about 3,000 to about 200,000, from about 4,000 to about 150,000, from about 5,000 to about 100,000, from about 10,000 to about 90,000, from about 20,000 to about 80,000, from about 30,000 to about 70,000, from about 40,000 to about 60,000, or about 50,000.

[0169] In various embodiments, the bioactive synthetic copolymer / homopolymer has a polydispersity index (PDI) of from about 1.0 to about 10.0. In various embodiments, PDI of the bioactive synthetic copolymer / homopolymer is about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5 or about 10.0. In various embodiments, the bioactive synthetic copolymer / homopolymer has a polydispersity index (PDI) of from about 1 .0 to about 3.0, from about 1 .05 to about 2.95, from about 1 .1 to about 2.9, from about 1 .2 to about 2.8, from about 1 .4 to about 2.6, from about 1 .6 to about 2.4, from about 1 .8 to about 2.2 or about 2.0. In various embodiments, the PDI of the bioactive synthetic copolymer / homopolymer is no more than 1 .50.

[0170] In various embodiments, the one or more repeating units represented by general formula (1), (2) and / or (3) are designed to link to the poly(norbornene) backbone via at least covalent interactions. In various embodiments, each repeating unit represented by general formula (1) is covalently bonded to the poly(norbornene) backbone and / or each repeating unit represented by general formula (2) is covalently bonded to the poly(norbornene) backbone. Advantageously, embodiments of the nanoparticles are adsorbed through the skin and may enter the circulatory system as a whole.

[0171] It will be appreciated that other interactions such as Van der Waals interactions may also be present within the copolymer. In various embodiments, the one or more repeating units represented by general formula (2) and / or (3) comprises two or more different types of bioactive moiety X1and / or X2. In various embodiments, the one or more repeating units represented by general formula (2) and / or (3) comprises 2, 3, 4, 5, 6, 7 or 8 different types of bioactive moiety X1and / or X2. For example, within a bioactive synthetic copolymer / homopolymer, there may be repeating units represented by general formula (2) and / or (3) comprising peptide as X1and / or X2and repeating units represented by general formula (I) comprising carbohydrate as X1and / or X2. Advantageously, in various embodiments, the bioactive synthetic copolymer imparts two or more different types of bioactivities.

[0172] In various embodiments, the one or more repeating units represented by general formula (1) and / or (3) comprises two or more different types of synthetic polymer Y1and / or Y2. In various embodiments, the one or more repeating units represented by general formula (1) and / or (3) comprises 2, 3, 4, 5, 6, 7 or 8 different types of synthetic polymer Y1and / or Y2.

[0173] Advantageously, the bioactive synthetic copolymer disclosed herein is highly customizable. Depending on the application that the bioactive synthetic copolymer is intended, X1and / or X2with the desired biological activity and Y2with the desired physical attributes may be selected to eventually obtain the bioactive synthetic copolymer with the desired repeating units represented by general formulae (I) and (II).

[0174] BIOACTIVE SYNTHETIC COPOLYMER

[0175] In various embodiments, the bioactive synthetic polymer comprises an amphiphilic bioactive synthetic copolymer with a poly(norbornene-dicarboximide) backbone having one or more repeating units represented by general formula (1) and one or more repeating units represented by general formula (2): wherein

[0176] R1, R2, R3, L1, X1, Y1, Z1and Z2contain one or more features and / or share one or more properties that are similar to those described above.

[0177] In various embodiments, the repeating units (1) and / or (2) are arranged / distributed sequentially / orderly within the bioactive synthetic copolymer. In various embodiments, the polymer comprises a regulated sequence of repeating units. For example, the repeating units (1) and / or (2) may be arranged in an ordered manner, e.g., in a fixed / pre-fixed order. In various embodiments, it will be appreciated that the repeating units (1) and / or (2) are not randomly distributed / arranged within the polymer.

[0178] In various embodiments, the bioactive synthetic copolymer comprises repeating units arranged in a A-b-B sequence, where b represents block; A represents one or more repeating units represented by general formula (1); and B represents one or more repeating units represented by general formula (2), and vice versa. For example, the bioactive synthetic copolymer may be a diblock copolymer comprising a structure represented by general formula (10):

[0179] -A-A-A-A-A-A-A-A-B-B-B-B-B-B-B-B- (10) In various embodiments, A represents one or more repeating units represented by general formula (1); and B represents one or more repeating units represented by general formula (2). In other embodiments, A represents one or more repeating units represented by general formula (2); and B represents one or more repeating units represented by general formula (1).

[0180] In various embodiments, Y1(or the synthetic polymer) comprises poly (lactic-co-glycolic acid) (PLGA) or derivatives thereof. In such embodiments, the nanoparticle comprises a bioactive PLGA copolymer with a poly(norbornene- dicarboximide) backbone having one or more repeating units represented by general formula (1 A) and one or more repeating units represented by general formula (2A): wherein R1, R2, R3, L1, X1, Z1and Z2contain one or more features and / or share one or more properties that are similar to those described above; m > 1 ; and n > 1. In various embodiments, m is a value from 1 to 5,000. For example, m may be a value from about 1 to about 5,000, from about 2 to about 4,500, from about 3 to about 4,000, from about 4 to about 3,500, from about 5 to about 3,000, from about 6 to about 2,500, from about 7 to about 2,000, from about 8 to about 1 ,500, from about 9 to about 1 ,000, from about 10 to about 950, from about 15 to about 900, from about 20 to about 850, from about 25 to about 800, from about 30 to about 750, from about 35 to about 700, from about 40 to about 650, from about 45 to about 600, from about 50 to about 550, from about 55 to about 500, from about 60 to about 450, from about 65 to about 400, from about 70 to about 350, from about 75 to about 300, from about 80 to about 250, from about 85 to about 200, from about 90 to about 150, from about 95 to about 145, from about 100 to about 140, from about 105 to about 135, from about 110 to about 130, or from about 115 to about 125, or about 120.

[0181] In various embodiments, n is a value from 1 to 5,000. For example, n may be a value from about 1 to about 5,000, from about 2 to about 4,500, from about 3 to about 4,000, from about 4 to about 3,500, from about 5 to about 3,000, from about 6 to about 2,500, from about 7 to about 2,000, from about 8 to about 1 ,500, from about 9 to about 1 ,000, from about 10 to about 950, from about 15 to about 900, from about 20 to about 850, from about 25 to about 800, from about 30 to about 750, from about 35 to about 700, from about 40 to about 650, from about 45 to about 600, from about 50 to about 550, from about 55 to about 500, from about 60 to about 450, from about 65 to about 400, from about 70 to about 350, from about 75 to about 300, from about 80 to about 250, from about 85 to about 200, from about 90 to about 150, from about 95 to about 145, from about 100 to about 140, from about 105 to about 135, from about 110 to about 130, or from about 115 to about 125, or about 120.

[0182] In various embodiments, Y1(or the synthetic polymer) comprises poly(caprolactone) (PCL) or derivatives thereof. In such embodiments, the nanoparticle comprises a bioactive PCL copolymer with a poly(norbornene- dicarboximide) backbone having one or more repeating units represented by general formula (I B) and one or more repeating units represented by general formula (2B):

[0183] (1 B) (2B) wherein R1, R2, R3, L1, X1, Z1and Z2contain one or more features and / or share one or more properties that are similar to those described above; and p > 1.

[0184] In various embodiments, p is a value from 1 to 5,000. For example, p may be a value from about 1 to about 5,000, from about 2 to about 4,500, from about 3 to about 4,000, from about 4 to about 3,500, from about 5 to about 3,000, from about 6 to about 2,500, from about 7 to about 2,000, from about 8 to about 1 ,500, from about 9 to about 1 ,000, from about 10 to about 950, from about 15 to about 900, from about 20 to about 850, from about 25 to about 800, from about 30 to about 750, from about 35 to about 700, from about 40 to about 650, from about 45 to about 600, from about 50 to about 550, from about 55 to about 500, from about 60 to about 450, from about 65 to about 400, from about 70 to about 350, from about 75 to about 300, from about 80 to about 250, from about 85 to about 200, from about 90 to about 150, from about 95 to about 145, from about 100 to about 140, from about 105 to about 135, from about 110 to about 130, or from about 115 to about 125, or about 120.

[0185] In various embodiments, Y1(or the synthetic polymer) comprises polyflactic acid) (PLA) or derivatives thereof. In such embodiments, the nanoparticle comprises a bioactive PLA copolymer with a polyfnorbornene- dicarboximide) backbone having one or more repeating units represented by general formula (1C) and one or more repeating units represented by general formula (2C): wherein

[0186] R1, R2, R3, L1, X1, Z1and Z2contain one or more features and / or share one or more properties that are similar to those described above; and q > 1.

[0187] In various embodiments, q is a value from 1 to 5,000. For example, q may be a value from about 1 to about 5,000, from about 2 to about 4,500, from about 3 to about 4,000, from about 4 to about 3,500, from about 5 to about 3,000, from about 6 to about 2,500, from about 7 to about 2,000, from about 8 to about 1 ,500, from about 9 to about 1 ,000, from about 10 to about 950, from about 15 to about 900, from about 20 to about 850, from about 25 to about 800, from about 30 to about 750, from about 35 to about 700, from about 40 to about 650, from about 45 to about 600, from about 50 to about 550, from about 55 to about 500, from about 60 to about 450, from about 65 to about 400, from about 70 to about 350, from about 75 to about 300, from about 80 to about 250, from about 85 to about 200, from about 90 to about 150, from about 95 to about 145, from about 100 to about 140, from about 105 to about 135, from about 110 to about 130, or from about 115 to about 125, or about 120.

[0188] In various embodiments, Y1(or the synthetic polymer) comprises polyfmethyl methacrylate) (PMMA) or derivatives thereof. In such embodiments, the nanoparticle comprises a bioactive PMMA copolymer with a poly(norbornene- dicarboximide) backbone having one or more repeating units represented by general formula (1 D) and one or more repeating units represented by general formula (2D):

[0189] (1D) (2D) wherein

[0190] R1, R2, R3, L1, X1, Z1and Z2contain one or more features and / or share one or more properties that are similar to those described above; and r > 1 .

[0191] In various embodiments, r is a value from 1 to 5,000. For example, r may be a value from about 1 to about 5,000, from about 2 to about 4,500, from about 3 to about 4,000, from about 4 to about 3,500, from about 5 to about 3,000, from about 6 to about 2,500, from about 7 to about 2,000, from about 8 to about 1 ,500, from about 9 to about 1 ,000, from about 10 to about 950, from about 15 to about 900, from about 20 to about 850, from about 25 to about 800, from about 30 to about 750, from about 35 to about 700, from about 40 to about 650, from about 45 to about 600, from about 50 to about 550, from about 55 to about 500, from about 60 to about 450, from about 65 to about 400, from about 70 to about 350, from about 75 to about 300, from about 80 to about 250, from about 85 to about 200, from about 90 to about 150, from about 95 to about 145, from about 100 to about 140, from about 105 to about 135, from about 110 to about 130, or from about 115 to about 125, or about 120.

[0192] In various embodiments, Y1(or the synthetic polymer) comprises polystyrene (PS) or derivatives thereof. In such embodiments, the nanoparticle comprises a bioactive PS copolymer with a poly(norbornene-dicarboximide) backbone having one or more repeating units represented by general formula (1 E) and one or more repeating units represented by general formula (2E):

[0193] (1E) (2E) wherein

[0194] R1, R2, R3, L1, X1, Z1and Z2contain one or more features and / or share one or more properties that are similar to those described above; and s > 1.

[0195] In various embodiments, s is a value from 1 to 5,000. For example, s may be a value from about 1 to about 5,000, from about 2 to about 4,500, from about 3 to about 4,000, from about 4 to about 3,500, from about 5 to about 3,000, from about 6 to about 2,500, from about 7 to about 2,000, from about 8 to about 1 ,500, from about 9 to about 1 ,000, from about 10 to about 950, from about 15 to about 900, from about 20 to about 850, from about 25 to about 800, from about 30 to about 750, from about 35 to about 700, from about 40 to about 650, from about 45 to about 600, from about 50 to about 550, from about 55 to about 500, from about 60 to about 450, from about 65 to about 400, from about 70 to about 350, from about 75 to about 300, from about 80 to about 250, from about 85 to about 200, from about 90 to about 150, from about 95 to about 145, from about 100 to about 140, from about 105 to about 135, from about 110 to about 130, or from about 115 to about 125, or about 120.

[0196] In various embodiments, the bioactive synthetic copolymer comprises at least about 1 .0 wt%, at least about 2.0 wt%, at least about 5.0 wt%, at least about 10.0 wt%, at least about 15.0 wt%, at least about 20.0 wt%, at least about 25.0 wt%, at least about 30.0 wt%, at least about 35.0 wt%, at least about 40.0 wt%, at least about 45.0 wt%, at least about 50.0 wt%, at least about 55.0 wt%, at least about 60.0 wt%, at least about 65.0 wt%, at least about 70.0 wt%, at least about 75.0 wt%, at least about 80.0 wt%, at least about 85.0 wt%, or at least about 90.0 wt% of the repeating units represented by general formula (2), (2A), (2B), (2C), (2D) and / or (2E).

[0197] In various embodiments, the bioactive synthetic copolymer comprises at least about 99.0 wt%, at least about 98.0 wt%, at least about 95.0 wt%, at least about 90.0 wt%, at least about 85.0 wt%, at least about 80.0 wt%, at least about 75.0 wt%, at least about 70.0 wt%, at least about 65.0 wt%, at least about 60.0 wt%, at least about 55.0 wt%, at least about 50.0 wt%, at least about 45.0 wt%, at least about 40.0 wt%, at least about 35.0 wt%, at least about 30.0 wt%, at least about 25.0 wt%, or at least about 20.0 wt%, at least about 15.0 wt%, or at least about 10.0 wt% of the repeating units represented by general formula (1), (1A), (1B), (1C), (1 D) and / or (1 E).

[0198] BIOACTIVE SYNTHETIC HOMOPOLYMER In various embodiments, In various embodiments, the bioactive synthetic polymer is an amphiphilic bioactive synthetic homopolymer with a poly(norbornene-dicarboximide) backbone having one or more repeating units represented by general formula (3): wherein R4, R5, R6, R7, L2, X2, Y2, and Z3contain one or more features and / or share one or more properties that are similar to those described above.

[0199] In various embodiments, Y2(or the synthetic polymer) comprises poly (lactic-co-glycolic acid) (PLGA) or derivatives thereof. In such embodiments, the nanoparticle comprises a bioactive PLGA homopolymer with a poly(norbornene-dicarboximide) backbone having one or more repeating units represented by general formula (3A): wherein

[0200] R4, R5, R6, R7, L2, X2, Z3, m and n contain one or more features and / or share one or more properties that are similar to those described above; and

[0201] R8is selected from a single bond, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl or optionally substituted alkylcarbonylalkyl. In various embodiments, Y1(or the synthetic polymer) comprises poly(caprolactone) (PCL) or derivatives thereof. In such embodiments, the nanoparticle comprises a bioactive PCL homopolymer with a polyfnorbornene- dicarboximide) backbone having one or more repeating units represented by general formula (3B): wherein

[0202] R4, R5, R6, R7, L2, X2, Z3, and p contain one or more features and / or share one or more properties that are similar to those described above; and R8is selected from a single bond, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl or optionally substituted alkylcarbonylalkyl. In various embodiments, Y1(or the synthetic polymer) comprises polyflactic acid) (PLA) or derivatives thereof. In such embodiments, the nanoparticle comprises a bioactive PLA homopolymer with a poly(norbornene- dicarboximide) backbone having one or more repeating units represented by general formula (3C): wherein

[0203] R4, R5, R6, R7, L2, X2, Z3and q contain one or more features and / or share one or more properties that are similar to those described above; and R8is selected from a single bond, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl or optionally substituted alkylcarbonylalkyl.

[0204] In various embodiments, Y1(or the synthetic polymer) comprises poly(methyl methacrylate) (PMMA) or derivatives thereof. In such embodiments, the nanoparticle comprises a bioactive PMMA homopolymer with a poly(norbomene-dicarboximide) backbone having one or more repeating units represented by general formula (3D): wherein R4, R5, R6, R7, L2, X2, Z3and r contain one or more features and / or share one or more properties that are similar to those described above.

[0205] In various embodiments, Y1(or the synthetic polymer) comprises polystyrene (PS) or derivatives thereof. In such embodiments, the nanoparticle comprises a bioactive PS homopolymer with a poly(norbornene-dicarboximide) backbone having one or more repeating units represented by general formula (3E):

[0206] wherein

[0207] R4, R5, R6, R7, L2, X2, Z3and s contain one or more features and / or share one or more properties that are similar to those described above; and R8is selected from a single bond, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl or optionally substituted alkylcarbonylalkyl. METHOD OF PREPARING POLYMERIC NANOPARTICLES

[0208] There is provided a method of preparing a plurality of bioactive synthetic polymeric nanoparticles as disclosed herein, the method comprising:

[0209] (a-i) mixing one or more bioactive synthetic polymer(s) with a poly(norbornene- dicarboximide) backbone having one or more repeating units selected from the group consisting of general formula (1), general formula (2) and general formula (3) in a suitable organic solvent to obtain a solution;

[0210] (a-ii) precipitating polymeric nanoparticles from the solution into a non-solvent to obtain a dispersion of polymeric nanoparticles; and

[0211] (a-iii) removing the organic solvent and non-solvent from the dispersion to obtain polymeric nanoparticles.

[0212] In various embodiments, the step (a-i) comprises mixing one or more, two or more, three or more, four or more, or five or more different polymers selected from the group consisting of:

[0213] • a bioactive synthetic copolymer having one or more repeating units represented by general formula (1); one or more repeating units represented by general formula (2); and one or more repeating units represented by general formula (3);

[0214] • a bioactive synthetic copolymer having one or more repeating units represented by general formula (1); and one or more repeating units represented by general formula (2);

[0215] • a bioactive synthetic copolymer having one or more repeating units represented by general formula (1); and one or more repeating units represented by general formula (3);

[0216] • a bioactive synthetic copolymer having one or more repeating units represented by general formula (2); and one or more repeating units represented by general formula (3); and

[0217] • a bioactive synthetic homopolymer having one or more repeating units represented by general formula (3). In various embodiments, the step (a-i) comprises a step of converting / returning a precipitate (e.g., bioactive synthetic polymer(s)) to a dissolved state (e.g., a solution containing bioactive synthetic polymer(s)). In various embodiments, the step (a-ii) comprises a step of precipitating a precipitate (e.g., bioactive synthetic polymeric nanoparticles) from the dissolved state (e.g., the solution containing bioactive synthetic polymer(s)). In various embodiments, the step (a-iii) comprises a step of removing precipitant and / or supernatant from the precipitate (e.g., bioactive synthetic polymeric nanoparticles). That is, in various embodiments, the method comprises a repetition of a precipitation process, whereby a precipitate is returned to a dissolved state and subjected to precipitation again. In various embodiments therefore, the method comprises reprecipitation to obtain / prepare / fabricate nanoparticles.

[0218] In various embodiments, the step (a-ii) comprises adding / feeding / passing / injecting the solution obtained from (a-i) into a non-solvent (e.g., aqueous medium). In various embodiments, the step (a-ii) comprises reprecipitation whereby planned repetition of a precipitation to remove impurities from a precipitate or improve its stoichiometry.

[0219] Advantageously, in various embodiments, the method is designed such that the size of the nanoparticles is adjustable and / or customizable by changing parameters / conditions of the method. For example, the size of the nanoparticles may be adjusted as desired by controlling / tu ning the volume ratio of the organic solvent to the non-solvent. The size of the nanoparticles may also be adjusted as desired by tuning / controlling the amount / concentration of the polymers in the solution. The size of the nanoparticles may also be adjusted as desired by tuning / controlling the rate / speed at which the solution obtained from (a-i) is added / fed / passed / injected into the non-solvent.

[0220] Advantageously, in various embodiments, the method is designed such that the particle size distribution is adjustable and / or customizable by changing parameters / conditions of the method. For example, the particle size distribution (e.g., uniformity) of the nanoparticles may be adjusted as desired by controlling / tuning the volume ratio of the organic solvent to the non-solvent. The particle size distribution (e.g., uniformity) may also be adjusted as desired by tuning / controlling the amount / concentration of the polymers in the solution. The particle size distribution (e.g., uniformity) may also be adjusted as desired by tuning / controlling the rate at which the solution obtained from (a-i) is added / fed / passed / injected into the non-solvent.

[0221] In various embodiments, the size of the nanoparticles is adjusted as desired by controlling the volume ratio of the organic solvent to the non-solvent. In various embodiments, smaller nanoparticles are prepared by decreasing the volume ratio of the organic solvent to the non-solvent. For example, the size of nanoparticles may be decreased by increasing volume of non-solvent and / or decreasing volume of organic solvent. In various embodiments, the volume ratio of the organic solvent to the non-solvent is from about 1 :1 to about 1 :10, about 1 :1 , about 1 :2, about 1 :3, about 1 :4, about 1 :5, about 1 :6, about 1 :7, about 1 :8, about 1 :9, or about 1 :10.

[0222] In various embodiments, the size of the nanoparticles is adjusted as desired by controlling the concentration / amount of the polymer in the organic solvent / solution. In various embodiments, smaller nanoparticles are prepared by decreasing amount / concentration of the polymers in the solvent / solution. For example, the size of nanoparticles may be decreased by decreasing amount / concentration of the polymers in the solvent / solution. In various embodiments, the amount / concentration of the one or more, two or more, three or more, four or more, or five or more different polymers in the solvent / solution is from about 0.1 w / v% to about 50.0 w / v%. For example, the amount / concentration of the one or more different polymers in the solvent / solution is from about 0.1 w / v% to about 50.0 w / v%, from about 0.2 w / v% to about 45.0 w / v%, from about 0.3 w / v% to about 40.0 w / v%, from about 0.4 w / v% to about 35.0 w / v%, from about 0.5 w / v% to about 30.0 w / v%, from about 0.6 w / v% to about 25.0 w / v%, from about 0.7 w / v% to about 20.0 w / v%, from about 0.8 w / v% to about 15.0 w / v%, from about 0.9 w / v% to about 10.0 w / v%, from about 1 .0 w / v% to about 9.5 w / v%, from about

[0223] 1 .5 w / v% to about 9.0 w / v%, from about 2.0 w / v% to about 8.5 w / v%, from about

[0224] 2.5 w / v% to about 8.0 w / v%, from about 3.0 w / v% to about 7.5 w / v%, from about

[0225] 3.5 w / v% to about 7.0 w / v%, from about 4.0 w / v% to about 6.5 w / v%, from about

[0226] 4.5 w / v% to about 6.0 w / v%, or from about 5.0 w / v% to about 5.5 w / v% of polymers. In various embodiments, the step (a-i) comprises adding polymers in an amount / concentration of from about 0.1 w / v% to about 50.0 w / v% to the organic solvent.

[0227] In various embodiments, the size of the nanoparticles is adjusted as desired by controlling the feeding / injection rate. In various embodiments, smaller nanoparticles are prepared by increasing the feeding / injection rate. For example, the size of the nanoparticles may be decreased by increasing the feeding / injection rate. Advantageously, in various embodiments, uniform particles (e.g., having uniform particle sizes / diameters / distribution / packing) are also prepared by increasing the feeding / injection rate (e.g., using high feeding / injection rate). In various embodiments, the step (a-ii) comprises adding / feeding / passing / injecting the solution obtained from (a-i) into a nonsolvent (e.g., aqueous medium) at a feeding / injection rate of from about 0.10 mL / s to about 5.00 mL / s. The feeding / injection rate of the solution obtained from (a-i) into a non-solvent may be from about 0.10 mL / s to about 5.00 mL / s, from about 0.20 mL / s to about 4.90 mL / s, from about 0.30 mL / s to about 4.80 mL / s, from about 0.40 mL / s to about 4.70 mL / s, from about 0.50 mL / s to about 4.60 mL / s, from about 0.60 mL / s to about 4.50 mL / s, from about 0.70 mL / s to about 4.40 mL / s, from about 0.80 mL / s to about 4.30 mL / s, from about 0.90 mL / s to about 4.20 mL / s, from about 1 .00 mL / s to about 4.10 mL / s, from about 1.10 mL / s to about 4.00 mL / s, from about 1.20 mL / s to about 3.90 mL / s, from about 1.30 mL / s to about 3.80 mL / s, from about 1.40 mL / s to about 3.70 mL / s, from about 1.50 mL / s to about 3.60 mL / s, from about 1.60 mL / s to about 3.50 mL / s, from about 1.70 mL / s to about 3.40 mL / s, from about 1.80 mL / s to about 3.30 mL / s, from about 1.90 mL / s to about 3.20 mL / s, from about 2.00 mL / s to about 3.10 mL / s, from about 2.10 mL / s to about 3.00 mL / s, about 2.20 mL / s to about 2.90 mL / s, from about 2.30 mL / s to about 2.80 mL / s, from about 2.40 mL / s to about 2.70 mL / s, from about 2.50 mL / s to about 2.60 mL / s, or about 2.55 mL / s.

[0228] In various embodiments, the one or more bioactive synthetic polymer(s) with a poly(norbornene-dicarboximide) backbone having one or more repeating units selected from the group consisting of general formula (1), (2) and (3) is / are mixed / dispersed in / added to a suitable organic solvent to obtain a solution. It will be appreciated that any suitable organic solvent that effectively serves as a medium to contain components of the solution may be used. In various embodiments, the organic solvent is selected from tetrahydrofuran (THF), benzene, toluene, acetonitrile (ACN), dichloromethane (DCM), dimethyl sulfoxide (DMSO), acetone, methyl ethyl ketone (MEK), ethyl vinyl ether, methanol, ethanol, butanol, or the like or combinations thereof. It is to be appreciated that the type of organic solvent used is not limited to the above so long as it does not react with or is substantially non-reactive to the polymers.

[0229] In various embodiments, the non-solvent comprises aqueous medium. The aqueous medium may be water (e.g., deionized water).

[0230] In various embodiments, the method further comprises, prior to the step (a-iii) of removing organic solvent and non-solvent / aqueous medium, a step of mixing, stirring, dissolving, sonicating and / or ultrasonicating the dispersion of nanoparticles. For example, the dispersion of nanoparticles may be further subjected to sonication and / or mixing using a stirrer.

[0231] In various embodiments, the step of mixing, stirring, dissolving, sonicating and / or ultrasonicating the dispersion of nanoparticles is performed at a temperature that is no more than about 35.0°C, no more than about 34.0°C, no more than about 33.0°C, no more than about 32.0°C, no more than about 31 ,0°C, no more than about 30.0°C, no more than about 29.0°C, no more than about 28.0°C, no more than about 27.0°C, no more than about 26.0°C, no more than about 25.0°C, no more than about 24.0°C, no more than about 23.0°C, no more than about 22.0°C, no more than about 21.0°C, no more than about 20.0°C, no more than about 19.0°C, no more than about 18.0°C, no more than about 17.0°C, no more than about 16.0°C, no more than about 15.0°C, no more than about 14.0°C, no more than about 13.0°C, no more than about 12.0°C, no more than about 11.0°C, no more than about 10.0°C, no more than about 9.0°C, no more than about 8.0°C, no more than about 7.0°C, no more than about 6.0°C, or no more than about 5.0°C. The step of mixing, stirring, dissolving, sonicating and / or ultrasonicating the dispersion of nanoparticles may be performed at a temperature that is no more than room temperature, from about 5.0°C to about 30.0°C, from about 10.0°C to about 30.0°C, from about 15.0°C to about 30.0°C, or from about 20.0°C to about 25.0°C. Advantageously, in various embodiments, keeping the temperature of the step of mixing, stirring, dissolving, sonicating and / or ultrasonicating the dispersion of nanoparticles to no more than about 35.0°C avoids polymer nanoparticles from aggregation / agglomeration and / or forming clusters, lumps or clumps.

[0232] In various embodiments, the step of mixing, stirring, dissolving, sonicating and / or ultrasonicating the dispersion of nanoparticles is performed over a time duration of from about 1 minute to about 24 hours, from about 2 minutes to about 23 hours, from about 3 minutes to about 22 hours, from about 4 minutes to about 21 hours, from about 5 minutes to about 20 hours, from about 10 minutes to about 19 hours, from about 15 minutes to about 18 hours, from about 20 minutes to about 17 hours, from about 25 minutes to about 16 hours, from about 30 minutes to about 15 hours, from about 35 minutes to about 14 hours, from about 40 minutes to about 13 hours, from about 45 minutes to about 12 hours, from about 50 minutes to about 11 hours, from about 55 minutes to about 10 hours, from about 1 hour to about 9 hours, from about 2 hours to about 8 hours from about 3 hours to about 7 hours from about 4 hours to about 6 hours, or about 5 hours.

[0233] In various embodiments, the step of mixing, stirring, dissolving, sonicating and / or ultrasonicating the dispersion of nanoparticles is performed at a stirring rate / speed of from about 100 rpm to about 5,000 rpm, from about 250 rpm to about 4,500 rpm, from about 500 rpm to about 4,000 rpm, from about 750 rpm to about 3,500 rpm, from about 1 ,000 rpm to about 3,000 rpm, from about 1 ,500 rpm to about 2,500 rpm, or about 2,000 rpm.

[0234] In various embodiments, the step (a-iii) of removing the organic solvent from the dispersion comprises placing the dispersion of polymeric nanoparticles in an environment under reduced pressure (e.g., under vacuum).

[0235] In various embodiments, the step (a-iii) of removing the organic solvent from the dispersion is performed at a temperature that is no more than about 35.0°C, no more than about 34.0°C, no more than about 33.0°C, no more than about 32.0°C, no more than about 31 .0°C, no more than about 30.0°C, no more than about 29.0°C, no more than about 28.0°C, no more than about 27.0°C, no more than about 26.0°C, no more than about 25.0°C, no more than about 24.0°C, no more than about 23.0°C, no more than about 22.0°C, no more than about 21.0°C, no more than about 20.0°C, no more than about 19.0°C, no more than about 18.0°C, no more than about 17.0°C, no more than about 16.0°C, no more than about 15.0°C, no more than about 14.0°C, no more than about 13.0°C, no more than about 12.0°C, no more than about 11 .0°C, no more than about 10.0°C, no more than about 9.0°C, no more than about 8.0°C, no more than about 7.0°C, no more than about 6.0°C, or no more than about 5.0°C. The step of removing the organic solvent from the dispersion may be performed at a temperature that is no more than room temperature, from about 5.0°C to about 30.0°C, from about 10.0°C to about 30.0°C, from about 15.0°C to about 30.0°C, or from about 20.0°C to about 25.0°C.

[0236] In various embodiments, the step (a-iii) of removing the non- solvent / aqueous medium (e.g., water) from the dispersion of polymeric nanoparticles comprises subjecting the dispersion to freeze-drying, lyophilization and / or cryodesiccation. In various embodiments, the nanoparticle is prepared via nanoprecipitation (which comprises the steps of sonication, evaporation and / or lyophilization).

[0237] POLYMERIC NANOPARTICLE COMPOSITION

[0238] There is provided a polymeric nanoparticle composition comprising a dispersion of a plurality of polymeric nanoparticles as disclosed herein. In various embodiments, the composition is a topical skin formulation. The topical skin formulation may be provided in the form of a liquid, gel or cream formulation.

[0239] In various embodiments, the polymeric nanoparticle composition further comprises:

[0240] (i) a buffer solution;

[0241] (ii) a thickening agent; and

[0242] (iii) optionally a stabilizer.

[0243] In various embodiments, the buffer solution has a pH of from about 6.5 to about 8.5, from about 6.6 to about 8.4, from about 6.7 to about 8.3, from about 6.8 to about 8.2, from about 6.9 to about 8.1 , from about 7.0 to about 8.0, from about 7.1 to about 7.9, from about 7.2 to about 7.8, from about 7.3 to about 7.7, from about 7.4 to about 7.6, or about 7.5. In various embodiments, the buffer solution comprises physiological and / or biological buffers. In various embodiments, the buffer solution comprises saline buffers. For example, the buffer solution may comprise phosphate buffered saline (PBS buffer), citrate buffered saline, borate buffered saline, Tris-buffered saline (TBS), Tris-HCI, NaCI, Tween buffered saline (TNT), Triton X-100 (PBT) or the like, or combinations thereof. The buffer solution may comprise one or more buffering agents selected from sodium chloride, potassium chloride, calcium chloride, magnesium chloride, disodium phosphate, monopotassium phosphate, 4-(2- hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES), piperazine-N,N’-bis(2- ethanesulfonic acid) (PIPES), tetramethyl ammonium chloride (TMAC), tris(hydroxymethyl)aminomethane (T ris) , tris(hydroxymethyl)-aminomethane (THAM), N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), saline sodium phosphate EDTA buffer (SSPE), citric acid, potassium phosphate, and sodium pyrophosphate. In various embodiments, the buffer solution comprises phosphate buffered saline (PBS buffer). It will be appreciated that the type of buffer solution used is not limited to the above and any suitable solution that effectively helps in regulating / maintaining / keeping the pH of the solution at a substantially constant value may be used. For example, suitable buffers that are used in human products or cosmetics may also be used.

[0244] In various embodiments, the thickening agent is selected from hydroxyethyl cellulose (HEC), xanthan gum, hydroxypropyl cellulose, or the like, or combinations thereof.

[0245] In various embodiments, the compatibilizer / stabilizer is selected from propylene glycol (PG; propane-1 ,2-diol or 1 ,2-propanediol), glycerol, glycerin, or the like, or combinations thereof.

[0246] In various embodiments, the composition has a dynamic viscosity of from about 30.0 Pa.s to about 500.0 Pa.s, from about 40.0 Pa.s to about 450.0 Pa.s, from about 50.0 Pa.s to about 400.0 Pa.s, from about 60.0 Pa.s to about 350.0 Pa.s, from about 70.0 Pa.s to about 300.0 Pa.s, from about 80.0 Pa.s to about 250.0 Pa.s, from about 90.0 Pa.s to about 200.0 Pa.s, or from about 100.0 Pa.s to about 150.0 Pa.s.

[0247] In various embodiments, the composition has a pH value of from about 6.5 to about 8.5, from about 6.6 to about 8.4, from about 6.7 to about 8.3, from about 6.8 to about 8.2, from about 6.9 to about 8.1 , from about 7.0 to about 8.0, from about 7.1 to about 7.9, from about 7.2 to about 7.8, from about 7.3 to about 7.7, from about 7.4 to about 7.6, or about 7.5. In various embodiment, the nanoparticle / composition / formulation / liquid / gel / cream is biocompatible, i.e. the nanoparticle / composition / formulation / liquid / gel / cream is compatible with biological systems or parts of the biological systems without substantially or significantly eliciting an adverse physiological response such as a toxic reaction / response, an immune reaction / response, an injury or the like when used on the human or animal body. In various embodiments, the nanoparticle / composition / formulation / liquid / gel / cream is substantially devoid of substances that elicit an adverse physiological response. Advantageously, in various embodiments, the nanoparticles show excellent biocompatibility and are capable of being utilized as bio additives.

[0248] In various embodiments, the nanoparticle / composition / formulation / liquid / gel / cream displays / exhibits high skin / dermal adsorption / penetration rate. In various embodiments, the nanoparticle / composition / formulation / liquid / gel / cream displays / exhibits high skin / epidermis moisture retention. In various embodiments, the nanoparticle / composition / formulation / liquid / gel / cream displays / exhibits low skin / epidermis water evaporation (e.g., low transepidermal water loss (TEWL)) rate.

[0249] Advantageously, in various embodiments, the composition is suitable for use as a wound care, skin care, consumer care, beauty, anti-aging, pet care, aesthetic products, the like, and combinations thereof.

[0250] Advantageously, in various embodiments, the structure of the nanoparticle eliminates the requirement of a steroid or steroid-based ingredients, which is otherwise necessary in a conventional formulation for treatment of skin conditions such as eczema and various forms of dermatitis such as atopic dermatitis, allergic dermatitis or the like. In various embodiments, the nanoparticle, composition, formulation, liquid, gel or cream is substantially devoid of steroid or steroid-based ingredients. Advantageously, in various embodiments, the structure of the nanoparticle eliminates the requirement of an immunosuppressing drug e.g., calcineurin inhibitor (CNI), which is otherwise necessary in a conventional formulation for treatment of skin conditions. In various embodiments, the nanoparticle, composition, formulation, liquid, gel or cream is substantially devoid of immunosuppressing drugs e.g., calcineurin inhibitor (CNI).

[0251] Advantageously, in various embodiments, the composition is suitable for use as a non-steroidal, non-immunosuppressive anti-inflammatory agent.

[0252] METHOD OF PREPARING A POLYMERIC NANOPARTICLE COMPOSITION

[0253] There is provided a method of preparing a polymeric nanoparticle composition, formulation, liquid, gel or cream comprising a dispersion of a plurality of polymeric nanoparticles as disclosed herein, the method comprising: (b-i) mixing polymeric nanoparticles with buffer solution to obtain a dispersion; and

[0254] (b-ii) mixing a thickening agent and optionally a stabilizer / compatibilizer with the dispersion to obtain a composition, formulation, liquid, gel or cream comprising polymeric nanoparticles.

[0255] In various embodiments, the step (b-i) comprises adding polymeric nanoparticles in an amount of from about 0.1 wt% to about 30.0 wt%. The polymeric nanoparticles may be mixed with or added to the buffer solution in an amount of from about 0.1 wt% to about 30.0 wt%, from about 0.2 wt% to about

[0256] 29.5 wt%, from about 0.5 wt% to about 29.0 wt%, from about 1 .0 wt% to about

[0257] 28.5 wt%, from about 1 .5 wt% to about 28.0 wt%, from about 2.0 wt% to about

[0258] 27.5 wt%, from about 2.5 wt% to about 27.0 wt%, from about 3.0 wt% to about

[0259] 26.5 wt%, from about 3.5 wt% to about 26.0 wt%, from about 4.0 wt% to about

[0260] 25.5 wt%, from about 4.5 wt% to about 25.0 wt%, from about 5.0 wt% to about

[0261] 24.5 wt%, from about 5.5 wt% to about 24.0 wt%, from about 6.0 wt% to about

[0262] 23.5 wt%, from about 6.5 wt% to about 23.0 wt%, from about 7.0 wt% to about 22.5 wt%, from about 7.5 wt% to about 22.0 wt%, from about 8.0 wt% to about

[0263] 21 .5 wt%, from about 8.5 wt% to about 21 .0 wt%, from about 9.0 wt% to about

[0264] 20.5 wt%, from about 9.5 wt% to about 20.0 wt%, from about 10.0 wt% to about

[0265] 19.5 wt%, from about 10.5 wt% to about 19.0 wt%, from about 11 .0 wt% to about

[0266] 18.5 wt%, from about 11 .5 wt% to about 18.0 wt%, from about 12.0 wt% to about

[0267] 17.5 wt%, from about 12.5 wt% to about 17.0 wt%, from about 13.0 wt% to about

[0268] 16.5 wt%, from about 13.5 wt% to about 16.0 wt%, from about 14.0 wt% to about

[0269] 15.5 wt%, or from about 14.5 wt% to about 15.0 wt%. For example, the dispersion may comprise from about 0.1 wt% to about 30.0 wt% of the polymeric nanoparticles.

[0270] In various embodiments, the step (b-ii) comprises adding thickening agent in an amount of from about 0.1 wt% to about 30.0 wt%. The thickening agent may be mixed with or added to the composition, formulation, liquid, gel or cream in an amount of from amount of from about 0.1 wt% to about 30.0 wt%, from about 0.2 wt% to about 29.5 wt%, from about 0.5 wt% to about 29.0 wt%, from about 1 .0 wt% to about 28.5 wt%, from about 1 .5 wt% to about 28.0 wt%, from about 2.0 wt% to about 27.5 wt%, from about 2.5 wt% to about 27.0 wt%, from about 3.0 wt% to about 26.5 wt%, from about 3.5 wt% to about 26.0 wt%, from about 4.0 wt% to about 25.5 wt%, from about 4.5 wt% to about 25.0 wt%, from about 5.0 wt% to about 24.5 wt%, from about 5.5 wt% to about 24.0 wt%, from about 6.0 wt% to about 23.5 wt%, from about 6.5 wt% to about 23.0 wt%, from about 7.0 wt% to about 22.5 wt%, from about 7.5 wt% to about 22.0 wt%, from about 8.0 wt% to about 21 .5 wt%, from about 8.5 wt% to about 21 .0 wt%, from about 9.0 wt% to about 20.5 wt%, from about 9.5 wt% to about 20.0 wt%, from about 10.0 wt% to about 19.5 wt%, from about 10.5 wt% to about 19.0 wt%, from about 11 .0 wt% to about 18.5 wt%, from about 11 .5 wt% to about 18.0 wt%, from about 12.0 wt% to about 17.5 wt%, from about 12.5 wt% to about 17.0 wt%, from about 13.0 wt% to about 16.5 wt%, from about 13.5 wt% to about 16.0 wt%, from about 14.0 wt% to about 15.5 wt%, or from about 14.5 wt% to about 15.0 wt%. For example, the composition, formulation, liquid, gel or cream may comprise from about 0.1 wt% to about 30.0 wt% of the thickening agent. In various embodiments, the step (b-ii) comprises adding a stabilizer / compatibilizer. Advantageously, in various embodiments, the presence of a stabilizer / compatibilizer improves / increases stability of the polymeric nanoparticles in the buffer solution and / or thickening agent. In various embodiments, the compatibilizer / stabilizer provides / increases dispersion stability of the polymeric nanoparticles in the buffer solution and / or thickening agent by promoting interfacial adhesion (or reducing interfacial tension) between the substantially immiscible contents (e.g., polymeric nanoparticles, thickening agent and / or buffer solution).

[0271] In various embodiments, the In various embodiments, the step (b-ii) comprises adding stabilizer / compatibilizer in an amount of from about 0.1 wt% to about 30.0 wt%. The stabilizer / compatibilizer may be mixed with or added to the composition, formulation, liquid, gel or cream in an amount of from amount of from about 0.1 wt% to about 30.0 wt%, from about 0.2 wt% to about 29.5 wt%, from about 0.5 wt% to about 29.0 wt%, from about 1 .0 wt% to about 28.5 wt%, from about 1 .5 wt% to about 28.0 wt%, from about 2.0 wt% to about 27.5 wt%, from about 2.5 wt% to about 27.0 wt%, from about 3.0 wt% to about 26.5 wt%, from about 3.5 wt% to about 26.0 wt%, from about 4.0 wt% to about 25.5 wt%, from about 4.5 wt% to about 25.0 wt%, from about 5.0 wt% to about 24.5 wt%, from about 5.5 wt% to about 24.0 wt%, from about 6.0 wt% to about 23.5 wt%, from about 6.5 wt% to about 23.0 wt%, from about 7.0 wt% to about 22.5 wt%, from about 7.5 wt% to about 22.0 wt%, from about 8.0 wt% to about 21 .5 wt%, from about 8.5 wt% to about 21 .0 wt%, from about 9.0 wt% to about 20.5 wt%, from about 9.5 wt% to about 20.0 wt%, from about 10.0 wt% to about 19.5 wt%, from about 10.5 wt% to about 19.0 wt%, from about 11 .0 wt% to about 18.5 wt%, from about 11 .5 wt% to about 18.0 wt%, from about 12.0 wt% to about 17.5 wt%, from about 12.5 wt% to about 17.0 wt%, from about 13.0 wt% to about 16.5 wt%, from about 13.5 wt% to about 16.0 wt%, from about 14.0 wt% to about 15.5 wt%, or from about 14.5 wt% to about 15.0 wt%. For example, the composition, formulation, liquid, gel or cream may comprise from about 0.1 wt% to about 30.0 wt% of the stabilizer / compatibilizer. In various embodiments, the volume / weight ratio of stabilizer / compatibilizer to buffer solution is from about 1 :1 to about 1 :30, from about 1 :2 to about 1 :29, from about 1 :3 to about 1 :28, from about 1 :4 to about 1 :27, from about 1 :5 to about 1 :26, from about 1 :6 to about 1 :25, from about 1 :7 to about 1 :24, from about 1 :8 to about 1 :23, from about 1 :9 to about 1 :22, from about 1 :10 to about 1 :21 , from about 1 :11 to about 1 :20, from about 1 :12 to about 1 :19, from about 1 :13 to about 1 :18, from about 1 :14 to about 1 :17, or from about 1 :15 to about 1 :16.

[0272] In various embodiments, the step (b-i) and / or (b-ii) comprises one or more of the following: mixing, stirring, dissolving, sonicating and / or ultrasonicating the dispersion. For example, the dispersion may be subjected to agitation using a sonicator.

[0273] In various embodiments the step (b-i) and / or (b-ii) is / are performed over a time duration of from about 1 minute to about 24 hours, from about 2 minutes to about 23 hours, from about 3 minutes to about 22 hours, from about 4 minutes to about 21 hours, from about 5 minutes to about 20 hours, from about 10 minutes to about 19 hours, from about 15 minutes to about 18 hours, from about 20 minutes to about 17 hours, from about 25 minutes to about 16 hours, from about 30 minutes to about 15 hours, from about 35 minutes to about 14 hours, from about 40 minutes to about 13 hours, from about 45 minutes to about 12 hours, from about 50 minutes to about 11 hours, from about 55 minutes to about 10 hours, from about 1 hour to about 9 hours, from about 2 hours to about 8 hours from about 3 hours to about 7 hours from about 4 hours to about 6 hours, or about 5 hours.

[0274] In various embodiments, the step (b-i) and / or (b-ii) is / are performed at a temperature that is from about 5.0°C to about 30.0°C, from about 10.0°C to about 29.0°C, from about 15.0°C to about 28.0°C, from about 16.0°C to about 27.0°C, from about 17.0°C to about 26.0°C, from about 18.0°C to about 25.0°C, from about 19.0°C to about 24.0°C, from about 20.0°C to about 23.0°C, or from about 21.0°C to about 22.0°C.

[0275] METHODS OF PREPARING COPOLYMER AND HOMOPOLYMER

[0276] There is provided a method of preparing a bioactive synthetic copolymer as disclosed herein, the method comprising:

[0277] (c) polymerizing one or more synthetic macromolecules represented by general formula (4) with one or more bioactive macromolecules represented by general formula (5) to obtain the bioactive synthetic copolymer:

[0278] (4) (5) wherein R1, R2, R3, L1, X1, Y1, Z1and Z2contain one or more features and / or share one or more properties that are similar to those described above.

[0279] In various embodiments, the polymerizing step (c) is designed to be performed sequentially or successively. Advantageously, in various embodiments, such a sequential / successive polymerization advantageously allow the preparation of a bioactive synthetic copolymer having repeating units that are arranged sequentially within the polymer in an ordered manner, e.g., in a fixed / pre-fixed order. In various embodiments, step (c) comprises sequential / successive addition of the one or more synthetic macromolecules represented by general formula (4) and the one or more bioactive macromolecules represented by general formula (5). That is, the one or more synthetic macromolecules and the one or more bioactive macromolecules may be added sequentially, successively, or one after the other.

[0280] For example, step (c) may comprise:

[0281] (c-i) polymerizing one or more synthetic macromolecules represented by general formula (4) to obtain a synthetic homopolymer; and

[0282] (c-ii) polymerizing one or more bioactive macromolecules represented by general formula (5) with the synthetic homopolymer obtained from step (c-i) to form a bioactive synthetic copolymer.

[0283] The order in which the synthetic macromolecules and bioactive macromolecules are added may be reversed, for example, step (c) may comprise:

[0284] (c-i) polymerizing one or more bioactive macromolecules represented by general formula (5) to obtain a bioactive homopolymer; and

[0285] (c-ii) polymerizing one or more synthetic macromolecules represented by general formula (4) with the bioactive homopolymer obtained from step (c-i) to form a bioactive synthetic copolymer.

[0286] In various embodiments, the step (c) of polymerizing comprises mixing one or more synthetic macromolecules in a first organic solvent prior to mixing with one or more bioactive macromolecules in a second organic solvent, wherein the first and second organic solvents are miscible with each other. In various embodiments, the synthetic macromolecules are miscible with / soluble in the first organic solvent and in the second organic solvent. In various embodiments, the bioactive macromolecules are miscible with / soluble in the second organic solvent but not miscible with / soluble in the first organic solvent. For example, step (c-i) comprises mixing / polymerizing one or more synthetic macromolecules in a first organic solvent, while step (c-ii) comprises providing one or more bioactive macromolecules in a second organic solvent and mixing / polymerizing said bioactive macromolecules in the second organic solvent with synthetic homopolymer obtained from step (c-i).

[0287] In various embodiments, the organic solvent used is different for step c-i) and c-ii). In various embodiments, the first organic solvent is different from the second organic solvent. In various embodiments, the first organic solvent has a polarity and / or dielectric constant that is lower than that of the second organic solvent. In various embodiments, the first and second organic solvents are each independently selected from chloroform, dichloromethane (DCM), toluene, N,N- dimethylformamide (DMF), ethyl acetate, or the like or combinations thereof. For example, the first organic solvent may be tetrahydrofuran (THF) while the second organic solvent may be dimethylformamide (DMF)

[0288] Advantageously, in various embodiments, such a sequential / successive addition of the macromolecules / macromonomers advantageously allow the preparation of a brush / block copolymer (e.g., diblock brush copolymer) having repeating units that are arranged sequentially / orderly e.g., in a A-b-B sequence. In various embodiments, by using different organic solvents for mixing / dissolving the bioactive macromolecules and synthetic macromolecules (e.g., using THF for synthetic macromolecules and DMF for bioactive macromolecules) during the sequential / successive polymerization, embodiments of the method advantageously allows for the incorporation of a large amount / concentration of hydrophilic groups (e.g., bioactive macromolecules / repeating units / polymer blocks) into the copolymer. Even more advantageously, the presence of large amounts of hydrophilic groups (e.g., bioactive macromolecules / repeating units / polymer blocks) in the copolymer aids in the conversion of the copolymer into nanoparticles by stabilizing particles in water during reprecipitation.

[0289] In various embodiments, the step (c) of polymerizing comprises adding the one or more synthetic macromolecules represented by general formula (4) and the one or more bioactive macromolecules represented by general formula (5) in a weight / mass / volume ratio of from about 50.0 - 200.0 : 25.0 - 100.0, about 75.0 - 150.0 : 50.0 - 75.0, or about 100.0 - 125.0 : 50.0 - 75.0.

[0290] There is provided a method of preparing a bioactive synthetic homopolymer as disclosed herein, the method comprising:

[0291] (d) polymerizing one or more bioactive macromolecules represented by general formula (6) to obtain the bioactive synthetic homopolymer: wherein R4, R5, R6, R7, L2, X2, Y2and Z3contain one or more features and / or share one or more properties that are similar to those described above.

[0292] In various embodiments, the polymerization reaction (c), (c-i), (c-ii) and / or (d) comprise one or more olefin metathesis chain-growth polymerization step(s). The olefin metathesis chain-growth polymerization may be ring opening metathesis polymerization (ROMP). In various embodiments, the ROMP reaction occurs at the reactive moiety of the macromonomers, for e.g., at the olefins / alkene / C=C moieties. Advantageously, ROMP allows quick development / construction of well-defined synthetic polymers with the desired bioactivities. In various embodiments, depending on the targeted application, an appropriate synthetic polymer and a biomolecule with the bioactivity of interest may be chosen and copolymerized together using ROMP.

[0293] In various embodiments, the polymerizing step (c), (c-i), (c-ii) and / or (d) is performed in the presence of a polymerisation initiator / catalyst / promoter. In various embodiments, the polymerisation initiator / catalyst / promoter comprises a metal complex. The metal complex may be a ruthenium (Ru), molybdenum (Mo) or tungsten (W) complex. In various embodiments, the polymerization is performed in the presence of a ruthenium complex. Advantageously, as compared to other transition metals (e.g., W and Mo), Ru is more stable in the presence of polar functional groups, thereby making Ru a suitable olefin metathesis catalyst for ROMP reactions that involve oligosaccharides or oligopeptides or drug molecules with acid, alcohol groups and / or derivatives thereof. In various embodiments, Ru is air-stable (i.e. stable in air) and thermally stable (i.e. stable at high temperatures) whilst being commercially available on a large scale, allowing ROMP to be carried out at elevated temperatures. The ruthenium complex may comprise a Grubbs catalyst selected from a first- generation Grubbs catalyst, second-generation Grubbs catalyst, Hoveyda- Grubbs’ catalyst, a third-generation Grubbs catalyst or derivatives thereof.

[0294] In various embodiments, steps (c), (c-i), (c-ii) and / or (d) is / are carried out or undertaken at a temperature in the range of from about 20 °C to about 100 °C. The temperature(s) at which (c), (c-i), (c-ii) and / or (d) is carried out may be independently selected from a temperature of about 20 °C, about 25 °C, about 30 °C, about 35 °C, about 40 °C, about 50 °C, about 60 °C, about 70 °C, about 80 °C, about 90 °C or about 100 °C.

[0295] In various embodiments, steps (c), (c-i), (c-ii) and / or (d) is / are carried out or undertaken for a time period in the range of from about 30 mins to about 3 days. The time period at which steps (c), (c-i), (c-ii) and / or (d) is carried out may be independently selected from a time period of about 30 mins, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 20 hours, 1 day, 2 days or 3 days. In various embodiments, steps (c), (c-i), (c-ii) and / or (d) is / are carried out in the presence of an organic solvent. The organic solvent may be a protic solvent, an aprotic solvent or combinations thereof. In various embodiments, the organic solvent(s) for (c), (c-i), (c-ii) and / or (d) is independently selected from the group consisting of tetrahydrofuran (THF), dimethylformamide (DMF), benzene, toluene, acetonitrile (ACN), dichloromethane (DCM), dimethyl sulfoxide (DMSO), acetone, methyl ethyl ketone (MEK), ethyl acetate and the like and combinations thereof. In various embodiments, the organic solvent used is different for step c-i) and c-ii). It is to be appreciated that the type of solvent used is dependent on the type of reactants used and is not limited to the above.

[0296] In various embodiments, steps (c), (c-i), (c-ii) and / or (d) is / are carried out in a mixture of organic solvents. The mixture of organic solvents may contain one or more aprotic organic solvents and one or more protic organic solvents. In various embodiments, the mixture of organic solvents for steps (c), (c-i), (c-ii) and / or (d) is selected from the group consisting of tetrahydrofuran (THF), dimethylformamide (DMF), benzene, toluene, acetonitrile (ACN), dichloromethane (DCM), dimethyl sulfoxide (DMSO), acetone, methyl ethyl ketone (MEK), ethyl vinyl ether, methanol, ethanol, butanol and the like and combinations thereof. It is to be appreciated that the type of solvent used is dependent on the type of reactants used and is not limited to the above.

[0297] Method of Preparing Bioactive Synthetic Macromolecule

[0298] There is provided a method of preparing a bioactive synthetic macromolecule / macromonomer represented by general formula (6) as disclosed herein, the method comprising:

[0299] (e-i) reacting a synthetic macromolecule / macromonomer represented by general formula (7) with a diamine represented by general formula (8) to obtain an amine represented by general formula (9):

[0300] wherein

[0301] R9is selected from H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; and

[0302] R4, R5, R6, R7, L2, Y2and Z3contain one or more features and / or share one or more properties that are similar to those described above.

[0303] (e-ii) reacting said amine represented by general formula (9) with an acid- containing bioactive moiety (e.g., X2-C(=O)OH) to obtain the bioactive synthetic macromolecule / macromonomer represented by general formula (6), wherein X2contain one or more features and / or share one or more properties that are similar to that described above. In various embodiments, the step (e-i) and / or (e-ii) is / are carried out in the presence of an organic solvent. The organic solvent may be a protic solvent, an aprotic solvent or combinations thereof. In various embodiments, the organic solvent(s) for step (b-i) and / or (b-ii) is independently selected from the group consisting of tetrahydrofuran (THF), benzene, toluene, acetonitrile (ACN), dichloromethane (DCM), dimethyl sulfoxide (DMSO), acetone, methyl ethyl ketone (MEK), formic acid, acetic acid and the like and combinations thereof.

[0304] In various embodiments, the step (e-i) and / or (e-ii) is / are performed at a temperature that is from about 15°C to about 50°C, from about 16°C to about 49°C, from about 17°C to about 48°C, from about 18°C to about 47°C, from about 19°C to about 46°C, from about 20°C to about 45°C, from about 21 °C to about 44°C, from about 22°C to about 43°C, from about 23°C to about 42°C, from about 24°C to about 41 °C, from about 25°C to about 40°C, from about 26°C to about 39°C, from about 27°C to about 38°C, from about 28°C to about 37°C, from about 29°C to about 36°C, from about 30°C to about 35°C, from about 31 °C to about 34°C, or from about 32°C to about 33°C.

[0305] BIOACTIVE SYNTHETIC MACROMOLECULE

[0306] There is provided a bioactive synthetic macromolecule / macromonomer represented by general formula (6) for preparing the bioactive synthetic homopolymer / nanoparticle as disclosed herein:

[0307] wherein

[0308] R4, R5, R6, R7, L2, X2, Y2and Z3contain one or more features and / or share one or more properties that are similar to those described above.

[0309] In various embodiments, the bioactive synthetic macromolecule / macromonomer comprises a pegylated macromonomer, e.g., a pegylated oligosaccharide / oligopeptide macromonomer. In various embodiments, Y2(or the synthetic polymer) comprises poly (lactic-co-glycolic acid) (PLGA) or derivatives thereof. In such embodiments, the bioactive synthetic macromolecule / macromonomer is bioactive PLGA macromolecule / macromonomer represented by general formula (6A):

[0310] wherein

[0311] R4, R5, R6, R7, R8, L2, X2, Z3, m and n contain one or more features and / or share one or more properties that are similar to those described above.

[0312] In various embodiments, Y1(or the synthetic polymer) comprises poly(caprolactone) (PCL) or derivatives thereof. In such embodiments, the bioactive synthetic macromolecule / macromonomer is bioactive PCL macromolecule / macromonomer represented by general formula (6B):

[0313] wherein

[0314] R4, R5, R6, R7, L2, X2, Z3, and p contain one or more features and / or share one or more properties that are similar to those described above; and R8is selected from a single bond, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl or optionally substituted alkylcarbonylalkyl. In various embodiments, Y1(or the synthetic polymer) comprises poly(lactic acid) (PLA) or derivatives thereof. In such embodiments, the bioactive synthetic macromolecule / macromonomer is bioactive PLA macromolecule / macromonomer represented by general formula (6C):

[0315] wherein

[0316] R4, R5, R6, R7, L2, X2, Z3and q contain one or more features and / or share one or more properties that are similar to those described above; and

[0317] R8is selected from a single bond, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl or optionally substituted alkylcarbonylalkyl.

[0318] In various embodiments, Y1(or the synthetic polymer) comprises poly(methyl methacrylate) (PMMA) or derivatives thereof. In such embodiments, the bioactive synthetic macromolecule / macromonomer is bioactive PMMA macromolecule / macromonomer represented by general formula (6D):

[0319] wherein

[0320] R4, R5, R6, R7, L2, X2, Z3and r contain one or more features and / or share one or more properties that are similar to those described above.

[0321] In various embodiments, Y1(or the synthetic polymer) comprises polystyrene (PS) or derivatives thereof. In such embodiments, the bioactive synthetic macromolecule / macromonomer is bioactive PS macromolecule / macromonomer represented by general formula (6E):

[0322] wherein

[0323] R4, R5, R6, R7, L2, X2, Z3and s contain one or more features and / or share one or more properties that are similar to those described above; and R8is selected from a single bond, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl or optionally substituted alkylcarbonylalkyl. In various embodiments, there is similarly also provided corresponding macromolecules / macromonomers for preparing formula (1A), (2A), (1 B), (2B), (1C), (2C), (1 D), (2D), (1 E), (2E). In various embodiments, said corresponding macromolecules / macromonomers for preparing formula (2A), (2B), (2C), (2D) and / or (2E) comprise pegylated macromolecules / macromonomers, e.g., pegylated oligosaccharide / oligopeptide macromolecules / macromonomers. In various embodiments, the corresponding macromolecules / macromonomers for preparing formula (1A), (2A), (1 B), (2B), (1C), (2C), (1 D), (2D), (I E) and (2E) are represented by corresponding macromolecules / macromonomers for preparing formula (11A), (12A), (11 B), (12B), (11C), (12C), (11 D), (12D), (11E), and (12E) respectively.

[0324] In various embodiments, there is provided a polymeric nanoparticle or a composition / formulation / liquid / gel / cream comprising a dispersion of a plurality of polymeric nanoparticles for use in medicine. In various embodiments, there is provided polymeric nanoparticles or a composition / formulation / liquid / gel / cream comprising a dispersion of a plurality of polymeric nanoparticles for use in accelerating / promoting / stimulating skin and / or tissue regeneration. In various embodiments, there is also provided use of polymeric nanoparticles or a composition / formulation / liquid / gel / cream comprising a dispersion of a plurality of polymeric nanoparticles in the manufacture of a medicament for accelerating / promoting / stimulating skin and / or tissue regeneration. In various embodiments, there is also provided a method of accelerating / promoting / stimulating skin and / or tissue regeneration, the method comprising administering / applying the polymeric nanoparticles or a composition / formulation / liquid / gel / cream disclosed herein to a human or animal body.

[0325] In various embodiments, there is provided polymeric nanoparticles or a composition / formulation / liquid / gel / cream comprising a dispersion of a plurality of polymeric nanoparticles for use in treating / relieving / reducing / controlling skin inflammation and / or irritation, rashes, eczema, psoriasis and / or aging skin, retaining / maintaining / increasing moisture level in the skin / skin barrier (e.g., epidermis), hydrating skin, and / or strengthening / improving / repairing / restoring skin barrier. In various embodiments, there is also provided use of polymeric nanoparticles or a composition / formulation / liquid / gel / cream comprising said polymeric nanoparticles in the manufacture of a medicament for treating / relieving / reducing / controlling skin inflammation and / or irritation, eczema, rashes, psoriasis and / or aging skin, retaining / maintaining / increasing moisture level in the skin / skin barrier (e.g., epidermis), hydrating skin, and / or strengthening / improving / repairing / restoring skin barrier. In various embodiments, there is also provided a method of treating / relieving / reducing / controlling skin inflammation and / or irritation, eczema, psoriasis, rashes and / or aging skin, retaining / maintaining / increasing moisture level in the skin / skin barrier (e.g., epidermis), hydrating skin, strengthening / improving / repairing / restoring skin barrier, the method comprising administering / applying the polymeric nanoparticles or a composition / formulation / liquid / gel / cream disclosed herein to a human or animal body.

[0326] In various embodiments, there is provided polymeric nanoparticles or a composition / formulation / liquid / gel / cream comprising a dispersion of a plurality of polymeric nanoparticles for use in treating / relieving / reducing eczema selected from the group consisting of atopic dermatitis / eczema, contact dermatitis, dyshidrotic eczema, neurodermatitis, nummular eczema, seborrheic dermatitis, stasis dermatitis, allergic dermatitis, the like, and combinations thereof. In various embodiments, there is also provided use of polymeric nanoparticles or a composition / formulation / liquid / gel / cream comprising said polymeric nanoparticles in the manufacture of a medicament for treating / relieving / reducing eczema selected from the group consisting of atopic dermatitis / eczema, contact dermatitis, dyshidrotic eczema, neurodermatitis, nummular eczema, seborrheic dermatitis, stasis dermatitis, allergic dermatitis, the like, and combinations thereof. In various embodiments, there is also provided a method of treating / relieving / reducing eczema selected from the group consisting of atopic dermatitis / eczema, contact dermatitis, dyshidrotic eczema, neurodermatitis, nummular eczema, seborrheic dermatitis, stasis dermatitis, the like, and combinations thereof, the method comprising administering / applying the polymeric nanoparticles or a composition / formulation / liquid / gel / cream disclosed herein to a human or animal body.

[0327] In various embodiments, there is also provided polymeric nanoparticles or a composition / formulation / liquid / gel / cream comprising a dispersion of a plurality of polymeric nanoparticles for use in the prophylaxis or treatment of wounds (e.g. acute wounds) such as burns, chronic and / or slow healing wounds such as pressure sores, venous ulcers, diabetic ulcers, foot ulcers, bed sores and the like, and combinations thereof. In various embodiments, there is also provided use of polymeric nanoparticles or a composition / formulation / liquid / gel / cream comprising said polymeric nanoparticles in the manufacture of a medicament for the prophylaxis or treatment of wounds (e.g. acute wounds) such as burns, chronic and / or slow healing wounds such as pressure sores, venous ulcers, diabetic ulcers, foot ulcers, bed sores and the like, and combinations thereof. In various embodiments, there is also provided a method of treating a wound, the method comprising contacting the polymeric nanoparticles disclosed herein with the wound. The wound may be a dermal wound or a skin wound. In various embodiments, the method comprises treating acute wounds such as burns, chronic and / or slow healing wounds such as pressure sores, venous ulcers, diabetic ulcers, foot ulcers, bed sores and the like, and combinations thereof.

[0328] In various embodiments, the composition / formulation / liquid / gel / cream is substantially or completely devoid of steroids (e.g. corticosteroids) and / or immunosuppressive agents (e.g. typically used for treating eczema).

[0329] In various embodiments, the administration is topical administration.

[0330] In various embodiments, the present technology is different (e.g., structurally different) from those of the art that contains randomly connected polymer blocks, e.g., random brush copolymer such as poly(lactic-co-glycolic acid) (PLGA) peptide and oligosaccharide brush polymers where their polymer blocks are randomly connected. It will be appreciated that the amount of hydrophilic group that may be incorporated in the structure of random brush polymers in the art is low (e.g., maximum of 10% even with a high amount of macromonomers added), and it is therefore impossible to form a hydrophilic shell of nanoparticle in such copolymer structure. It will be appreciated that, due to lack of or an insufficient amount of hydrophilic groups, it is not possible to stabilize particles during nanoparticle fabrication by reprecipitation. It will also be appreciated that stable and spheric particle formation, as well as particle size control are not possible when working with random brush polymers of the art.

[0331] In various embodiments, the present technology comprises two types of hydrophobic and hydrophilic amphiphilic brush polymers, e.g., A-b-B diblock brush copolymers having structures in which the hydrophobic block (I) and the hydrophilic block (II) are sequentially connected. Advantageously, embodiments of the present technology allow an incorporation of more than about 25%, more than about 30 wt%, more than about 35 wt%, more than about 40 wt more than about 45 wt%, or more than about 50 wt% of hydrophilic group in the structure of the brush polymers. In various embodiments, in the process of preparing nanoparticles by reprecipitation in water, the hydrophobic block (I) is rearranged as core, and the hydrophilic block (II) forms a shell around the core to become a core-shell structure as it faces / is exposed to water. In various embodiments, the hydrophilic block (II) stabilizes the particles in the water. In various embodiments, embodiments of the method advantageously allows the formation of uniform particles and control of the particle size.

[0332] In various embodiments, the present technology also comprises amphiphilic poly(PLGA-PEG-peptide) and poly(PLGA-PEG-oligosaccharide) brush homopolymers (III) that are not disclosed or suggested in the art.

[0333] In various embodiments, the present technology comprises two types of hydrophobic and hydrophilic amphiphilic brush polymers. In various embodiments, two different macromonomers (i.e. hydrophobic macromonomer and hydrophilic macromonomer) are used for synthesis of A-b-B diblock brush copolymers. In various embodiments, in the step of polymerization, the hydrophilic macromonomer concentration is increased to form a hydrophobic and hydrophilic amphiphilic diblock brush copolymer structure, thereby allowing the incorporation of a concentration of about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more of the hydrophilic block (II).

[0334] In various embodiments, the present technology is different (e.g., structurally different) from those of the art that performs direct grafting of synthetic polymer on bioactive molecules to obtain a linear polymer (e.g., direct grafting of poly(lactic-co-glycolic acid) (PLGA) onto linear hyaluronic acid (HA) to form PLGA-grafted HA copolymer). It will be appreciated that it is impossible to synthesize A-b-B diblock brush copolymer structure from such a direct grafting method (as the resultant linear polymer can no longer be further polymerized).

[0335] In various embodiments, the brush copolymers of the present technology comprise A-b-B diblock copolymers containing hydrophobic block and hydrophilic block that can form hydrophobic-hydrophilic core-shell nanoparticles. Advantageously, the present application has demonstrated successful preparation of hydrophobic PLGA core (A) and hydrophilic bioactive shell (B) nanoparticles using embodiments of the A-b-B diblock brush copolymers. Even more advantageously, the present application has shown that embodiments of said nanoparticles are biocompatible.

[0336] In various embodiments, the present technology is different (e.g., structurally different) from those of the art that prepare nanoparticles by coupling synthetic polymer with bioactive molecules to form a linear polymer before allowing to self-assembly into nanoparticle, for e.g., a poly(lactic-co-glycolic acid) (PLGA)-polyethylene glycol (PEG)-folic acid (FA) copolymer linear structure which was synthesized using coupling reaction between PLGA and FA-PEG-NH2 and self-assembled into nanoparticles. It will be appreciated that as synthetic polymer PLGA has no polymerizable function, it cannot function as a monomer or macromonomer for subsequent polymerization such as homo- or block- or random copolymer. Accordingly, in various embodiments, it is impossible to synthesize A-b-B diblock brush copolymer structure from such a method.

[0337] In various embodiments, in the synthesis of amphiphilic poly(PLGA-PEG- peptide) and poly(PLGA-PEG-oligosaccharide) brush homopolymers (III) of the present application, PLGA is first synthesized using norbornene functionalized initiator to form norbornene-PLGA and this norbornene group makes PLGA polymerizable via ROMP (ring opening metathesis polymerization) in order to form brush polymer. In various embodiments, norbornene-PLGA is then coupled with PEG followed by peptide or oligosaccharide to form norbornene-PLGA-PEG- peptide or norbornene-PLGA-PEG-oligosaccharide respectively. In various embodiments, the final structure of poly(PLGA-PEG-peptide) and poly(PLGA- PEG-oligosaccharide) are brush polymers containing hydrophobic block and hydrophilic block in one brush each that are capable of forming hydrophobic- hydrophilic core-shell nanoparticles. Advantageously, the present application has demonstrated successful preparation of hydrophobic PLGA core and a hydrophilic bioactive shell nanoparticles using embodiments of poly(PLGA-PEG- peptide and poly(PLGA-PEG-oligosaccharide) brush polymer.

[0338] In various embodiments, the present technology is different from those of the art that comprise single particles with no core-shell configuration.

[0339] In various embodiments, the present application has demonstrated fabrication of hydrophobic and hydrophilic core-shell nanoparticle from hydrophobic and hydrophilic PLGA brush polymers such as A-b-B diblock brush copolymer (that is copolymerized from hydrophilic macromonomer and hydrophilic macromonomer) and homopolymer (that is polymerized from a macromonomer comprising hydrophobic and hydrophilic components). In various embodiments, the reprecipitation conditions employed during the reactions of hydrophobic and hydrophilic amphiphilic brush polymers of the present application such as polymer concentration, feeding speed and agitation time allow the core-shell nanoparticle size to be controlled. The control of the coreshell nanoparticle size of the presently disclosed nanoparticles through reprecipitation conditions are not disclosed or suggested in the art.

[0340] In various embodiments, the present technology is different from those of the art that fabricate PLGA nanoparticles by emulsification and nanoprecipitation techniques. It will be appreciated that emulsification and nanoprecipitation require polyvinyl alcohol (PVA) to form and stabilize hydrophobic PLGA nanoparticles in water medium. On the other hand, embodiments of the present technology use nanoprecipitation, but do not involve any additional emulsifier such as PVA to form and stabilize the nanoparticles. In various embodiments, the present technology relates to a new structure of poly(lactic-co-glycolic acid) (PLGA) peptide and oligosaccharide A-b-B diblock brush copolymer. In various embodiments, PEGylated peptide and oligosaccharide blocks in the diblock brush copolymer structure are hydrophilic and this hydrophilic block advantageously stabilizes the hydrophobic PLGA block during nanoparticle formation in the water medium.

[0341] In various embodiments, bioactive PLGA polymer nanoparticles were prepared by reprecipitation method.

[0342] BRIEF DESCRIPTION OF FIGURES

[0343] FIG. 1 is a schematic diagram showing the synthesis of a bioactive synthetic copolymer (e.g., PLGA brush block copolymer) for preparing bioactive synthetic polymeric nanoparticles in accordance with various embodiments disclosed herein. Synthesis of the amphiphilic brush polymer is carried out via ROMP.

[0344] FIG. 2 is a schematic diagram showing the synthesis of a bioactive synthetic homopolymer (e.g., PLGA brush homopolymer) for preparing bioactive synthetic polymeric nanoparticles. Synthesis of the amphiphilic brush polymer is carried out via ROMP.

[0345] FIG. 3 is a schematic diagram of a method of preparing bioactive synthetic polymeric nanoparticles in accordance with various embodiments disclosed herein.

[0346] FIG. 4A is a dynamic light scattering (DLS) histogram of PLGA-b-HA nanoparticles prepared in accordance with various embodiments disclosed herein. The average size of nanoparticles was determined as 300 - 500 nm from DLS analysis. FIG. 4B is a dynamic light scattering (DLS) histogram of PLGA-b-RGD nanoparticles prepared in accordance with various embodiments disclosed herein. The average size of nanoparticles was determined as 300 - 500 nm from DLS analysis.

[0347] FIG. 5 is a dynamic light scattering (DLS) histogram of PCL-b-PEGRGD nanoparticles prepared in accordance with various embodiments disclosed herein. The average size of nanoparticles was determined as 300 - 500 nm from DLS analysis.

[0348] FIG. 6 is a dynamic light scattering (DLS) histogram of PLA-b-PEGRGD nanoparticles prepared in accordance with various embodiments disclosed herein. The average size of nanoparticles was determined as 300 - 500 nm from DLS analysis.

[0349] FIG. 7 is a dynamic light scattering (DLS) histogram of PMMA-b-PEGRGD nanoparticles prepared in accordance with various embodiments disclosed herein. The average size of nanoparticles was determined as 200 - 500 nm from DLS analysis.

[0350] FIG. 8 is a dynamic light scattering (DLS) histogram of PS-b-PEGRGD nanoparticles prepared in accordance with various embodiments disclosed herein. The average size of nanoparticles was determined as 200 - 500 nm from DLS analysis.

[0351] FIG. 9 is a schematic diagram of a method of preparing bioactive synthetic polymeric nanoparticles for biocompatibility testing in accordance with various embodiments disclosed herein.

[0352] FIG. 10 is a photograph showing 5 wt% HEC / PBS gel. FIG. 11 is a photograph of MTT assay used for testing biocompatibility on 3D human skin ex vivo models in accordance with various embodiments disclosed herein.

[0353] FIG. 12 is a graph showing the cell viability (%) of bioactive PLGA nanoparticle gel formulation on 3D skin model in accordance with various embodiments disclosed herein.

[0354] FIG. 13A is an image obtained from histology study (hematoxylin-eosin (H&E) staining) of the skin of AD mice (MC903) with no treatment. Scale bar is 50 pm.

[0355] FIG. 13B is an image obtained from histology study (hematoxylin-eosin (H&E) staining) of the skin of AD mice (MC903) after treatment with 0.5% PLGA- RGD. Scale bar is 50 pm.

[0356] FIG. 13C is an image obtained from histology study (hematoxylin-eosin (H&E) staining) of the skin of AD mice (MC903) after treatment with 2.0% PLGA- RGD. Scale bar is 50 pm.

[0357] FIG. 13D is an image obtained from histology study (hematoxylin-eosin (H&E) staining) of the skin of AD mice (MC903) after treatment with a commercial steroid hydrocortisone (HC). Scale bar is 50 pm.

[0358] FIG. 14A is a graph showing the measurement results of thickness of ear skin of AD mice (MC903) without treatment and with various treatments over 26 days (i.e. using HEC gel, 2 wt% PLGA-RGD gel, 4 wt% PLGA-RGD gel, and a commercial steroid hydrocortisone (HC)), in accordance with various embodiments disclosed herein.

[0359] FIG. 14B is a graph showing the measurement results of thickness of ear skin of AD mice (MC903) without treatment and with various treatments on Day 20 (i.e. using HEC gel, 2 wt% PLGA-RGD gel, 4 wt% PLGA-RGD gel, and a commercial steroid hydrocortisone (HC)), in accordance with various embodiments disclosed herein.

[0360] FIG. 15A is a graph showing the measurement results of transepidermal water loss (TEWL) of AD mice (MC903) without treatment and with various treatments over 26 days (i.e. using HEC gel, 2 wt% PLGA-RGD gel, 4 wt% PLGA- RGD gel, and a commercial steroid hydrocortisone (HC)), in accordance with various embodiments disclosed herein.

[0361] FIG. 15B is a graph showing the measurement results of transepidermal water loss (TEWL) of AD mice (MC903) without treatment and with various treatments on Day 20 (i.e. using HEC gel, 2 wt% PLGA-RGD gel, 4 wt% PLGA- RGD gel, and a commercial steroid hydrocortisone (HC)), in accordance with various embodiments disclosed herein.

[0362] FIG. 16A is an image obtained from histology study (hematoxylin-eosin (H&E) staining) of the skin of AD mice (MC903) with ethanol (no treatment).

[0363] FIG. 16B is an image obtained from histology study (hematoxylin-eosin (H&E) staining) of the skin of AD mice (MC903) with ethanol (after treatment using HEC gel).

[0364] FIG. 16C is an image obtained from histology study (hematoxylin-eosin (H&E) staining) of the skin of AD mice (MC903) with no treatment.

[0365] FIG. 16D is an image obtained from histology study (hematoxylin-eosin (H&E) staining) of the skin of AD mice (MC903) after treatment using HEC gel.

[0366] FIG. 16E is an image obtained from histology study (hematoxylin-eosin (H&E) staining) of the skin of AD mice (MC903) after treatment using 2 wt% PLGA-RGD. FIG. 16F is an image obtained from histology study (hematoxylin-eosin (H&E) staining) of the skin of AD mice (MC903) after treatment using 4 wt% PLGA-RGD.

[0367] FIG. 16G is an image obtained from histology study (hematoxylin-eosin (H&E) staining) of the skin of AD mice (MC903) after treatment using a commercial steroid hydrocortisone (HC).

[0368] EXAMPLES

[0369] Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following examples, tables and if applicable, in conjunction with the figures. It should be appreciated that other modifications related to structural, and chemical changes may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new example embodiments. The example embodiments should not be construed as limiting the scope of the disclosure.

[0370] The method of preparing bioactive synthetic polymeric nanoparticles in accordance with various embodiments disclosed herein involve fabricating nanoparticles from bioactive synthetic polymers (e.g., amphiphilic bioactive synthetic brush block copolymers and brush homopolymers). These amphiphilic polymer structures provide core-shell type nanoparticles with a PLGA hydrophobic core and a HA (or RGD) hydrophilic shell, using reprecipitation method. These amphiphilic polymer structures may be fabricated into nanoparticles (e.g., core-shell type nanoparticles with a hydrophobic (synthetic polymer) core and a hydrophilic bioactive shell), using a reprecipitation method. The bioactive synthetic polymeric nanoparticles may be subsequently suspended or dispersed in liquid, gel, and cream formulation / form for further application in skin tissue regeneration, wound healing, treating skin inflammation, eczema, psoriasis, irritation, and anti-aging treatment as well as formulate into skin care, consumer care or aesthetic products such as anti-aging gels. For example, the nanoparticles may be formulated into gels using hydroxyethylcellulose in PBS buffer solution. The gels advantageously showed excellent biocompatibility and thus potential in applications such as wound care, skin care, consumer care and aesthetic products.

[0371] Synthetic polymers that may be used include poly(caprolactone) (PCL), polyesters such as poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA), polystyrene (PS), polyacrylates, poly(meth)acrylates such as polyfmethyl methacrylate) (PMMA) and polyamides (PA). Bioactives that may be used include oligosaccharides and oligopeptides.

[0372] Example 1 : Preparation of Bioactive Synthetic Polymers

[0373] 1.1. Bioactive Synthetic Copolymers

[0374] The method of preparing a bioactive synthetic copolymer in accordance with various embodiments disclosed herein involve creating hydrophobic macromonomers (containing synthetic polymer) and hydrophilic macromonomers (containing bioactive molecules) separately and using ring opening metathesis polymerization (ROMP) techniques to link these otherwise mutually incompatible molecules together. The result is a brush copolymer bearing both the hydrophobic component (i.e. synthetic polymers) and the hydrophilic component (i.e. bioactive molecules).

[0375] Using PLGA hydrophobic macromonomer and PEG(RGD) hydrophilic macromonomer as examples, FIG. 1 provides an illustration on the preparation of a bioactive synthetic copolymer 104 (e.g., PLGA brush block copolymer) by using ring opening metathesis polymerization (ROMP). Hydrophobic macromonomer 100 and hydrophilic macromonomer 102 are copolymerized via ROMP to obtain a brush copolymer 104 bearing both hydrophobic and hydrophilic components. As shown in FIG. 1 , hydrophobic macromonomer 100 and hydrophilic macromonomer 102 each comprises a norbornenyl group 106 and 110 respectively, which is the ROMP site. Hydrophobic macromonomer 100 comprises a biocompatible hydrophobic polymer 108, e.g., PLGA. Hydrophilic macromonomer 102 comprises a biocompatible hydrophilic polymer 112, i.e. PEG and a bioactive molecule 114, e.g., RGD or HA.

[0376] 1.1.1. Bioactive PLGA Brush Block Copolymers

[0377] PLGA is used as an example of synthetic polymer. Hyaluronic acid (HA) and arginyl-glycyl-aspartic acid (RGD) are used as examples of biomolecules or bioactive molecules. To develop new skin tissue regenerative bioactive PLGA polymer nanoparticles using hyaluronic acid (HA) and arginyl-glycyl-aspartic acid (RGD) as biomolecules, HA and RGD containing PLGA brush block copolymer and PLGA brush homopolymer were created. Hydrophilic macromonomers containing pegylated HA and RGD biomolecules are copolymerized with hydrophobic synthetic biocompatible polymers PLGA to form bioactive amphiphilic poly(lactic-co-glycolic acid) PLGA brush block copolymers via ring opening metathesis polymerization (ROMP). Similar strategies may also be adopted for other oligosaccharides and oligopeptides to create different bioactive nanoparticles for formulating various topical application products in wound care, consumer care, skin care, pet care and aesthetic space.

[0378] To create HA (or RGD) containing amphiphilic PLGA brush block copolymers, HA (or RGD) biomacromonomers are prepared using earlier disclosed strategies in PCT / SG2020 / 050621 , which is herein fully incorporated by reference.

[0379] By connecting HA (or RGD) to norbornene linkers via polyethylene glycol (PEG) units, a library of biomacromonomers was created that can be used to form amphiphilic bioactive PLGA brush block copolymers. The same strategy can be used with other oligosaccharides and oligopeptides.

[0380] The HA (or RGD) containing biomacromonomers are synthesized by amine coupling reaction between NBPEG-NH2 and HA (or RGD) (Scheme 1 and 2). Synthesis of NBPEG-NH2 has been disclosed earlier in PCT / SG2020 / 050621 , which is herein fully incorporated by reference.

[0381] To illustrate use of HA and RGD biomacromonomers in brush copolymer structure, a brush block copolymer and brush homopolymer were prepared with PLGA synthetic macromonomer. The NPH-PLGA macromonomers are created and its preparation have been disclosed earlier in PCT / SG2020 / 050621 , which is herein fully incorporated by reference.

[0382] Final bioactive brush type copolymer is prepared by ROMP of synthetic macromonomer with HA and RGD biomacromonomers using Grubbs catalyst (Schemes 3-4, 9). Synthesis of NB-PEGHA has been disclosed earlier in PCT / SG2023 / 050288, which is herein fully incorporated by reference. Once both HA and RGD biomacromonomers and PLGA macromonomer have been synthesized, ring opening metathesis polymerization (ROMP) was carried out on them, using Grubbs catalyst (Scheme 9) to obtain desired HA (or RGD) based PLGA brush block copolymer (Schemes 3-4). The purified copolymer showed an average of 25% HA (or RGD) incorporation in the polymer, despite a reaction condition of [PLGA macromonomer] : [HA (or RGD)-based macromonomer] = 100 : 50.

[0383] It will be appreciated that PLGA may be replaced with other biocompatible hydrophobic polymers such as POL, PLA, PMMA and PS etc.

[0384]

[0385] Scheme 1 . Synthesis of NBPEG macromonomer containing hyaluronic acid (NBPEGHA)

[0386]

[0387] Scheme 2. Synthesis of NBPEG macromonomer containing RGD (NBPEGRGD)

[0388]

[0389] Scheme 3. Synthesis of HA based PLGA brush block copolymer (PLGA-b- PEGHA) by ROMP

[0390]

[0391] Scheme 4. Synthesis of RGD based PLGA brush block copolymer (PLGA-b-PEGRGD) by ROMP

[0392] 1.2. Bioactive Synthetic Homopolymers

[0393] The method of preparing a bioactive synthetic homopolymer in accordance with various embodiments disclosed herein involve creating amphiphilic macromonomers (containing both synthetic polymer and bioactive molecule) and using ring opening metathesis polymerization (ROMP) techniques to link the amphiphilic macromonomers together. The result is a brush homopolymer bearing both the hydrophobic component (i.e. synthetic polymers) and the hydrophilic component (i.e. bioactive molecules).

[0394] Using PLGA-PEG(RGD or HA) amphiphilic macromonomer as an example, FIG. 2 provides an illustration on the preparation of a bioactive synthetic homopolymer 202 (e.g., PLGA brush homopolymer) by using ring opening metathesis polymerization (ROMP). Amphiphilic macromonomers 200 are polymerized (e.g., homopolymerized) via ROMP to obtain a brush homopolymer 202 bearing both hydrophobic and hydrophilic components. As shown in FIG. 2, amphiphilic macromonomer 200 comprises a norbornenyl group 204, which is the ROMP site. Amphiphilic macromonomer 200 also comprise a biocompatible hydrophobic polymer 206, e.g., PLGA; a biocompatible hydrophilic polymer 208, i.e. PEG; and a bioactive molecule 210, e.g., RGD or HA.

[0395] 1.2.1. Bioactive PLGA Brush Homopolymers

[0396] PLGA is used as an example of synthetic polymer. Hyaluronic acid (HA) and arginyl-glycyl-aspartic acid (RGD) are used as examples of biomolecules or bioactive molecules.

[0397] To create HA (or RGD) containing amphiphilic PLGA brush homopolymers, two step coupling reactions were carried out to create both PLGA and PEG-HA (or PEG-RGD) containing amphiphilic macromonomers; first coupling reaction between NPH-PLGA and PEG diamine to form NPH-PLGA- PEGNH2 and second coupling reaction between NPH-PLGAPEGNH2 and HA (or RGD) biomolecules to form NPH-PLGA-PEG-HA (or NPH-PLGA-PEGRGD) amphiphilic macromonomers to prepare P(PLGA-PEG-HA) and P(PLGA-PEG- RGD) brush homopolymers. The same strategy can be used with other oligosaccharides with up to 20 disaccharide units or oligopeptides with 3 - 20 amino acids.

[0398] A series of hyaluronic acid (HA) and arginyl-glycyl-aspartic acid (RGD) peptides containing bioactive amphiphilic poly(lactic-co-glycolic acid) PLGA brush homopolymers have been synthesized via ring opening metathesis polymerization (ROMP).

[0399] New biomacromonomers, NB-PEGA-PEG-HA and NB-PLGA-PEG-RGD, containing PLGA, PEG and biomolecules (HA or RGD) in one macromonomer structure were synthesized by two step coupling reactions (Schemes 5 and 6). Both NB-PEGA-PEG-HA and NB-PLGA-PEGRGD biomacromonomers were homopolymerized by ring opening metathesis polymerization (ROMP) using Grubbs catalyst (Scheme 9) to obtain desired HA (or RGD) based PLGA brush homopolymer (Schemes 7-8). It is believed that the synthesis of norbornenyl group modified amphiphilic biomacromonomer, NB-PLGA-PEG-HA (or NB- PLGA-PEG-RGD) synthesis has not been reported before.

[0400] It will be appreciated that PLGA may be replaced with other biocompatible hydrophobic polymers such as PCL, PLA, PMMA and PS etc.

[0401]

[0402] Scheme 5. Synthesis of NB-PLGA-PEG-HA macromonomer

[0403]

[0404] [NPH.PLOA-PEGNH^

[0405] |N8-Pt.GA-PEG-RGO]

[0406] Scheme 6. Synthesis of NB-PLGA-PEG-RGD macromonomer

[0407]

[0408] Scheme 7. Synthesis of HA based PLGA brush homopolymer (P(PLGA-PEG-HA)) by ROMP

[0409]

[0410] Scheme 8. Synthesis of RGD based PLGA brush homopolymer (P(PLGA-PEG-RGD)) by ROMP

[0411]

[0412] Scheme 9. Grubbs type catalyst for ROMP reactions.

[0413] Example 2: Method of Preparing Bioactive Synthetic Polymeric Nanoparticles from Bioactive Synthetic Polymers

[0414] FIG. 3 is a schematic diagram of a method of preparing bioactive synthetic polymeric nanoparticles in accordance with various embodiments disclosed herein. As shown in FIG. 3, the amphiphilic polymers (e.g., bioactive amphiphilic brush block copolymers or bioactive amphiphilic brush homopolymer) are mixed / dissolved in an organic solvent (e.g., THF) before being precipitated into DI water while undergoing sonication to obtain a dispersion of core-shell type nanoparticles 300. The resulting nanoparticle dispersion is subjected to freeze drying to obtain amphiphilic polymeric nanoparticles 302. Polymer chains in the amphiphilic polymers undergo self assembly, arranging the polymers into coreshell structures, and thereby forming nanoparticles. Each core-shell type nanoparticle 300 comprises a core 304 (i.e. hydrophobic core comprising synthetic polymer such as PLGA); an inner shell 306 (i.e. hydrophilic inner shell comprising a linker linking the core to an outer shell 308 such as PEG); and an outer shell 308 (i.e. hydrophilic outer shell comprising a bioactive molecule such as HA or RGD). Ill

[0415] In various embodiments, bioactive PLGA polymer nanoparticles were prepared by reprecipitation method.

[0416] 1. 1 g of PLGA-b-PEGRGD brush block copolymers were dissolved in THF (50 mL) as solvent at room temperature. The polymer solution was injected rapidly into Dl-water (150 mL) as non-solvent under magnetic stirring at 900-1000 rpm for 30 min at room temperature.

[0417] When solvent (THF) / non-solvent (Dl-water) volume ratio increases while the amount of polymer is fixed, the size of particle increases. For example, if volume of THF is increased, the polymer concentration decreases and the particle size decreases. If the concentration of polymer solution is increased or volume of THF is decreased while the amount of Dl-water is fixed, the size of the produced polymer nanoparticles is non-uniform and larger than 400 nm.

[0418] Eg-1 ) When 1 g polymer in 50mL THF is injected into 100mL Dl-Water (solvent / non-solvent=1 / 2), size of nanoparticles is ranged from about 400nm to about few microns.

[0419] Eg-2) When 1 g polymer in 50mL THF is injected into 150mL Dl-Water (solvent / non-solvent=1 / 3), size of nanoparticles is ranged from about 200nm to about 500nm.

[0420] Eg-3) When 1 g polymer in 50mL THF is injected into 200mL Dl-Water (solvent / non-solvent=1 / 4), size of nanoparticles is ranged from about 100nm to about 300nm.

[0421] By controlling the solvent / non-solvent ratio, the size of nanoparticles can be varied from about 10nm to about 1 ,000nm.

[0422] -> The speed of polymer solution injection is also an important factor in determining particle size. When injecting a polymer solution into Dl-water, slow injection causes the formation of non-uniform particles with particle size range from about 500nm to about few microns. Therefore, it will be appreciated that, in various embodiments, polymer solution is injected into Dl-water rapidly.

[0423] 2. The dispersion of nanoparticles was sonicated further for 30min at room temperature or below. In various embodiments, the temperature of sonication bath is no higher than room temperature. It will be appreciated that, in various embodiments, performing sonication at (or using a sonication bath having) a temperature that is higher than room temperature causes the polymer nanoparticles to aggregate.

[0424] 3. THF was evaporated under reduced pressure at max 35 °C and the resulting nanoparticle dispersion was lyophilized. The average size of these nanoparticles was determined as 200 - 500 nm from DLS analysis.

[0425] 2.1. Bioactive PLGA polymeric nanoparticles

[0426] Bioactive PLGA polymer nanoparticles were prepared by reprecipitation method through tuning the concentration of bioactive amphiphilic PLGA brush block copolymers and bioactive amphiphilic PLGA brush homopolymer for skin tissue regenerative and / or anti-inflammatory materials. Bioactive polymers were dissolved in THF and precipitated into DI water while being agitated in a sonicator. The dispersion of nanoparticles was carried out by sonication for a further 20 min followed by additional stirring for another 20 min on a magnetic stirrer. THF was evaporated under reduced pressure and the resulting nanoparticle dispersion was lyophilized. The average size of these nanoparticles was determined as 300 - 500 nm from DLS analysis (FIG. 4A and FIG. 4B).

[0427] 2.2. Bioactive PCL polymeric nanoparticles

[0428] PCL-b-PEGRGD polymer nanoparticles were prepared by reprecipitation method through tuning the concentration of amphiphilic PCL-b-PEGRGD brush block copolymers. The average size of these nanoparticles was determined as 300 - 500 nm from DLS analysis (FIG. 5).

[0429] 2.3. Bioactive PLA polymeric nanoparticles

[0430] PLA-b-PEGRGD polymer nanoparticles were prepared by reprecipitation method through tuning the concentration of amphiphilic PLA-b-PEGRGD brush block copolymers. The average size of these nanoparticles was determined as 300 - 500 nm from DLS analysis (FIG. 6) 2.4. Bioactive PMMA polymeric nanoparticles

[0431] PMMA-b-PEGRGD polymer nanoparticles were prepared by reprecipitation method through tuning the concentration of amphiphilic PMMA-b- PEGRGD brush block copolymers. The average size of these nanoparticles was determined as 200 - 500 nm from DLS analysis (FIG. 7).

[0432] 2.5. Bioactive PS polymeric nanoparticles

[0433] PS-b-PEGRGD polymer nanoparticles were prepared by reprecipitation method through tuning the concentration of amphiphilic PS-b-PEGRGD brush block copolymers. The average size of these nanoparticles was determined as 200 - 500 nm from DLS analysis (FIG. 8)

[0434] Example 3: Biocompatibility of Bioactive Synthetic Polymeric Nanoparticles

[0435] To evaluate the biocompatibility of the bioactive PLGA nanoparticles, bioactive PLGA nanoparticles (2 wt%) were formulated using a base gel comprising 5 % hydroxyethyl cellulose (HEC) in PBS buffer solution (FIG. 9). FIG. 10 is a photograph showing 5 wt% HEC / PBS gel. Table 1 shows the dynamic viscosity and pH of 7.5 wt% HEC in PBS and 5 wt% HEC in PBS respectively.

[0436] Table 1. Dynamic viscosity and pH of HEC (varying wt%) in PBS

[0437] Viscosity measurement via rotational viscometer

[0438] > Spindle type: RH7

[0439] > Rotational speed: 20.0 rpm

[0440] > Temperature: room temperature The gels were tested on 3D human skin ex vivo models and cell viability was determined using the MTT assay (Table 2 and FIG. 11 ) The cell viability results showed that all the bioactive PLGA nanoparticle gels are non-irritant based on OECD Test Guideline 439 and have good biocompatibility after 72 h (Table 3 and FIG. 12). This demonstrates non-toxicity of bioactive PLGA nanoparticle gel formulation on 3D skin model. Importantly, PLGA-RGD nanoparticles showed slight cell proliferation at 72 h with 111 % viable cells relative to control of no treatment.

[0441] Table 2. 96-well sample map for Absorbance measurement at 570 nm

[0442] Table 3. Cell viability assay of bioactive PLGA nanoparticle gel formulation on 3D skin model.

[0443] Cell viability is calculated as OD value of each treated sample (ODT) based on OD value of untreated 3D skin model (ODNC).

[0444] Example 4: In Vivo Study with Atopic Dermatitis (AD) Mice Models

[0445] Biocompatibility of PLGA polymer nanoparticles (PLGA NPs) using hyaluronic acid (HA) and arginyl-glycyl-aspartic acid (RGD) as biomolecules has been established with 3D human skin ex vivo models. In this example, use of these nanoparticle formulations was tested in eczema animal models to demonstrate the application of such formulations in treatment of skin conditions. Gel formulations were improved using a new protocol where the concentration of hydroxycellulose (HEC) was reduced and the stirring of Np with HEC was longer and standardized, to improve the texture of the gel. The gel formulation was also optimized through the addition of 1 ,2-propanediol as a stabilizer that helps to prevent phase separation of the HEC and nanoparticles.

[0446] Mice with MC903-induced atopic dermatitis (AD mice) were used as in vivo eczema models. The AD mice to be tested were intentionally created with atopic dermatitis before application of gel samples, PLGA-RGD NPs gel, and control samples. The negative control is AD mice with no treatment and the positive control is AD mice treatment with hydrocortisone (HC) which is a commercially available steroid cream for eczema treatment.

[0447] 2.0 wt% and 4.0 wt% PLGA-RGD polymer NPs powders were formulated with 2 wt% HEC in PBS (pH 7.4) buffer solution and compared with hydrocortisone (HC).

[0448] FIG. 13A, FIG. 13B, FIG. 13C and FIG. 13D show the images obtained from histology (hematoxylin-eosin (H&E) staining) study of the AD mouse skin. AD mice treated with both 2 wt% PLGA-RGD gel and 4 wt% PLGA-RGD gel show reduced epidermal thickness and less immune cells infiltration in dermis compared to untreated AD mice and mice treated with HEC gel only. This indicates reduced swelling and inflammation in mice treated with the PLGA-RGD gels. Based on biocompatibility and inflammation reduction AD mice studies, it can be concluded that embodiments of the bioactive PLGA nanoparticles gel formulation are capable of reducing skin swelling and inflammation in atopic dermatitis as compared to both positive control of corticosteroid and negative control of no treatment. As shown, inflammation reduction in skin was observed, as a proxy to showing nanoparticle absorption.

[0449] Thickness of ear skin of AD mice (MC903) was recorded daily to assess skin inflammation via ear swelling (FIG. 14A and FIG. 14B). AD mice (MC 903) without treatment shows the highest skin swelling due to high inflammation and HEC gel without bioactive PLGA-RGD NP treated mice shows some reduced skin swelling compared to no treatment due to moisturizing effect of HEC gel.

[0450] 2.0 wt% and 4.0 wt% PLGA-RGD gel treated AD mice (MC903) shows good skin thickness reduction with similar rate of reduction. Although PLGA- RGD gels showed a slower reduction rate compared to AD mice treated with the commercial steroid hydrocortisone (HC), AD mice treated with 4.0 wt% PLGA-RGD gel showed significant reduction of skin thickness by Day 20 compared to hydrocortisone (HC) treated mice. This study shows more effective inflammation reduction of 4.0 wt% PLGA-RGD gel treatment compared to HC treatment.

[0451] Hydration of eczema skin is important to protect and restore the skin barrier. Transepidermal water loss (TEWL) measurement in skin of AD mice was carried out to study the amount of water evaporation through skin to external environment (FIG. 15A and FIG. 15B). Barrier function of the skin and its ability of retain moisture in the epidermis can be assessed by TEWL measurement.

[0452] TEWL measurement results showed the TEWL of 4.0 wt% PLGA-RGD gel treated mice was lowest amongst the AD mice group at Day 20. This indicates that 4.0 wt% PLGA-RGD can retain moisture in the epidermis well. In fact, TEWL of mice treated with 4 wt% PLGA-RGD showed lower TEWL than AD mice with no treatment, indicating that the gel in accordance with various embodiments disclosed herein is able to strengthen skin barrier in eczema mice. AD mice treated with HC shows significant increases in TEWL in Day 26 compared to Day 1 , indicating weakening and drying of epidermis by hydrocortisone.

[0453] FIG. 16A, FIG. 16B, FIG. 16C, FIG. 16D, FIG. 16E, FIG. 16F, and FIG. 16G show the images obtained from histology (hematoxylin-eosin (H&E) staining) study of the AD mouse skin. AD mice treated with both 2 wt% PLGA-RGD gel and 4 wt% PLGA-RGD gel show reduced epidermal thickness and less immune cells infiltration in dermis compared to untreated AD mice and mice treated with HEC gel only. This indicates reduced swelling and inflammation in mice treated with the PLGA-RGD gels.

[0454] Based on biocompatibility and inflammation reduction AD mice studies, it is concluded that the bioactive PLGA nanoparticles gel formulation prepared in accordance with various embodiments disclosed herein is capable of reducing skin swelling and inflammation in atopic dermatitis as compared to both positive control of corticosteroid and negative control of no treatment.

[0455] The examples have thus demonstrated effective treatment of eczema using PLGA-RGD nanoparticle formulations and the gel's ability to strengthen skin barrier as compared to corticosteroids. The nanoparticle formulation of PLGA-RGD in hydroxyethylcellulose and PBS buffer solution is an effective formulation for eczema treatment.

[0456] Example 5: Experimental Methods

[0457] 5.1. General procedure

[0458] Ring opening metathesis polymerization (ROMP) reactions and peptide / saccharide-based macromonomer synthesis were carried out in a vacuum atmosphere glovebox under nitrogen atmosphere. PLGA macromonomer (NPH-PLGA) synthesis were carried out using standard Schlenk line techniques under nitrogen atmosphere. NBPEG and NPH synthesis was carried out in a fume hood under atmospheric conditions, following procedures provided in PCT / SG2020 / 050621 , which is herein fully incorporated by reference. Hyaluronic acid (HA) with average Mw 3,000 - 5,000 was purchased from Glentham Life Sciences Ltd. Arginyl-Glycyl-Aspartic acid (RGD) peptide was purchased from Biomatik Inc. PEG diamine (Mw 3,400) and c / s-5-norbornene- exo-2,3-dicarboxylic anhydride (NB) were purchased from Alfa Aesar. Hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (NHS), 1 -Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDO), N,N-diisopropylethylamine ('PrzEtN) and Grubbs 2ndgeneration catalyst were purchased from Sigma Aldrich. Hydroxyethyl cellulose (HEC) (4,500-6,500 mPa.s, 2% in water at 25°C) was purchased from TCI. All solvents used in the glovebox are anhydrous and all purchased reagents were used without further purification.

[0459] 1H NMR spectra were recorded on a JEOL 500 MHz NMR spectrometer using deuterium oxide and DMSO-De as solvent for peptide / saccharide-based macromonomer. CDCh was used as solvent for PLGA macromonomer and its copolymers. Gel permeation chromatography (GPC) was carried out on a Waters Aquity APC System equipped with Acquity APC XT 45, XT 200 and XT 450 columns, Acquity Rl detector. THF was used in sample preparation and a flow rate of 0.5 ml / min at 40 °C was used.

[0460] Synthesis of NB-PEG, NB-PEGRGD, NPH-PLGA, NPH-PCL and NPH- PLA, NB-PMMA and NB-PS have been described earlier in PCT / SG2020 / 050621 , which is herein fully incorporated by reference. Synthesis of NB-PEGHA has been described earlier in PCT / SG2023 / 050288, which is herein fully incorporated by reference.

[0461] Mouse eczema treatment studies are carried out under IACLJC protocol

[0462] 211610. 5.2. Synthesis of NPH-synthetic polymer macromonomer

[0463] 5.2.1. Synthesis of NPH-PLGA macromonomer

[0464] NPH-PLGA macromonomers were prepared by ring opening polymerization (ROP). As an example, a 25 mL Schlenk tube was charged with NPH initiator (55 mg, 0.25 mmol), D, L-lactide (864 mg, 6.0 mmol), glycolide (174 mg, 1.5 mmol), Sn(0ct)2 (2 mg), and a stir bar. The tube was evacuated and backfilled with nitrogen four times, and was then immersed in an oil bath at 135 °C. After 3 h, the contents were cooled to room temperature, diluted with dichloromethane, and precipitated into cold MeOH. The mother liquor was decanted and the residue washed with MeOH, followed by drying in vacuum oven.

[0465] 1H NMR (500 MHz, CDCIs): 5 6.28 (br t, 2H), 5.27-5.08 (m, PLA), 4.85-4.65 (m, PLGA) 4.35 (m, 1 H), 4.19 - 4.02 (m, 2H), 3.62 - 3.44 (m, 2H), 3.27 (s, 2H), 2.69 (m, 2H), 1 .97-1 .47 (m, PLA), 1 .19 (d, 1 H).

[0466] 5.2.2. Synthesis of NPH-PCL macromonomer

[0467] NPH-PCL macromonomers were prepared by ring opening polymerization (ROP). As an example, e-caprolactone (6.0 mL, 54.0 mmol) was added to a 20 mL scintillation vial containing alcohol initiator (0.398 g, 1 .8 mmol) and dissolved in toluene (4 mL). Sn(Oct)2 (29 mg, 72 pmol) was added to the mixture and the resultant solution was stirred at 110 °C. After 2 h, the contents were cooled to room temperature and precipitated into MeOH. The mother liquor was decanted and the residue washed with MeOH, followed by drying in vacuum oven.

[0468] 1H NMR (500 MHz, CDCh): 56.28 (br t, 2H, NB), 4.06-4.03 (m, PCL) 3.64 (t, 2H), 3.54 (t, 2H), 3.27 (s, 2H), 2.67 (s, 2H), 2.31 -2.28 (m, PCL), 1.92-1.87 (m, 2H), 1 .66-1 .61 (m, PCL), 1 .39-1 .35 (m, PCL), 1 .22 (d, 1 H, J=9.5Hz).

[0469] 5.2.3. Synthesis of NPH-PLA macromonomer

[0470] NPH-PLA macromonomers were prepared by ring opening polymerization (ROP). As an example, a 25 mL Schlenk tube was charged with NPH initiator (150 mg, 0.677 mmol), D, L-lactide (1.95 g, 13.54 mmol), Sn(0ct)2 (6 mg), and a stir bar. The tube was evacuated and backfilled with nitrogen four times, and was then immersed in an oil bath at 125 °C. After 3 h, the contents were cooled to room temperature, diluted with dichloromethane, and precipitated into cold MeOH. The mother liquor was decanted and the residue washed with MeOH, followed by drying in vacuum oven.

[0471] 1H NMR (500 MHz, CDCh): 6 6.28 (br t, 2H, NB), 5.30-5.08 (m, 1 H, PLA), 4.38 (m, 1 H), 4.19-4.02 (m, 2H), 3.62-3.44 (m, 2H), 3.27 (s, 2H), 2.68 (m, 2H), 1.97- 1.47 (m, 3H, PLA), 1.19 (d, 1 H).

[0472] 5.3. Synthesis of HA macromonomer (NB-PEGHA)

[0473] Hyaluronic acid (HA) (100 mg, 0.025 mmol) and HOBT (5.06 mg, 0.0375 mmol) were dissolved in deionized (DI) water (10 mL), followed by addition of EDC (5.8 mg, 0.0375 mmol) and stirred for 1 hr. NBPEG3400NH2 (88.8 mg, 0.025 mmol) and 'Pr2EtN (3.87 mg, 0.03 mmol) were added to the reaction mixture and stirred at room temperature overnight. The solution was dialyzed against DI water for 48 h using 5,000 Da molecular weight cut off cellulose membrane to remove uncoupled NBPEG3400NH2 and HA. A pale yellow powder was obtained after the freeze-drying process. (Yield 90%)

[0474] 1H NMR (500MHz, D2O): 5 6.37 (t, 2H), 3.64 (s, 4H, PEG), 2.01 (s, 3H, HA)

[0475] 5.4. Synthesis of RGD macromonomer (NB-PEGRGD)

[0476] RGD (0.0937 g, 0.26 mmol), was dissolved in MeOH (2.5 ml) in a 4 ml vial, in the glovebox. 'Pr2EtN (91 pL, 0.52 mmol) was added and the mixture stirred

[0477] (A). HBTU (0.0992 g, 0.26 mmol) were dissolved in MeOH (12.5 ml) in a 20 ml vial at 40°C, followed by addition of the RGD solution from (A), to give solution

[0478] (B). Solution B is then added to NBPEG3400NH2 (0.77 g, 0.218 mmol) in a 40 ml vial and stirred at rt overnight. The resultant mixture was then evaporated and dialyzed against DI water for 48 h using 1 ,000 Da molecular weight cut off cellulose membrane to remove uncoupled NBPEG3400NH2 and RGD. A pale yellow powder was obtained after the freeze-drying process. (Yield 95 %)

[0479] 1H NMR (D2O, 500MHz): 5 = 6.37 (s, 2H, NB), 4.02 (m, RGD), 3.78 (s, RGD), 3.71 (m, PEG), 3.64 (d, 4H, J=5Hz), 3.22 (m, 4H, RGD), 3.01 (d, J=5Hz, RGD), 2.86 (s, 2H), 1 .96 (m, RGD), 1 .70 (m, RGD), 1 .51 (d, 1 H, J=10Hz).

[0480] 5.5. Synthesis of Synthetic Polymer-b-PEGRGD brush block copolymer

[0481] 5.5.1. Synthesis of PLGA-b-PEGRGD brush block copolymer

[0482] NPH-PLGA macromonomer (200 mg, 0.037 mmol) was dissolved in THF and a solution of catalyst 3 in THF (2 mol%, 0.04 mmol) was added to the solution and the mixture was stirred for 2 hr at room temperature. NB-PEGRGD (0.5 eq) in THF (0.02 M) was added to reaction mixture and stirred overnight at room temperature before the reaction was terminated by adding ethyl vinyl ether. The polymer solution was precipitated in cold methanol. The polymer mixture was centrifuged and supernatant was decanted. The residue was repeatedly washed with DI water to remove unreacted NB-PEGRGD and followed by washing with cold methanol before drying under vacuum at 45 °C overnight.

[0483] The experiment may be repeated by replacing NB-PEGRGD in THF with NB-PEGHA in DMF to obtain PLGA-b-PEGHA brush block copolymer.

[0484] 1H NMR (500MHz, DMSO-D6): 5 5.26-5.09 (m, 1 H, PLA), 4.95-4.81 (m, 2H, PGA), 3.54-3.47 (s, 4H, PEG), 1.53-1.37 (m, PLA), GPC analysis (THF): Mn = 162,724 PDI = 1.132

[0485] 5.5.2. Synthesis of PCL-b-PEGRGD brush block copolymer

[0486] NPH-PCL macromonomer (1 g, 0.2 mmol) was dissolved in THF (0.04M) and a solution of catalyst 3 in THF (3.19 mg / 100pL) added to the solution and the mixture was stirred for 2 hr at room temperature. NB-PEGRGD (0.5 eq) in DMF (0.04 M) was added to reaction mixture and stirred for 3hour at room temperature before the reaction was terminated by adding ethyl vinyl ether. The polymer solution was precipitated in cold methanol. The polymer mixture was centrifuged and supernatant was decanted. The residue was repeatedly washed with DI water to remove unreacted NB-PEGRGD and followed by washing with cold methanol before drying under vacuum at 45 °C overnight.

[0487] 1H NMR (500MHz, CDCI3): 5 4.05 (m, 2H, PCL), 3.63 (s, 4H, PEG), 2.30 (t, 2H, PCL), 1 .50-1 .70 (m, 4H, PCL), 1 .25-1 .45 (m, 2H, PCL)

[0488] 5.5.3. Synthesis of PLA-b-PEGRGD brush block copolymer

[0489] NPH-PLA macromonomer (700 mg, 0.175 mmol) was dissolved in THF (0.04M) and a solution of catalyst 3 in THF (2.5mg / 100 iL) was added to the solution and the mixture was stirred for 2 hr at room temperature. NB-PEGRGD (0.5 eq) in DMF (0.04 M) was added to reaction mixture and stirred overnight at room temperature before the reaction was terminated by adding ethyl vinyl ether. The polymer solution was precipitated in cold methanol. The polymer mixture was centrifuged and supernatant was decanted. The residue was repeatedly washed with DI water to remove unreacted NB-PEGRGD and followed by washing with cold methanol before drying under vacuum at 45 °C overnight.

[0490] 1H NMR (500MHz, CDCh): 5 5.26-5.09 (m, 1 H, PLA), 3.64 (s, 4H, PEG), 1.40- 1.70 (m, 3H, PLA)

[0491] 5.5.4. Synthesis of PMMA-b-PEGRGD brush block copolymer

[0492] NB-PMMA macromonomer (50 mg, 0.0125 mmol) was dissolved in THF (0.04M) and a solution of catalyst 3 in THF (0.14 mg / 100pL) added to the solution and the mixture was stirred for 2 hr at room temperature. NB-PEGRGD (0.5 eq) in DMF (0.04 M) was added to reaction mixture and stirred for 3hour at room temperature before the reaction was terminated by adding ethyl vinyl ether. The polymer solution was precipitated in methanol. The polymer mixture was centrifuged and supernatant was decanted. The residue was repeatedly washed with DI water to remove unreacted NB-PEGRGD and followed by washing with methanol before drying under vacuum at 45 °C overnight (Scheme 10).1H NMR (500MHz, CDCh): 3 3.64 - 3.60 (m, PMMA and PEG), 2.00 - 1 .70 (m,

[0493] PMMA), 1.1 1 - 0.73 (m, PMMA) Scheme 10. Synthesis of PMMA-b-PEGRGD brush block copolymer

[0494] 5.5.5. Synthesis of PS-b-PEGRGD brush block copolymer

[0495] NB-PS macromonomer (40 mg, 0.01 mmol) was dissolved in THF (0.04M) and a solution of catalyst 3 in THF (0.14mg / 100 iL) was added to the solution and the mixture was stirred for 2 hr at room temperature. NB-PEGRGD (0.5 eq) in DMF (0.04 M) was added to reaction mixture and stirred overnight at room temperature before the reaction was terminated by adding ethyl vinyl ether. The polymer solution was precipitated in methanol. The polymer mixture was centrifuged and supernatant was decanted. The residue was repeatedly washed with DI water to remove unreacted NB-PEGRGD and followed by washing with methanol before drying under vacuum at 45 °C overnight (Scheme 11 ).1H NMR (500MHz, CDCh): 66.99-7.15 (m, 3H, Ph), 6.4-6.8 (m, 2H, Ph), 3.64 (s, 4H, PEG), 1.93 (quintet, 1 H, PS), 1.42 (t, 2H, PS). Scheme 11 . Synthesis of PS-b-PEGRGD brush block copolymer

[0496] 5.6. Synthesis of NB-PLGA-PEG-HA and NB-PLGA-PEG-RGD macromonomer

[0497] 5.6.1. Synthesis of NB-PLGA-PEGNH2 NB-PLGA (200 mg, 0.037 mmol), NHS (13 mg, 0.113 mmol) and EDC (17.5 mg, 0.131 mmol) were dissolved in DCM (5 mL) and stirred for 1 hr at room temperature. PEG diamine (384.5 mg, 0.113 mmol) with few drops of TEA in DCM (3 mL) was added to reaction mixture and stirred for 3 hr at room temperature. DCM was removed under reduced pressure after reaction and the residue was diluted with DMSO (3 mL). The solution was dialyzed against DI water for 48 h using 7,000 Da molecular weight cut off cellulose membrane to remove uncoupled PEG diamine. A pale yellow powder was obtained after the freeze-drying process. (Yield 95%)1H NMR (500MHz, DMSO-De): 3 6.29 (s, 2H, NB), 5.26-5.06 (m, 1 H, PLA), 4.95- 4.81 (m, 2H, PGA), 3.59-3.44 (s, 4H, PEG), 1.55-1.34 (m, PLA). GPC analysis (THF): Mn = 6,300 PDI = 1.53

[0498] 5.6.2. Synthesis of NB-PLGA-PEG-HA

[0499] HA (67.2 mg, 0.016 mmol), NHS (2.6 mg, 0.016 mmol) and EDC (1 .93 mg, 0.016 mmol) were dissolved in DMSO (5mL) and stirred for 1 hr at room temperature. NB-PLGANH2 (150 mg, 0.016 mmol) in DMSO (1 mL) was added to reaction mixture and stirred for 3 hr at room temperature. The reaction solution was dialyzed against DI water for 48 h using 7,000 Da molecular weight cut off cellulose membrane to remove uncoupled HA. A pale yellow powder was obtained after the freeze-drying process. (Yield 90%)

[0500] 1H NMR (500MHz, DMSO-De): 6 6.30 (s, 2H, NB), 5.26-5.08 (m, 1 H, PLA), 4.93- 4.81 (m, 2H, PGA), 3.58-3.49 (s, 4H, PEG), 2.01 (s, 3H, HA), 1 .56-1 .38 (m, PLA)

[0501] 5.6.3. Synthesis of NB-PLGA-PEG-RGD

[0502] RGD (6 mg, 0.016 mmol), NHS (2.6 mg, 0.016 mmol) and EDC (1.93 mg, 0.016 mmol) were dissolved in DMSO (2mL) and stirred for 1 hr at room temperature. NB-PLGANH2 (150 mg, 0.016 mmol) in DMSO (1 mL) was added to reaction mixture and stirred for 3 hr at room temperature. The reaction solution was dialyzed against DI water for 48 h using 7,000 Da molecular weight cut off cellulose membrane to remove uncoupled HA. A pale yellow powder was obtained after the freeze-drying process. (Yield 90%)

[0503] 1H NMR (500MHz, DMSO-De): 6 6.30 (s, 2H, NB), 5.29-5.09 (m, 1 H, PLA), 4.95- 4.83 (m, 2H, PGA), 3.55-3.45 (s, 4H, PEG), 3.18 (d, RGD), 1.53-1.34 (m, PLA), 2.09 (m, RGD), 1.80 (m, RGD)

[0504] 5.7. Synthesis of Poly(PLGA-PEG-HA) brush homopolymer

[0505] NB-PLGA-PEG-HA macromonomer (140 mg, 0.011 mmol) was dissolved in cosolvent (4.5mL, THF:DMF=1 :2 (volume)) and stirred for 1 hr at room temperature. A solution of catalyst 3 in THF (5 mol %) was added to the solution and the mixture was stirred for 3hr at room temperature before the reaction was terminated by adding ethyl vinyl ether. The polymer solution was precipitated in cold Methanol. The polymer mixture was centrifuged and supernatant was decanted. The residue was washed repeatedly with MeOH followed by drying under vacuum. The obtained polymer is white powder.

[0506] 1H NMR (500MHz, DMSO-De): 5 5.26-5.08 (m, 1 H, PLA), 4.94-4.81 (m, 2H, PGA), 3.55-3.46 (s, 4H, PEG), 1 .90 (s, 3H, HA), 1 .51 -1 .40 (m, PLA)

[0507] 5.8. Synthesis of Polv(PLGA-PEG-RGD) brush homopolymer

[0508] NB-PLGA-PEG-RGD macromonomer (132 mg, 0.015 mmol) was dissolved in THF (0.02 M) and stirred for 1 hr at room temperature. A solution of catalyst 3 in THF (5 mol %) was added to the solution and the mixture was stirred for 3hr at room temperature before the reaction was terminated by adding ethyl vinyl ether. The polymer solution was precipitated in cold Methanol. The polymer mixture was centrifuged and supernatant was decanted. The residue was washed repeatedly with MeOH followed by drying under vacuum. The obtained polymer is white powder.

[0509] 1H NMR (500MHz, DMSO-D6): 5 5.27-5.08 (m, 1 H, PLA), 4.91 -4.79 (m, 2H, PGA), 3.55-3.45 (s, 4H, PEG), 3.17 (d, RGD), 1 .79 (m, RGD), 1 .56-1 .38 (m, PLA)

[0510] 5.9. Preparation of bioactive PLGA nanoparticle gel

[0511] Lyophilized bioactive PLGA nanoparticles (120 mg, 2 wt%) were dispersed in phosphate buffered saline (PBS, pH 7.4) (5 mL) by sonication for 10-20 min, followed by addition of hydroxyethyl cellulose (HEC) (250 mg, 5 wt%) and stirred for 30 min at room temperature until a consistent, homogeneous gel is obtained. The rheological property of gel was measured by rotational viscometer (Visco QC 100) using spindle type RH7 and rotational speed 20.0 rpm. Dynamic viscosity of 5 wt% HEC gel is 78 Pa.s. .10. Bioactive PLGA nanoparticle preparation

[0512] Bioactive PLGA polymer nanoparticles were prepared by reprecipitation method. 1 g of PLGA-RGD brush block copolymers were dissolved in THF (50 mL) and the polymer solution was injected rapidly into DI water (150 mL) while stirring at 900-1000 rpm using a magnetic stirrer for 30 min. The dispersion of nanoparticles was sonicated further for 30min. THF was evaporated under reduced pressure and the resulting nanoparticle dispersion was lyophilized. The average size of these nanoparticles was determined as 200 - 500 nm from DLS analysis.

[0513] Bioactive PLGA-RGD gels were prepared in two different concentrations, PLGA-RGD gel-1 (2 wt% PLGA-RGD NPs in gel) and PLGA-RGD gel-2 (4 wt% PLGA-RGD NPs in gel). Lyophilized bioactive PLGA nanoparticles were dispersed in phosphate buffered saline (PBS, pH 7.4) (10 mL) by sonication for 30 min, followed by addition of hydroxyethyl cellulose (HEC) (5 wt% for PLGA- RGD gel-1 and 2 wt% for PLGA-RGD gel-2) and stirred for 30 min at room temperature until a consistent, homogeneous gel is obtained.

[0514] 1 ,2-propanediol, also known as propylene glycol (PG), was used for gel formulation at to increase the stability of bioactive PLGA nanoparticles in PBS buffer. It can provide moisturizing properties to bioactive PLGA nanoparticle gel formulations, providing a smooth, dewy finish.

[0515] Bioactive PLGA nanoparticles gel formulation with various concentration of PLGA NPs were successfully scaled up from 10 mL to 100 mL. 3, 4 and 5 wt% of PLGA NPs were dispersed in phosphate buffered saline (PBS, pH 7.4) (100 mL) by sonication for 30 min, followed by addition of hydroxyethyl cellulose (HEC) (2 wt%) and stirred less than 1 hour at room temperature until a consistent, homogeneous gel is obtained. PG was then added to increase the dispersion stability of PLGA NPs in HEC gel. Specifically, PG to PBS buffer was used in 1 : 10 ratio but this ratio may be varied from 0 - 50 % (1 : 1 ). 5.11. Ear thickness measurement of mouse model

[0516] Ear thickness measurements of mice ear were done daily by calipers.

[0517] 5.12. Transepidermal water loss (TEWL) measurement of mouse model

[0518] TEWL is the amount of water that passively evaporates through the skin to the external environment. It is widely used measurement to assess the barrier function of the skin and its ability to retain moisture in the epidermis. Atopic dermatitis (AD) skin is characterized by skin barrier dysfunction and increased TEWL. In the examples, TEWL were measured with a vapometer (Delfin Technologies, Finland).

[0519] Example 6: Summary

[0520] In summary, HA and RGD based bioactive amphiphilic PLGA brush block copolymers and brush homopolymers were prepared. These bioactive amphiphilic PLGA brush polymers were fabricated into nanoparticles by reprecipitation method and formulated into gels using hydroxyethyl cellulose in PBS buffer solution. Biocompatibility studies on the gels using ex vivo human skin models showed good biocompatibility and non-toxicity of bioactive nanoparticles gel formulation. The gels may potentially be used for wound healing, reducing skin inflammation, eczema, rashes, psoriasis treatment, skin irritation management, and anti-aging. Similar synthesis and formulation methods may be used on nanoparticles created using other oligopeptides and oligosaccharides.

[0521] The examples also show that embodiments of the bioactive nanoparticle formulations disclosed herein exhibit anti-inflammatory and skin regenerative effect and can function as a non-steroidal and non-immunosuppressive agent for treating atopic dermatitis. For example, in vivo study using MC903-induced atopic dermatitis mouse model showed effective reduction of inflammation without weakening of skin barrier, which is a common side effect of steroid treatment. Advantageously, embodiments of the bioactive nanoparticle formulations may be able to replace steroid-based treatments as topical steroid usage often causes skin thinning, infection, discolorations, stretch marks (striae), easy bruising, thin spidery blood vessels (telangiectasias), worsening of acene / rosacea / perioral dermatitis and tendency of developing Cushing’s syndrome. It will be appreciated by a person skilled in the art that other variations and / or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.

Claims

CLAIMS1 . A polymeric nanoparticle comprising:(i) a hydrophobic synthetic polymer core; and (ii) a hydrophilic bioactive shell, wherein the hydrophobic synthetic polymer core and hydrophilic bioactive shell are parts of a bioactive synthetic polymer with a poly(norbornene-dicarboximide) backbone having one or more repeating units selected from the group consisting of general formula (1), (2) and (3):and whereinR1and R4are each independently selected from a single bond, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl or optionally substituted alkylcarbonylalkyl;R2and R6are each optionally substituted alkyl;R3, R5and R7are each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl;L1and L2are each heteroalkylene;X1and X2each independently comprise a bioactive moiety selected from the group consisting of proteins, peptides, oligopeptides, carbohydrates, oligosaccharides, sugar, collagen, hyaluronic acid, therapeutic / drug molecules and derivatives thereof;Y1and Y2each independently comprise a synthetic polymer selected from the group consisting of poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid-co-lactic acid) (PGLA), poly(glycolic acid) (PGA), polystyrene (PS), poly(lactic acid) (PLA), poly(caprolactone) (PCL), polyacrylates, poly(meth)acrylates, polyesters, derivatives thereof and parts thereof; and Z1, Z2and Z3are each independently selected from CRaRb, O, NRa, SiRaRb, PRaor S, wherein Raand Rbare each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl and optionally substituted alkynyl.

2. The nanoparticle of claim 1 , wherein the hydrophilic bioactive shell is at least 1 wt% of the nanoparticle.

3. The nanoparticle of any one of the preceding claims, wherein the hydrophobic synthetic polymer core is at least 10 wt% of the nanoparticle.

4. The nanoparticle of any one of the preceding claims, wherein the nanoparticle has an average size falling in the range of from 10 nm to 1 ,000 nm.

5. The nanoparticle of any one of the preceding claims, wherein the repeating units represented by general formula (1 ); (2); and / or (3) are sequentially distributed within the bioactive synthetic copolymer.

6. The nanoparticle of any one of the preceding claims, wherein L1and L2are each polyethylene glycol (PEG).

7. The nanoparticle of any one of the preceding claims, wherein R1and R4are each independently selected from C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C1-C20 alkoxyalkyl, C2-C20 alkylcarbonyl or C3-C20 alkylcarbonylalkyl; R2and R6are each independently selected from straight or branched C1-C20 alkyl; and R3, R5and R7are each independently selected from H, C1-C20 alkyl, C2-C20 alkenyl or C2-C20 alkynyl.

8. A method of preparing a plurality of polymeric nanoparticles as claimed in any one of claims 1 to 7, the method comprising:(a-i) mixing one or more bioactive synthetic polymer(s) with a poly(norbornene-dicarboximide) backbone having one or more repeating units selected from the group consisting of general formula (1), (2) and (3) in a suitable organic solvent to obtain a solution;(a-ii) precipitating polymeric nanoparticles from the solution into a nonsolvent to obtain a dispersion of polymeric nanoparticles; and(a-iii) removing the organic solvent and non-solvent from the dispersion to obtain polymeric nanoparticles.

9. The method of claim 8, wherein the volume ratio of the organic solvent to the non-solvent is from 1 :1 to 1 :10.

10. The method of any one of claims 8 to 9, wherein the step (a-i) comprises adding from 0.1 wt% to 50 wt% of the polymers.11 . The method of any one of claims 8 to 10, wherein the step (a-ii) comprises adding the solution obtained from (a-i) into a non-solvent at a rate of from 0.1 mL / s to 5 mL / s.

12. The method of any one of claims 8 to 11 , wherein the organic solvent is selected from tetrahydrofuran (THE), benzene, toluene, acetonitrile (ACN), dichloromethane (DCM), dimethyl sulfoxide (DMSO), acetone, methyl ethylketone (MEK), ethyl vinyl ether, methanol, ethanol, butanol and combinations thereof.

13. The method of any one of claims 8 to 12, wherein the non-solvent comprises an aqueous medium.

14. The method of any one of claims 8 to 13, wherein the method further comprises, prior to the step (a-iii) of removing organic solvent and non- solvent / aqueous medium, a step of sonicating the dispersion of nanoparticles under one or more of the following conditions:(i) at a temperature that is no more than 35°C;(ii) over a time duration of from 1 minute to 24 hours; and(iii) at a stirring rate / speed of from 100 rpm to 5,000 rpm.

15. A polymeric nanoparticle composition comprising: a dispersion of a plurality of polymeric nanoparticles as claimed in any one of claims 1 to 7.

16. The polymeric nanoparticle composition of claim 15 further comprising:(i) a buffer solution;(ii) a thickening agent; and(iii) optionally a stabilizer.

17. The composition of claim 16, wherein the thickening agent is selected from the group consisting of hydroxyethyl cellulose (HEC), xanthan gum, hydroxypropyl cellulose and combinations thereof.

18. The composition of any one of claims 16 to 17, wherein the stabilizer is selected from the group consisting of propylene glycol, glycerol, glycerin and combinations thereof.

19. The composition of any one of claims 15 to 18, wherein the composition is a topical skin formulation.

20. The composition of claim 19, wherein the topical skin formulation is in the form of a liquid, gel or cream formulation.21 . A composition of any one of claims 15 to 20 for use in stimulating skin and / or tissue regeneration.

22. A composition of any one of claims 15 to 20 for use in the treatment of a skin condition.

23. Use of a composition of any one of claims 15 to 20 in the manufacture of a medicament for stimulating skin and / or tissue regeneration.

24. Use of a composition of any one of claims 15 to 20 in the manufacture of a medicament for treatment of a skin condition.

25. A method of stimulating skin and / or tissue regeneration in a subject in need thereof, the method comprising applying the composition of any one of claims 15 to 20 to a body part of the subject in need thereof.

26. A method of treating a skin condition, the method comprising applying the composition of any one of claims 15 to 20 to a body part of a subject in need thereof.

27. The composition of claim 22, the use of claim 24 or the method of claim 26, wherein the skin condition is selected from the group consisting of skin wounds, skin inflammation, psoriasis, rashes, aging skin eczema, atopic dermatitis / eczema, contact dermatitis, allergic dermatitis, dyshidrotic eczema, neurodermatitis, nummular eczema, seborrheic dermatitis, stasis dermatitis, and combinations thereof.

28. A bioactive synthetic macromonomer represented by general formula (6) for preparing a bioactive synthetic polymer:wherein R4is selected from a single bond, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyalkyl, optionally substituted alkylcarbonyl or optionally substituted alkylcarbonylalkyl;R5and R7are each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl;R6is optionally substituted alkyl;L2is heteroalkylene;X2comprises a bioactive moiety selected from the group consisting of proteins, peptides, oligopeptides, carbohydrates, oligosaccharides, sugar, collagen, hyaluronic acid, therapeutic / drug molecules and derivatives thereof;Y2comprises a synthetic polymer selected from the group consisting of poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid-co-lactic acid) (PGLA), poly(glycolic acid) (PGA), polystyrene (PS), polyflactic acid) (PLA),poly(caprolactone) (PCL), polyacrylates, polyesters, derivatives thereof and parts thereof; andZ3is selected from CRaRb, O, NRa, SiRaRb, PRaor S, wherein Raand Rbare each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl and optionally substituted alkynyl.

29. A method of preparing a bioactive synthetic macromonomer represented by general formula (6) of claim 30, the method comprising:(e-i) reacting a synthetic macromonomer represented by general formula (7) with a diamine represented by general formula (8) to obtain an amine represented by general formula (9):whereinR9is selected from H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl.(e-ii) reacting said amine represented by general formula (9) with an acidcontaining bioactive moiety (e.g., X-C(=O)OH) to obtain thebioactive synthetic macromonomer represented by general formula (6).

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