WO2021108853A1

WO2021108853A1 - Polymer nanoparticles

Info

Publication number: WO2021108853A1
Application number: PCT/AU2020/051311
Authority: WO
Inventors: Chunxia ZHAO; Yun Liu; Guangze YANG
Original assignee: The University Of Queensland
Priority date: 2019-12-02
Filing date: 2020-12-02
Publication date: 2021-06-10

Abstract

The present invention relates to a method of forming a polymer nanoparticle encapsulating an active agent including the steps of: (a) dissolving at least one polymer and at least one active agent in at least one organic solvent to form an active solution; (b) selecting the salt concentration of an aqueous salt solution such that it is suitable to cause, upon contact with the active solution, the active agent to precipitate from solution substantially simultaneously with or prior to precipitation of the polymer; and (c) adding the aqueous salt solution of step (b) to the active solution to precipitate the active agent and the polymer; to thereby form a polymer nanoparticle encapsulating an active agent. The invention also relates to a polymer nanoparticle when produced by the method, a method of delivering an active agent to a target, and a method of prevention or treatment.

Description

POLYMER NANOPARTICLES FIELD OF THE INVENTION

[0001 ] The invention relates to the field of polymer particles as delivery agents. More particularly, this invention relates to a method of synthesising a polymer nanoparticle encapsulating an active agent, the active agent-encapsulated polymer nanoparticle thereby produced and its use in delivery of said active agent.

BACKGROUND TO THE INVENTION

[0002] Any reference to background art herein is not to be construed as an admission that such art constitutes common general knowledge in Australia or elsewhere.

[0003] Drug-loaded polymer nanoparticles may be considered as a polymeric nanoparticle with at least two defined regions and with one region, the core, more or less encapsulated by another region, the polymer shell. They can provide for improved properties and ensuing benefits not attainable from either component individually.

[0004] In biomedical applications the core region may be an active agent to be delivered to a patient. In such applications, polymer nanoparticles can be particularly useful in formulating poorly soluble drugs for improved bioavailability, safety, tolerability and efficacy. Approximately 40% of approved drugs and 90% of pipeline drugs exhibit poor water solubility, and so there is a significant need for new systems, such as polymer nanoparticles, which can deliver hydrophobic drugs to the appropriate biological sites at adequate therapeutic levels.

[0005] One of the major challenges hindering the practical application of most nanoparticle delivery systems is the low drug loading typically achieved. Among many nano-systems, drug loading is usually below 10% and polymeric nanoparticles having drug loading lower than 5% or even less than 1 % are not uncommon.

[0006] The drug loading is strongly impacted by the method of formation of the polymer nanoparticle. A variety of approaches have been tested in the art with emulsion polymerisation, dispersion polymerisation and precipitation polymerisation being three of the most common. [0007] Nanoprecipitation, is perhaps the most straightforward method for preparing drug-loaded polymer nanoparticles. Typically, a polymer and a drug are dissolved in an organic solvent, and then this solution is rapidly added to an ‘antisolvent’, for example water, to form the drug-loaded polymer nanoparticles. Fast mixing, to encourage a short precipitation time, is suitable for making uniform and monodispersed nanoparticles. The drug loading efficiency (DLE: drug mass / drug-loaded polymer nanoparticle mass) of the polymer nanoparticles using such an approach is generally low, with most systems offering under 5% DLE. This is mainly due to the significant difference in the precipitation time of the drug and the polymer which means that, if the drug precipitates significantly faster than the polymer, the precipitated drug will form larger aggregate structures leading to the subsequent formation of polymeric nanoparticles with very low drug loading. Clearly if the polymer precipitates prior to the drug then drug loading will be extremely low.

[0008] The applicant has addressed this problem, in one manner, as described in PCT application no. PCT/AU2019/050557 the contents of which are hereby incorporated by reference in their entirety.

[0009] The solution presented in PCT/AU2019/050557 is based on the insight that the timing of the precipitation of an active agent and polymer from solution, following contact with an antisolvent, can be controlled by use of an organic solvent system comprising two or more organic solvents. This allows for a high degree of control, as compared with the use of a single solvent, and enables the active agent to be precipitated more or less simultaneously with, or preferably just prior to, precipitation of the polymer.

[0010] It would be desirable to provide an approach to the synthesis of polymer nanoparticles encapsulating an active agent formed in a controlled manner to optimise drug loading for the delivery of active agents, such as drug molecules, which ameliorates, overcomes or circumvents one or more of the problems of the approaches of the prior art or at least provides a useful alternative. SUMMARY OF INVENTION

[0011] According to a first aspect of the invention, there is provided a method of forming a polymer nanoparticle encapsulating an active agent including the steps of:

(a) dissolving at least one polymer and at least one active agent in at least one organic solvent to form an active solution;

(b) selecting the salt concentration of an aqueous salt solution such that it is suitable to cause, upon contact with the active solution, the active agent to precipitate from solution substantially simultaneously with or prior to precipitation of the polymer; and

(c) adding the aqueous salt solution of step (b) to the active solution to precipitate the active agent and the polymer; to thereby form a polymer nanoparticle encapsulating an active agent.

[0012] A second aspect of the invention resides in a polymer nanoparticle, encapsulating an active agent, when produced by the method of the first aspect.

[0013] A third aspect of the invention resides in a method of delivering an active agent to a target by administering or contacting a polymer nanoparticle of the second aspect to or with the target.

[0014] A fourth aspect of the invention resides in a method of preventing or treating a disease or condition including the step of administering a therapeutically effective amount of a polymer nanoparticle of the second aspect to a subject in need thereof.

[0015] A fifth aspect of the invention resides in the use of a polymer nanoparticle of the second aspect in the manufacture of a medicament for the treatment of a disease or condition.

[0016] A sixth aspect of the invention resides in a polymer nanoparticle of the second aspect for use in preventing or treating a disease or condition.

[0017] The various features and embodiments of the present invention, referred to in individual aspects above apply, as appropriate, to other aspects, mutatis mutandis. Consequently, features specified in one aspect may be combined with features specified in other aspects, as appropriate.

[0018] Further features and advantages of the present invention will become apparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order that the invention may be readily understood and put into practical effect, preferred embodiments will now be described by way of example with reference to the accompanying figures wherein:

[0020] FIG 1 is a schematic indicating the approach of the present invention whereby a salt solution, having been optimised in a process of selecting the optimal salt concentration to achieve either simultaneous precipitation of active agent and polymer or precipitation of polymer just subsequent to precipitation of the active agent, is added to an active solution comprising the active agent and polymer to form drug-loaded polymer nanoparticles;

[0021] FIG 2 is a series of schematic representations and images of a salt concentration screening method including precipitation diagrams of a drug and a polymer; (a) employing a sub-optimal salt concentration whereby the polymer precipitates significantly faster than the drug to form empty polymer nanoparticles with the drug precipitating subsequently to form drug aggregates due to the lack of a stabilising polymer coating; (b) employing an optimal salt concentration whereby the drug precipitates to form drug nanoparticles just prior to the polymer precipitating to coat and stabilize the drug nanoparticles, resulting in high drug loading polymer nanoparticles;

[0022] FIG 3 is a series of graphical images showing dynamic light scattering (DLS) results of the analysis of curcumin (CCM) -loaded nanoparticles formed by testing of different concentrations of PBS solutions and, particularly; (a1 ) CCM-loaded single-polymer PLGA-io_k-PEGs_k (10k) nanoparticles with 50% drug-loading in DMF precipitated with a PBS concentration from Ox to 10^c (broad range) after 7 hours; (a2) CCM-loaded single-polymer PLGA-io_k-PEGs_k NPs of 50% drug-loading in DMF precipitated with a PBS concentration from 3.6^c to 4.5^c (narrow range) after 1 day; and (b) CCM-loaded single-polymer PLGA-io_k-PEGs_k nanoparticles with 50% drug-loading in DMSO precipitated with PBS concentrations from Ox to 10^c after 1 day. PBS concentrations highlighted with a square represent optimal PBS concentrations. Samples observed with large drug aggregates were recorded as those having a PDI=1 and particle size (Z-average) =1000 nm. Black bars represent Z-average values and white bars represent PDI values;

[0023] FIG 4 is a TEM image of the curcumin (CCM) loaded nanoparticles of FIG 3 formed from PLGA-io_k-PEGs_k, DMF, 4^c PBS salt solution concentration and showing 50% drug loading;

[0024] FIG 5 is a series of graphical images showing DLS results of the analysis of ibuprofen-loaded nanoparticles formed by testing of different concentrations of PBS solutions and, particularly; (a) Ibuprofen-loaded single-polymer PLGA-io_k-PEGs_k nanoparticles with 40% drug-loading in DMF precipitated with PBS concentrations from Ox to 20x after 1 day; (b) Ibuprofen-loaded single-polymer PLGAss_k-PEGs_k (55k) nanoparticles with 40% drug-loading in DMF precipitated with PBS concentrations from Ox to 20 x after 1 day;

[0025] FIG 6 is a series of graphical images showing DLS results of the analysis of ketamine-loaded nanoparticles formed by testing of different concentrations of PBS solutions and, particularly; (a) ketamine-loaded single-polymer PLGA-io_k-PEGs_k nanoparticles with 50% drug-loading in DMF precipitated with PBS concentrations from Ox to 20x after 1 day; (b) ketamine-loaded single-polymer PLGAss_k-PEGs_k nanoparticles with 50% drug-loading in DMF precipitated with PBS concentrations from Ox to 20^c after 1 day;

[0026] FIG 7 is a graphical image showing DLS results of the analysis of Dil-loaded nanoparticles (Dil is also known as DilCis(3) and is a fluorescent lipophilic cationic indocarbocyanine dye) formed by testing of different concentrations of PBS solutions with single-polymer PLGA-io_k-PEGs_k nanoparticles with 40% drug-loading in DMF precipitated with PBS concentrations from Ox to 20^c after 1 day; [0027] FIG 8 is a graphical image showing DLS results of the analysis of Amphotericin B (AMB)-loaded nanoparticles formed by testing of different concentrations of PBS solutions with single-polymer PLGAss_k-PEGs_k nanoparticles with 50% drug-loading in DMSO precipitated with PBS concentrations from Ox to 20^c after 1 day;

[0028] FIG 9 is a graphical image showing DLS results of the analysis of Paclitaxel (PTX)-loaded nanoparticles formed by testing of different concentrations of PBS solutions with double-polymer PLGA-io_k-PEGs_k/shellac (SH; w:w = 4:1 ) nanoparticles with 40% drug-loading in DMF precipitated with PBS concentrations from Ox to 10^c after 1 day;

[0029] FIG 10 is a series of graphical images showing DLS results of the analysis of docetaxel (DTX)-loaded nanoparticles formed by testing of different concentrations of PBS solutions and, particularly; single-polymer PLGA-io_k-PEGs_k (a) and PLGAss_k-PEGs_k (b) nanoparticles of 40% drug-loading in DMF precipitated with PBS concentrations from Ox to 20x after 1 day; DTX-loaded double-polymer PLGA-io_k-PEGs_k/shellac (SH; w:w = 4:1 ) nanoparticles (c) and PLGAss_k-PEGs_k/shellac (SH; w:w=4:1 ) nanoparticles (d) of 40% drug-loading in DMF precipitated with PBS concentrations from Ox to 20^c after 1 day (e) DTX-loaded double-polymer PLGA-io_k-PEGs_k/shellac (SH; w:w = 4:1 ) nanoparticles of 40% drug-loading in DMSO precipitated with PBS concentrations from Ox to 10^c after 1 day;

[0030] FIG 11 is a series of graphical images showing DLS results of the analysis of docetaxel (DTX)-loaded nanoparticles formed by testing of different concentrations of PBS solutions and, particularly; (a) DTX-loaded single-polymer PLGA-io_k-PEGs_k (a1 ) and PLGAss_k-PEGs_k (a2) nanoparticles of 50% drug-loading in DMF precipitated with PBS concentrations from Ox to 20^c after 1 day; (b) DTX-loaded single-polymer PLGAio_k-PEGs_k nanoparticles of 60% drug-loading in DMF precipitated with PBS concentrations from Ox to 20^c (b1 ) broad range screening, and with PBS concentrations from 9.2x to 10.8x (b2) narrow range screening based on the result of (b1 ) after 1 day; (c) DTX-loaded single-polymer PLGA-io_k-PEGs_k NPs of 70% drug-loading in DMF precipitated with PBS concentrations from Ox to 20^c after 1 day; and [0031] FIG 12 is a TEM image of DTX-loaded nanoparticles formed using - PLGAio_k-PEG5_k, DMF, 4* PBS and demonstrating 50% drug loading.

DETAILED DESCRIPTION

[0032] The present invention is predicated, at least in part, on the finding that the timing of the precipitation of an active agent and polymer from an organic solvent solution, following contact with a salt solution, can be effectively controlled by selection of the concentration of the salt in the salt solution. This selection step allows for a high degree of control and tailoring of the approach to a wide variety of polymer and active agent combinations and enables the active agent to be precipitated more or less simultaneously with, or, preferably, just prior to precipitation of the polymer. This is a preferred sequence as the precipitated active agent is available for encapsulation but has not been precipitated for a sufficient amount of time to form larger active agent-aggregate structures, which can be detrimental to the formation of highly loaded polymer nanoparticles. Larger drug nanoparticles may also be sub-optimal for drug delivery applications when subsequently encapsulated in the polymer nanoparticles.

[0033] It is also important that the salt solution is added to the organic solvent comprising the dissolved active agent and polymer (the active solution), rather than the other way around. Put another way, when the first portion of salt solution makes contact with the active solution, the active solution should be of a greater volume than that initial portion of the salt solution. That is, the active solution is the bulk solution to which the salt solution is to be added. This has been found to assist in minimising aggregation of the active agent prior to encapsulation within the polymeric nanoparticle and so to result in higher loading of the formed nanoparticles. It also avoids what can otherwise be instantaneous precipitation of both active agent and polymer in an uncontrolled manner.

[0034] In this patent specification, adjectives such as first and second, left and right, front and back, top and bottom, etc., are used solely to define one element or method step from another element or method step without necessarily requiring a specific relative position or sequence that is described by the adjectives unless such is clear from the context.

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as would be commonly understood by those of ordinary skill in the art to which this invention belongs.

[0036] As used herein, the term “polymer nanoparticle” refers generally to a polymeric particle having an active agent substantially surrounded with a polymeric layer. In certain embodiments the particle size/diameter of the polymer nanoparticle may be between 10 to 1000 nm, more preferably between about 15 to 500 nm, even more preferably between about 20 to 300 nm. In preferred embodiments, the polymer nanoparticles encapsulating the active agent will substantially fall within the 25 to 200 nm size range. In embodiments, the active agent will be in nanoparticulate form. In certain embodiments, the active agent will be completely encapsulated by the polymer. In embodiments, the polymer nanoparticle produced according to the method of the invention may have a polydispersity index (PDI) of less than 0.2.

[0037] In one embodiment, the polymer nanoparticle is a core shell polymer nanoparticle. In another embodiment, the polymer nanoparticle is a single core shell polymer nanoparticle. In a further embodiment, the polymer nanoparticle is a multi-core shell polymer nanoparticle.

[0038] The term “active agent” will be used herein largely to refer to therapeutic agents, fluorescent or radio-labelled agents, and particularly to small molecule therapeutic drugs. It will be appreciated, however, that any compound which can be precipitated to form nano-sized particles capable of being encapsulated by self-assembly of the precipitated polymer may be suitable for use as the active agent. This means that industrial chemicals such as anti-oxidants, anti-fouling agents and the like, which may be added to paints and other industrial formulations, may be used as the active agent and such agents and their encapsulation using the present method are explicitly considered within the scope of the present invention. [0039] As used herein, the word “mixing” may refer to any means of causing agitation, perturbation, blending or other dynamic movement of the active solution with the antisolvent during the mixing leading to precipitation. Stirring, pipette mixing, injection, continuous flow techniques, micromixing and mechanical mixing are preferred means of agitating the fluids although, sonication, shaking and other means may be acceptable.

[0040] In a first aspect of the invention, there is provided a method of forming a polymer nanoparticle encapsulating an active agent including the steps of:

[0041] In embodiments wherein the polymer nanoparticle is to be delivered to a subject, the polymer may be a natural or synthetic biocompatible polymer.

[0042] The natural polymer may be a resin.

[0043] In one embodiment, the resin may be shellac or rosin.

[0044] In embodiments, the polymer may be a block copolymer.

[0045] In certain embodiments, the polymer may be an amphiphilic block copolymer.

[0046] In further embodiments, the polymer may be an amphiphilic di-block copolymer.

[0047] In embodiments, the polymer is not water soluble.

[0048] The natural polymer may comprise polyhydroxy acids and/or esters and/or polyesters thereof. [0049] The polymer may be formed from monomers selected from the group consisting of lactic acids, glycolic acids, lactide, glycols, alkene oxides, acrylates, hydroxyalkanoates, terephthalates, and succinates.

[0050] The polymer may be or may comprise a polymer selected from the group consisting of poly(lactide-co-glycolide)-b-poly(ethylene glycol), shellac, PLGA, poly(D,L-lactide)-b-poly(ethylene glycol), poly(L-lactide)-b-poly(ethylene glycol), poly(caprolactone)-b-Poly(ethylene glycol), poly(acrylic acid), poly(ethylene oxide), polyethylene glycol), poly(methyl methacrylate), polystyrene, poly(pyridyldisulfide ethylmethacrylate), poly(N-isopropylacrylamide), poly(methacrylic acid), poly(lactic-co-glycolic acid), polylactic acid, polyglycolic acid, polycaprolactone, polylysine, polyglutamic acid, polyarginine, polylysine, polyhistidine, poly-ornithine, polyethyleneimine, polypropyleneimine, poly(allylamine), polystyrene-maleic acid, gelatin, polycrotonic acid, polyaspartic acid, hyaluronic acid, alginic acid, polystyrene sulfonate, carrageenan, poly(methylene-co-guanidine), polyphosphoric acid, pamidronic acid, polycarbophil, poly(methylvinyl ether-co-maleic anhydride), shellac, agar, pectin, polyvinyl acetate phthalate, guar gum, polyethylene glycol, polydextrose, poly-L lactide (PLLA), poly-DL-lactide (PDLLA), L-lactide/DL-lactide copolymers, lactide/tetramethyl-glycolide copolymers, poly-valerolacton (PVL), poly-hydroxy butyrate (PHB), poly vinyl alcohol (PVA) poly-hydroxyvalerate (PHV), polyvinylpyrrolidone (PVP), pollulan, and blends thereof.

[0051] In certain embodiments, the polymer may be selected from the group consisting of poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), poly(lactic-co-ethylene glycol) (PLA-PEG), poly(lactic-co-glycolic acid)ethylene glycol (PLGA-PEG) and shellac. References to poly(lactic acid) within a homopolymer or copolymer context include both D- and L- forms either separately or within the one polymer.

[0052] In embodiments, the polymer dissolved in the active solution may be 2 or more individual polymers selected from any of those classes and examples discussed above. The polymers may be in any ratio from 10: 1 to 1 : 10 including 6:1 to 1 :6. For example, in an active solvent comprising PLGA-PEG and a second polymer then the ratio may be from 6:1 PLGA-PEG: second polymer to 1 :6 PLGA-PEG:second polymer. The second polymer may be selected from those previously discussed and including, for example, shellac, PLGA, PLLA, PLA, polycaprolactone (PCL), PCL-PEG and PLLA-PEG.

[0053] In certain embodiments, the method may include the step of selecting a molecular weight range of the polymer. Many commercially available polymers are provided in specified molecular weight ranges. The molecular weight of the polymer may affect its precipitation time within the solvent system. For example, in certain solvents PLGA with a lower molecular weight has a longer precipitation time. Selecting the molecular weight range of the polymer can therefore provide a further layer of control in the present approach.

[0054] In one embodiment, the active agent may be selected from the group consisting of a small molecule drug, a chemotherapy drug, a radiotherapy drug, a photodynamic therapy drug, an anesthetic, an anti-inflammatory, and an imaging agent.

[0055] In particular embodiments, the active agent may be selected from the group consisting of an anti-infective, antimalarial, antiviral, antibiotic, antifungal, antioxidant, antiprotozoal, antineoplastic, cardiovascular agent, antihypertensive, analgesic, anticoagulant, antidepressant, antiarthritic, antipsychotic, neuroprotective, radiologic, respiratory agent, anti-cancer, anti-migraine, a fluorescent agent, a radio-labelled agent and antipyretic.

[0056] In embodiments, the active agent may be selected from taxol (paclitaxel), taxol derivatives including docetaxel, doxorubicin, bulleyaconitine A, amphotericin B, scutellarin, quercetin, silibinin, oleanolic acid, betulinic acid, honokiol, camptothecin, camptothecin derivatives, curcumin and curcumin derivatives, ibuprofen and ketamine.

[0057] It will be appreciated that the active agent, when it is a therapeutic molecule, may be a pharmaceutically acceptable prodrug, salt or ester or isomer or derivative of the biologically active molecule.

[0058] In certain embodiments, the active agent is a hydrophobic active agent. Put another way, in certain embodiments, the active agent is non-polar. [0059] Therefore, in embodiments, the active agent will have poor water solubility. The level of water solubility can be tested by well-known means of finding out the amount of the substance which can dissolve in water at a given temperature. In one embodiment, ‘poor water solubility’ may be considered to be attributed to any substance which requires greater than 50, preferably greater than 100, more preferably greater than 500, even more preferably greater than 1000, such as greater than 5000, mass parts of water to dissolve one part of said substance.

[0060] In certain embodiments, the active agent has a molecular mass of less than 5,000 Daltons, or less than 3,000 Daltons or less than 2,000 Daltons, or less than 1000 Daltons, and in another embodiment, the active agent has a molecular mass of less than 950 or 850 Daltons. Any of these values may be coupled with a lower molecular mass value of 20, 30 or 50 Daltons to form a molecular mass range such as 20 to 5,000, 20 to 3,000, 20 to 2,000, 20 to 1000, 20 to 950 or 20 to 850 Daltons.

[0061] The w/w ratio of the active agent to the polymer (being the total polymer content) in the active solution may be between 5:1 to 1 :5. Suitably, the w/w ratio of the active agent to the polymer is between 3:1 to 1 :3 including 2:1 to 1 :2 and is preferably about 1 :1.

[0062] It will be appreciated that, in certain embodiments, the active agent may comprise two or more active agents including 2, 3 or 4 active agents within the nanoparticle. This may be useful in a multi-drug administration treatment. Generally, however, it is preferred that a single active agent is present in the active solution when the nanoparticles are formed.

[0063] It will further be appreciated that the polymer nanoparticles are formed in the presence of the organic solvent in which the polymer and active agent were dissolved. That is, it is not necessary or desirable to remove the organic solvent from the active solution or the mixed active solution and aqueous salt solution of step (c) for the final polymer nanoparticles encapsulating the active agent to form.

[0064] Additionally, the present method does not require the formation of a drug-polymer emulsion prior to formation of the polymer nanoparticles encapsulating the active agent. Therefore, the steps of the present method, such as step (c), do not necessarily pass through and do not require a drug-polymer emulsion to form to generate the polymer nanoparticles encapsulating the active agent.

[0065] The active agent may be present in the active solution at a concentration between about 1 g/L to about 50 g/L, preferably between about 1 g/L to about 40 g/L, more preferably between about 1 g/L to about 30 g/L, even more preferably between about 1 g/L to about 20 g/L. Such ranges include between about 2 g/L to about 50 g/L, preferably between about 2 g/L to about 40 g/L, more preferably between about 2 g/L to about 30 g/L, even more preferably between about 2 g/L to about 20 g/L and between about 3 g/L to about 50 g/L, preferably between about 3 g/L to about 40 g/L, more preferably between about 3 g/L to about 30 g/L, even more preferably between about 3 g/L to about 20 g/L inclusive of between about 3 g/L to about 10 g/L. The polymer may be present in the active solution at the same concentration ranges.

[0066] It will be understood that, as the active solution is formed from at least one organic solvent, the active solution will generally not comprise any significant amounts of water or another non-organic solvent. It is preferred that the active solution is formed from the dissolution of the polymer and active agent in a suitable single organic solvent.

[0067] The nature of the organic solvent may largely be chosen based on the active agent and polymer both having an appropriate level of solubility in said solvent.

[0068] The organic solvent in which the polymer and active agent are dissolved to form the active solution may be selected from the group consisting of a formamide, a sulfoxide, an alcohol, a nitrile, an aliphatic ether, a cyclic ether, an ester, an alkane, a haloalkane, an amine, a ketone and an aromatic.

[0069] The organic solvent may be selected from the group consisting of DMSO, DMF, methanol, ethanol, and acetone.

[0070] As discussed, to achieve drug-encapsulated polymer nanoparticles, the aim is generally to match the precipitation time of the drug and the polymer, although ideally the present inventors have found that the drug should precipitate slightly earlier than the polymer, so that before the small drug particles assemble into larger aggregates they can be stabilized by the formation of the self-assembled polymer layer on and around the drug nanoparticles. This approach is indicated schematically in FIG 1 whereby an optimal salt concentration (in this instance a PBS solution) is selected and this salt solution added to the active solution to form an optimal drug-loaded nanoparticle.

[0071] FIG 2 indicates the importance of employing the correct salt concentration for the aqueous salt solution, for the method of the first aspect. The graph and images under (a) indicate the outcome when an inappropriate salt concentration is chosen and the polymer precipitates prior to the drug and so empty polymer nanoparticles are formed with the drug subsequently precipitating and forming larger drug aggregate structures. This is apparent from the photograph showing clumps of these structures having precipitated out of solution while the electron micrograph shows the polymer nanoparticles, without significant drug encapsulation, and the aggregate structures subsequently formed by the drug. The photograph shows the clumps of aggregates. The graph and images under (b) indicate the outcome according to the first aspect when an optimal salt concentration is chosen and the drug precipitates just prior to the polymer. This allows the drug to form nanoparticle structures but there is insufficient time for larger aggregates to form before the polymer coats and stabilises the drug nanoparticles to form the polymer nanoparticles encapsulating the drug with a high loading.

[0072] To achieve this optimal outcome, the step of selecting the salt concentration of the aqueous salt solution such that it is suitable to cause, upon contact with the active solution, the active agent to precipitate from solution substantially simultaneously with or just prior to precipitation of the polymer is a critical step.

[0073] The salt concentration of the aqueous salt solution may be selected such that the resulting polymer nanoparticles encapsulating the active agent have a polydispersity index (PDI) of less than 0.4, preferably less than 0.3 and more preferably less than 0.2.

[0074] Additionally, or alternatively, the salt concentration of the aqueous salt solution may be selected such that the resulting polymer nanoparticles encapsulating the active agent have an average particle diameter of less than 500 nm, preferably less than 400 nm, more preferably less than 300 nm, even more preferably less than 250 nm and more preferably still less than 200 nm.

[0075] The PDI and nanoparticle diameters may be measured by approaches which are well known in the art and including dynamic light scattering (DLS) and nanosight particle analysis.

[0076] The step of selecting the salt concentration of the aqueous salt solution may be a step of selecting between at least two differing salt concentrations. The step of selecting the salt concentration of the aqueous salt solution may be a step of selecting between more than 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 differing salt concentrations.

[0077] The step of selecting the salt concentration of the aqueous salt solution may comprise contacting a plurality of individual isolated portions of the relevant active solution with salt solutions of differing concentrations to identify an optimised salt solution concentration. It is this optimised salt solution which is then used in step (c) to precipitate the polymer and active agent, from the bulk active solution, and form the polymer nanoparticles encapsulating the active agent. In embodiments, more than one optimised salt solution may be identified for particular active agent/polymer combinations. That is, an optimised salt solution may be salt solutions of two or more different concentrations which bring about substantially the same optimal result in terms of the polymer nanoparticles encapsulating the active agent which are being formed.

[0078] The plurality of individual isolated portions of the relevant active solution will have substantially identical concentrations of polymer and active agent dissolved within them. Ideally, the portions will be sub-portions of a single batch of the active solution.

[0079] The contacting with the aqueous salt solutions will be aqueous salt solutions with the same solvent and the same salt(s) dissolved therein, with only the concentration of the salt(s) varying between the salt solutions to thereby identify the optimised salt solution concentration.

[0080] The variation in the concentrations of the salt solutions may be stepped in that there may be a repeating difference between the concentration of one salt solution to the next, for testing against the various active solution portions. The steps may be made in relation to baseline salt concentration which is arbitrarily defined with steps up, for example, doubling said baseline concentration and steps down halving that concentration.

[0081] In some embodiments, the above steps may be repeated with additional salt solution concentration variation around an identified optimised salt solution concentration to further identify a highly optimised salt solution concentration.

[0082] In determining which salt solution concentrations are optimised and/or highly optimised, the individual batches may be analysed and those having a PDI and/or nanoparticle diameter as set out above may be so identified. Salt solution concentrations which result in polymer nanoparticles encapsulating the active agent having a PDI of less than about 0.3, preferably less than about 0.2 and particle diameter of less than about 300 nm, preferably less than about 200 nm, would be considered optimised and/or highly optimised solutions.

[0083] In practical terms, it will be appreciated that the identification of an optimal salt solution concentration for a particular polymer/active agent/organic solvent combination is a relatively straightforward process in light of the information presently disclosed. When a polymer and active agent have been chosen and dissolved in a suitable organic solvent, to form a bulk active solution, then portions of this can be drawn off and put into separate vials. A salt is then chosen for the aqueous salt solution and a number of small batches of differing concentrations of that salt solution made up. For example, if a PBS solution is selected then a batch of close to saturated PBS may be made up and this can be simply diluted down at appropriate stepped levels to give the desired number of solutions for testing. It may be beneficial, initially, to have relatively large steps between the salt concentrations to identify an initial preferred salt solution concentration. Further salt solution concentrations can then be prepared with smaller steps in concentration around that initially identified concentration to indicate a highly optimised salt solution. In testing the salt solutions, they can be simply added to the active solutions and mixed and left for a period of time. The resulting solution or suspension can then be tested by appropriate means, such as DLS, to ascertain the PDI and average particle size diameter and so identify optimal outcomes. [0084] Traditional precipitation methods are based on fast precipitation of both active and polymer by adding a relatively small volume of the active solution, containing both active and polymer, to a large volume of antisolvent, thereby rapidly precipitating both to form drug-encapsulated nanoparticles, but with very low drug loading. The present approach is to add anti-solvent with a pre-selected and optimised salt concentration to the active solution comprising both active agent and polymer, which allows the formation of drug nanoparticles followed quickly by the precipitation of polymer forming the polymer shell. This optimised salt induced precipitation approach makes it possible to optimise the precipitation time for a wide variety of combinations of polymer and active agent providing for a controlled precipitation approach to predictably provide for optimal encapsulation of the active agent by the forming polymer nanoparticle. All of this can be achieved by simple, quick and non-onerous experimentation which lends itself to straightforward automation.

[0085] The provision of this level of fine control is particularly useful when forming polymer nanoparticles with certain active agents. For example, some nano-sized drugs, such as curcumin, may be stable for up to 48 hours. However, other nano-sized drugs, such as paclitaxel and ibuprofen, are very unstable, once formed, and will aggregate in less than a second. The present approach caters for this by identification of an optimal salt concentration to precipitate the active agent and polymer within a short space of time.

[0086] Therefore, in one embodiment, the method includes the step of causing the active agent to substantially precipitate prior to the polymer.

[0087] In some embodiments, the method may include the step of causing the active agent to substantially precipitate immediately prior to the polymer. The term “immediately prior’ in this context may mean the majority of, or substantially all of, the polymer will precipitate, following precipitation of the majority of, or substantially all of, the active agent, within less than 30 seconds, or less than 20 seconds, or less than 10 seconds, or less than 5 seconds, or less than 2 seconds or within about 1 second. [0088] Due to the efficiency of nanoparticle formation, it is a further advantage of the present method that a separate step of stabilising the polymer nanoparticle is not required.

[0089] Therefore, in one embodiment, the method does not include addition of a separate cross-linking or stabilising agent to stabilise the nanoparticle.

[0090] In one embodiment, the method does not include addition of a separate polymer and/or surfactant to stabilise the formed nanoparticle.

[0091 ] Suitably, the aqueous salt solution is a solution of one or more salts dissolved in water as the only or substantially the only solvent.

[0092] The salt or salts forming the aqueous salt solution may be selected from a wide range of salts so long as they do not interfere with the active agent or polymer or formation of the polymer nanoparticles.

[0093] The salt or salts forming the aqueous salt solution may be selected from the group consisting of a Group 1 and/or Group 2 metal salt and/or a heterocyclic ring-containing salt. That is, they may be salts of an alkali metal and/or an alkaline earth metal. The metals of the metal salts may be selected from the group consisting of lithium, sodium, potassium, magnesium and calcium.

[0094] The metal may form a halide, phosphate, hydrogen phosphate, dihydrogen phosphate, carbonate, hydrogencarbonate, nitrate, nitrite, sulfite, thiosulfate, acetate, sulfate, hydrogen sulfate, sulfonic acid, alkanesulfonic acid or other common salt anion.

[0095] In one embodiment, the aqueous salt solution is an aqueous buffer solution.

[0096] The aqueous buffer solution may be a solution comprising one or more of potassium chloride, sodium chloride, disodium hydrogen phosphate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and potassium dihydrogen phosphate.

[0097] Preferably, the aqueous buffer solution is a phosphate-buffered saline (PBS) or a HEPES buffered saline solution. [0098] The aqueous salt solution may have a pH of between about 3 to about 9, including between about 5 to about 8.

[0099] The concentration of the salt in the aqueous salt solution will clearly vary depending on the nature of the active agent, polymer and organic solvent but, in general terms, a suitable concentration may be between about 1 mM to about 4 M, preferably between about 50 mM to about 3.5 M, more preferably between about 100 mM to about 3 M.

[00100] The final ratio of the volume of the aqueous salt solution to the volume of the active solution, following the addition of the aqueous salt solution and at the point of active agent precipitation, is at least about 3:1 , preferably at least about 4:1 , more preferably at least about 5:1 or about 10:1. The ratio is not likely to need to be greater than 50:1 , or 40:1 or 30:1.

[00101] The aqueous salt solution may be added to the active solution in more than one portion, interspersed with mixing. In one embodiment, the aqueous salt solution volume may be added in two or three or more separate portions. In a further embodiment, the aqueous salt solution volume may be added by a controlled continuous flow. Continuous flow addition may be particularly useful during large-scale production of the polymer nanoparticles. In embodiments, the aqueous salt solution volume may be added dropwise, although it is preferable that mixing is not too slow, and so this may not be a preferred approach.

[00102] It is preferred that the aqueous salt solution is added to and becomes interspersed within the active solution in a controlled, gradual manner. This means the drug and polymer solubility in the organic solvent is reduced gradually allowing for controlled precipitation of drug and polymer in line with the optimised salt concentration. Approaches of the prior art which add all of one of the active solution or anti-solvent into the other do not achieve this level of control as the rapid drop in solubility results in both drug and polymer crashing out of solution in an uncontrolled manner, providing for sub-optimal encapsulation and nanoparticle formation. [00103] Therefore, the method may include adding the aqueous salt solution or the optimised salt solution to the active solution in a controlled and/or portion wise manner over a period of time. Portion wise means at least two separate portions are added at distinctly separate points in time. The period of time may be less than 30 s, less than 20 s, less than 10 s, less than 8 s, less than 6 s, less than 5 s, less than 4 s, less than 3 s, less than 2 s or less than 1 s.

[00104] In one embodiment, the active solution and the aqueous salt solution are mixed by mechanical mixing including stirring or pipette mixing, injection, confined impinging jet mixing, vortex mixing, multi-injection vortex mixing, microfluidic mixing or continuous flow mixing.

[00105] In one embodiment, the method includes the step of adding water to the mixture formed in step (c) to dilute further the level or organic solvent. The addition of water may reduce the organic solvent concentration to below 20%, 15%, 10% or at about or below 5% to minimise the Ostwald ripening effect.

[00106] A second aspect of the invention resides in a polymer nanoparticle, encapsulating an active agent, when produced by the method of the first aspect.

[00107] Preferably, the particle diameter is between 10 to 1000 nm, more preferably between about 15 to 500 nm, even more preferably between about 20 to 300 nm. In preferred embodiments, the polymer nanoparticles encapsulating the active agent will substantially fall within the 25 to 200 nm size range.

[00108] The drug loading efficiency of the polymer nanoparticles may be greater than 10%, or greater than 20%, or greater than 30%, or greater than 40%, or greater than 50%.

[00109] The PDI of the polymer nanoparticles synthesised by the method of the first aspect may have a PDI of less than 0.5, less than 0.4, less than 0.3 or less than 0.2.

[00110] The polymer nanoparticle of the second aspect may be a single drug core surrounded by a polymer shell. That is, the present approach results in a majority, or substantially all, of the formed polymer nanoparticles having a single, as opposed to multiple, drug cores within the polymer shell.

[00111] It is an advantage of the present method that the control of precipitation leads to an optimised sequence of active agent precipitation followed closely by polymer precipitation, prior to formation of any significant amount of aggregation of the active agent, such that the active agent is immediately ready and of an optimal nano-size for efficient polymer encapsulation.

[00112] The polymer nanoparticle comprising an active agent of the second aspect may be formed according to, or may have the properties of, the method or output as described in any statements for the first aspect as if they were reproduced herein in relation to the second aspect.

[00113] A third aspect of the invention resides in a method of delivering an active agent to a target by administering or contacting a polymer nanoparticle of the second aspect to or with the target.

[00114] The target may be a target subject or a target area. If the target is a target area then it may be an area of land, for example agricultural land, a building or particular flora. The administering to or contacting with may be to treat the target area to eliminate or reduce the number of certain pests or diseases or to apply beneficial nutrients or protective agents. Such applications are common in industry and it will be appreciated the same active agents can be employed and delivered in the same manner as is current best practice but simply with the active agent within the polymer nanoparticle shell.

[00115] A fourth aspect of the invention resides in a method of preventing or treating a disease or condition including the step of administering a therapeutically effective amount of a polymer nanoparticle of the second aspect to a subject in need thereof.

[00116] A fifth aspect of the invention resides in the use of a polymer nanoparticle of the second aspect in the manufacture of a medicament for the treatment of a disease or condition. [00117] A sixth aspect of the invention resides in a polymer nanoparticle of the second aspect for use in preventing or treating a disease or condition.

[00118] The polymer nanoparticle comprising an active agent for any of the third to sixth aspects may be that of the second aspect and may be formed, or may be as defined, as described in any embodiment of the first aspect or any combination of such aspects.

[00119] The use of the third aspect may be in relation to active delivery for theranostic applications. In such applications the active agent may be or may comprise liposomes, dendrimers, imaging agents, agrichemicals, insecticides, pesticides, herbicides, plant nutrients, antioxidants, colourants, metallic nanoparticles, quantum dots and carbon nanotubes. The applications may be, for example, pharmacogenetics, proteomics and biomarker profiling as well as diagnostics generally.

[00120] It will be appreciated by those skilled in the art that any composition formulated for the purposes of the third, fourth, fifth or sixth aspect may be formulated using any number or combination of excipient materials. These excipient materials may be included in a formulation for any number of reasons well known to those skilled in the art including, but not limited to, providing a stable formulation, improving flowability, adjusting pH, allowing easy reconstitution, stabilising the particles, minimising adverse toxicological responses, improving manufacturability, increasing stability or lifetime or allowing easier administration, storage or transportation. Such excipient materials are widely known in the art and are readily available through commonly used commercial channels.

[00121] By way of example only, excipients that could be used to formulate the present polymer nanoparticles into a composition to deliver to a subject may include, but are not limited to, acetone, alcohol, anhydrous lactose, castor oil, cellulose acetate phthalate, dextrose, D-fructose, D-mannose, FD&C Yellow #6 aluminium lake dye, fetal bovine serum, human serum albumin, magnesium stearate, micro-crystalline cellulose, plasdone C, polacrilin potassium, sodium bicarbonate, sucrose, aluminium hydroxide, amino acids, benzethonium chloride, formaldehyde, inorganic salts and sugars, vitamins, asparagine, citric acid, lactose, glycerin, iron ammonium citrate, magnesium sulfate, potassium phosphate, aluminium phosphate, formaldehyde, glutaraldehyde, 2-phenoxyethanol, glutaraldhyde, polysorbate 80, aluminium potassium sulfate, ammonium sulfate, bovine extract, gelatin, peptone, sodium phosphate, thimerosal, calf serum, glutaraldehyde, lactalbumin hydrolysate, neomycin sulfate, polymyxin B, lactalbumin hydrolysate, yeast extract, MRC-5 cellular protein, neomycin, polymyxin B sulphate, aluminium hydroxyphosphate sulphate, hemin chloride, mineral salts, nicotinamide adenine dinucleotide, potassium aluminium sulfate, sodium borate, soy peptone, phosphate buffers, polsorbate 20, sodium borate, lipids, sodium dihydrogen phosphate dehydrate, carbohydrates, L-histidine, Beta-propiolactone, calcium chloride, dibasic sodium phosphate, egg protein, monobasic potassium phosphate, monobasic sodium phosphate, polymyxin B, potassium chloride, sodium taurodeoxychoalate, gentamicin sulfate, hydrocortisone, octoxynol-10, a-tocopheryl hydrogen succinate, sodium deoxycholate, ovalbumin, nonylphenol ethoxylate, octylphenol ethoxylate (Triton X-100), arginine, dibasic potassium phosphate, egg protein, ethylene diamine tetraacetic acid, gentamicin sulfate, hydrolyzed porcine gelatin, monobasic potassium phosphate monosodium glutamate, protamine sulfate, sodium metabisulphite, phenol, casamino acid, sodium citrate, sodium phosphate monobasic monohydrate, sodium hydroxide, calcium carbonate, dextran, sorbitol, trehalose, sugar alcohols, polysaccharides, glucosamine, mannitol, polymers and xanthan.

[00122] An appropriate dosage may be simply determined by calculating the loading of the active agent within the polymer nanoparticle, or the loading efficiency, and then using an amount of said loaded nanoparticle which is broadly equivalent to the dosage of the free active agent which would typically be given to a patient.

[00123] As discussed previously, more than one active agent may be coated within a polymer nanoparticle formulation either at the time of formation (by having the actives within solution at the same time and assuming a similar solubility profile) or separate formulations of different active agents may be made up and then mixed. The dual active formulations may then be used in a co-treatment regime. It will be appreciated that any treatment regime can be mimicked by the present approach as it simply requires the forming of the polymer nanoparticles encapsulating the active agent(s) of interest and then treatment can be approached in a broadly equivalent manner to that using the free actives.

[00124] As used herein, the terms "subject" or "individual" or "patient" may refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired. Suitable vertebrate animals include, but are not restricted to, primates, avians, livestock animals (e.g., sheep, cows, horses, donkeys, pigs, fish), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs) and captive wild animals (e.g., foxes, deer, dingoes). A preferred subject is a human.

[00125] The disease or condition may be any one or more of those treated by the active agents listed in relation to the first aspect. Such conditions may include, pain, fever, cancer, arthritis, and inflammation.

[00126] In embodiments, the disease or condition being treated or prevented may be selected from the group consisting of infections (bacterial and/or viral and/or fungal and/or protozoal), malaria, antioxidant, antiprotozoal, neoplastic, cardiovascular, hypertension, pain, blood coagulation, depression, arthritis, psychosis, respiratory, migraine, fever and inflammation.

EXPERIMENTAL

General Approach to Optimising Nanoparticle Formation

[00127] It will be appreciated that the present method requires a degree of optimisation of the concentration of salt(s) in the aqueous salt solution for each drug/polymer combination. A distinct advantage, however, is that this can be accomplished in a reliable and methodical manner, without undue burden or the need for inventive skill, as will be described below.

[00128] To find the optimal salt solution concentration, as previously discussed, a simple screening method is employed. Briefly, an active solution comprising a drug and a polymer are prepared according to their solubility and desirable concentration range (an optimal drug concentration range to form drug nanoparticles is typically from 3~10 g/L in the organic solvent; e.g. 5 g/L docetaxel and 5 g/L PLGA-io_k-PEGs_k in DMF). The aqueous salt solution described herein is phosphate-buffered saline (PBS) of different concentrations (for example, from 0.1 ^c to 20*PBS, their pH values are kept the same at pH 7.4 so only the salt concentrations are different; the 20* PBS has the highest salt concentration; the Ox PBS is pure water) but this could be replaced with a wide variety of simple salts.

[00129] In relation to the use of ‘x’ nomenclature e.g. Ό.1 ^c to 20xPBS’, this is a relative multiplier in relation to a PBS concentration standard as set out below in Table 1 i.e. this is the baseline ‘1 xPBS’. 50 pL of the active solution containing the drug and polymer is added into a glass vessel, followed by adding 950 pl_ of the appropriate aqueous salt solution (PBS with different concentrations, respectively) with pipette mixing, leading to the formation of the polymer nanoparticles encapsulating the drug. Then, the final salt concentration is adjusted to 1 ^c PBS, if not at that value already, either by mixing with an appropriate amount of water for more than 1 xPBS or a high salt concentration PBS for those test samples with less than 1 xPBS; or dialyse against 1 ^c PBS using a molecular weight cut-off 10,000 daltons dialysis membrane. The obtained nanoparticle suspension is stored under room temperature and its DLS result is monitored over time. The optimal salt concentration should result in high drug-loaded nanoparticles (drug-loading >40%) which are stable for at least 24 hours with a polydispersity index (PDI) lower than 0.2 and an average size (diameter) less than 200 nm.

[00130] The aqueous salt solution is not restricted to PBS, theoretically, it can be any aqueous salt solution containing different salt concentrations (e.g. NaCI or NaaSC ). Herein, PBS is employed since it is a buffer which can maintain a physiological pH value at different salt concentrations. Additionally, after nanoprecipitation, all samples can be adjusted to 1 ^c PBS which is the ideal buffer condition for further biologic applications.

Table 1 . The composition of PBS (1 *).

[00131] To avoid the usage of large amounts of salt, it will be appreciated that the screening method for high salt concentrations (e.g. >5* PBS) can also be conducted as a two-step process. Firstly, 50 pL of the solvent containing the polymer and drug is added into a glass vessel, followed by adding 200 pl_ of aqueous salt solution (PBS with differing concentrations, respectively) with pipette mixing. Then, 750 mI_ of water is added with pipette mixing. According to precipitation diagrams of various drugs and polymers identified by the present applicant in such testing, the aqueous salt solution:active solvent ratio > 4:1 is enough to completely precipitate all drugs and polymers. The additional 750 mI_ water is purely for reducing the solvent concentration to 5% to reduce the Ostwald ripening effect as a result of the presence of the organic solvent in the solution (Voorhees, P. W. The theory of Ostwald ripening. J. Stat. Phys. 38, 231-252 (1985); and also Liu, Y., Kathan, K., Saad, W. & Prud’homme, R. K. Ostwald ripening of b-carotene nanoparticles. Phys. Rev. Lett. 98, 036102 (2007)).

Exemplary Approach Using Curcumin (CCM) and PLGA-PEG in DMF\

[00132] The following example shows the approach taking with one drug/polymer combination. It will be appreciated however that the same approach is followed, in combination with the trialling of the nanoprecipitation achieved using different salt combinations, for any drug/polymer combination.

[00133] This example employed curcumin (CCM) loaded PLGA-io_k-PEGs_k (10k) nanoparticles showing 50% drug-loading with DMF used to form the active solution. Briefly, the active solution is prepared by dissolving 5 mg CCM and 5 mg PLGA-io_k-PEGs_k in 1 mL DMF (5 g/L CCM and 5 g/L PLGA-io_k-PEGs_k concentrations in DMF). The aqueous salt solutions used are PBS buffer with different salt concentrations (0~10^c). 50 mI_ of the CCM/PLGA-PEG active solution is added into a number of separate glass vessels, followed by adding 950 mI_ of the PBS salt solution of the appropriate concentration, with pipette mixing. Then the salt concentration is adjusted to 1 ^c PBS. The obtained nanoparticle suspensions are stored under room temperature and the DLS results (PDI and particle diameter) are monitored over time. The salt concentration screen process can be designed to screen the broad range first (e.g. 0~1 * PBS with an interval of 0.1 , and 1~10x PBS with an interval of 1 ), then screen the narrow range around the optimal salt concentration according to the result of the broad screening.

[00134] According to the DLS result of CCM-loaded PLGA-io_k-PEGs_k nanoparticles with 50% of drug-loading after 7 h (FIG 3a1 ), it was found that the suspensions precipitated with a PBS concentration less than 0.5^c and higher than 5^c were aggregated drug structures (PDI«1 ). The suspensions precipitated with a PBS concentration of 4^c was the optimal one with the lowest PDI and particle size. Following this broad screen, a narrow range of the PBS concentration around 4^c (from 3.6 to 4.5^c PBS with an interval of 0.1 ) was further screened and it was found that all the PBS concentrations tested were stable after 1 day with good PDI values (FIG 3a2).

[00135] CCM loaded PLGA-io_k-PEGs_k NPs of 50% drug-loading with DMSO as a solvent were also screened (FIG 3b). The active solution contained 5 g/L CCM and 5 g/L PLGAio_k-PEGs_k in DMSO. The suspension precipitated with a PBS concentration of 0.9^c was identified by DLS as the optimal one with the lowest PDI and particle size after 1 day.

[00136] Essentially the same process was carried out with different combinations of drug and polymer with the optimised aqueous salt solution concentrations identified as follows: ibuprofen:PLGA-PEG in DMF - FIG 5; ketamine:PLGA-PEG in DMF - FIG 6; Dil:PLGA-PEG in DMF - FIG 7; AMB:PLGA-PEG in DMSO - FIG 8; paclitaxel:PLGA-PEG/shellac in DMF - FIG 9; docetaxel:PLGA-PEG in DMF, docetaxel:PLGA-PEG/shellac in DMF, and docetaxel:PLGA-PEG/shellac in DMSO - FIG 10; docetaxel:PLGA-PEG in DMF 50% drug loading and a TEM of the DTX-loaded nanoparticles are shown in FIG 12, docetaxel:PLGA-PEG in DMF 60% drug loading, and docetaxel:PLGA-PEG in DMF 70% drug loading - FIG 11 .

High-loading Using Docetaxel (DTX) and PLGA-PEG in DMF\

[00137] Briefly, the active solution is prepared by dissolving 5 mg DTX and 2.14 mg PLGAio_k-PEG5_k in 1 ml_ DMF (5 g/L DTX and 2.14 g/L PLGA-io_k-PEGs_k concentration in DMF; initial drug-loading is 70%). The aqueous salt solution used is 14* PBS buffer. 50 pL of the active solution is added into a glass vessel, followed by adding 200 pl_ of 14* PBS buffer with pipette mixing. Then, 750 mI_ of water is added with further pipette mixing, followed by dialysis against 1 *PBS using the 10,000 Daltons membrane overnight at 4 °C. It was found that the DTX loaded PLGA-io_k-PEGs_k nanoparticles were stable after 2 days at room temperature without any aggregates and with an unchanged PDI value (~0.2) and particle size (~65 nm). To the best of the inventors’ knowledge, this is the highest DTX drug-loading (70%) achieved among all DTX-loaded nanoparticles made by any method. The graphs showing testing results are shown in FIG 11c.

[00138] In the claims which follow and in the preceding description of the invention, except where the context clearly requires otherwise due to express language or necessary implication, the word “comprise”, or variations thereof including “comprises” or “comprising”, is used in an inclusive sense, that is, to specify the presence of the stated integers but without precluding the presence or addition of further integers in one or more embodiments of the invention.

Claims

1. A method of forming a polymer nanoparticle encapsulating an active agent including the steps of:

2. The method of claim 1 wherein the polymer is a natural or synthetic biocompatible polymer.

3. The method of claim 2 wherein the natural polymer is a resin.

4. The method of any one of the preceding claims wherein the aqueous salt solution is added to the active solution in a controlled manner, preferably not in one addition.

5. The method of claim 4 wherein the aqueous salt solution is added to the active solution in at least two separate additions.

6. The method of any one of the preceding claims wherein the active agent is selected from an anti-infective, antimalarial, antiviral, antibiotic, antifungal, antioxidant, antiprotozoal, antineoplastic, cardiovascular agent, antihypertensive, analgesic, anticoagulant, antidepressant, antiarthritic, antipsychotic, neuroprotective, radiologic, respiratory agent, anti-cancer, anti-migraine and antipyretic.

7. The method of claim 6 wherein the active agent is selected from taxol (paclitaxel), taxol derivatives including docetaxel, doxorubicin, bulleyaconitine A, amphotericin B, scutellarin, quercetin, silibinin, oleanolic acid, betulinic acid, honokiol, camptothecin, camptothecin derivatives, curcumin and curcumin derivatives, ibuprofen and ketamine.

8. The method of any one of the preceding claims wherein the active solution comprises a single organic solvent.

9. The method of any one of the preceding claims wherein the organic solvent forming the active solution is selected from the group consisting of a formamide, a sulfoxide, an alcohol, a nitrile, an aliphatic ether, a cyclic ether, an ester, an alkane, a haloalkane, an amine, a ketone and an aromatic.

10. The method of any one of the preceding claims wherein the organic solvent forming the active solution is DMSO or DMF.

11. A polymer nanoparticle, encapsulating an active agent, when produced by the method of any one of claim 1 to claim 10.

12. The polymer nanoparticle of claim 11 wherein the particle size is between 15 to 500 nm.

13. The polymer nanoparticle of claim 11 or claim 12 wherein the drug loading efficiency within the polymer nanoparticle is greater than 10%.

14. A method of delivering an active agent to a target by administering or contacting a polymer nanoparticle of any one of claim 11 to claim 13 to or with the target to the subject.

15. A method of preventing or treating a disease or condition including the step of administering a therapeutically effective amount of a polymer nanoparticle of any one of claim 1 to claim 10 to a subject in need thereof.

16. The method of claim 15 wherein the subject is a mammalian subject.

17. The method of claim 15 or claim 16 wherein the disease or condition is selected from the group consisting of pain, fever, cancer, arthritis, malaria, neoplastic, inflammation, infection, hypertension, psychosis, migraine, depression, coagulation, cardiovascular and respiratory conditions.