Polymer particles

Abstract

The present invention relates to a process for producing dispersible polymer particles comprising a releasable agent, the process comprising: (i) providing a polymer particle forming stock liquid comprising one or both of polymer in solution, polymer in a colloidal state, and (ii) combining with the polymer particle forming stock liquid an ionic nucleant that promotes precipitation of the polymer; wherein as the polymer precipitates it coats the releasable agent to form the dispersible polymer particles comprising the releasable agent.

Description

[0001] POLYMER PARTICLES

[0002] FIELD OF THE INVENTION

[0003] The present invention relates in general to polymer particles. More specifically the invention relates to a process for producing dispersible polymer particles comprising a releasable agent. As the dispersible polymer particles produced in accordance with the invention are well suited for comprising releasable biologically active agents and being used in therapeutic applications it will be convenient to describe the invention with an emphasis relating to that application. However, it is to be appreciated the dispersible polymer particles produced in accordance with the invention are well suited for use in a diverse range of other applications.

[0004] BACKGROUND OF THE INVENTION

[0005] The delivery of an agent to a specific location is of critical importance in numerous and diverse applications. For example, such applications range from the delivery of a biologically active agent to a subject for the treatment of disease or condition, the delivery of a pesticide or herbicide to a crop for combatting pest or weed infestation, through to the delivery of a fragrance on a substrate to alter the odour of or around the substrate.

[0006] While in principle it is possible to deliver an agent per se to a desired location, in practice that approach presents a number of short comings. For example, in some applications the environment on the way to a desired location can cause the agent to degrade before it reaches the desired location. In other applications it may be required that an agent is to be released progressively at a relatively low concentration over time, or it is only to be released in response to a specific set of conditions, the likes of which can simply not be achieved by just providing the agent per se at the desired location.

[0007] Various delivery vehicles for carrying and subsequently releasing agents have been developed. Such delivery vehicles serve to safely deliver the agent to a desired location and are often tailored to release the agent in a manner suitable for the intended application.

[0008] For example, biologically active agents can be incorporated into various delivery vehicles for administration across different routes, with the choice of delivery vehicle typically determined by the intended route of administration.The need for delivery vehicles in the context of biologically active agents is particularly important as it well know the efficacy of certain biologically active agents can be reduced or lost when administered via certain routes. For instance, many biologically active agents are susceptible to degradation by proteolytic enzymes and / or stomach acid, or they may be insufficiently absorbed into the systemic circulation by restrictions such as molecular weight and / or charge, in particular when administered by oral, transmucosal or transdermal routes.

[0009] Many biologically active agents also require repeated administration over a period of time to achieve or maintain a desired therapeutic response. This is evident, for example, with immunotherapy, where immunisation generally requires multiple vaccinations, boosters and / or high doses of vaccine compositions to be administered, resulting in increased economic costs to patients and the healthcare sector.

[0010] Various techniques have consequently been developed to assist with the administration of biologically active agents. For example, biologically active agents have been incorporated in delivery vehicles such as tablets, capsules, sprays, ointments or patches for delivery by routes such as intravascular (e.g., intravenous), subcutaneous, intraperitoneal, intramuscular, oral, sublingual, transmucosal and transdermal routes of administration.

[0011] One approach for the delivery of releasable agents that has undergone considerable research and development over the years involves the use of polymer as the delivery vehicle. While polymer can serve as an excellent delivery vehicle, there are certain challenges on how to suitably combine the polymer and the agent.

[0012] A thermoplastic polymer may be melt processed with a releasable agent so as to form a bulk polymer mass comprising the agent. A bulk polymer mass comprising releasable agent can also be produced when a thermoplastic polymer is dissolved in a solvent and combined with the agent, with the solvent then being removed to form the polymer mass. However, many agents will degrade under the relatively high temperatures of melt processing. It can prove difficult using either approach to distribute the agent throughout the so formed bulk polymer mass and provide for a uniform product. The resulting bulk polymer mass comprising the agent can also have limited applications in that polymer delivery vehicles are often required to be in the form of dispersible polymer particles.

[0013] Techniques have been developed for preparing dispersible polymer particles that comprise a releasable agent. For example, an agent can be introduced into a polymerisation process such that polymerisation of monomer forms polymer around particles of the agent that becomeencapsulated within so formed polymer particles. Such encapsulation techniques, while effective, can be quite complex and costly to scale. Also, some agents will be unstable and degrade when subjected to the polymerisation reaction conditions / environment.

[0014] An opportunity therefore remains to develop a technique for producing dispersible polymer particles comprising a releasable agent that overcomes or ameliorates one or more of the problems known in the art.

[0015] SUMMARY OF THE INVENTION

[0016] The present invention provides a process for producing dispersible polymer particles comprising a releasable agent, the process comprising:

[0017] (i) providing a polymer particle forming stock liquid comprising (a) polymer in solution, polymer in a colloidal state, or a combination thereof, and (b) both a solvent and a non-solvent for the polymer, and

[0018] (ii) combining with the polymer particle forming stock liquid an ionic nucleant that promotes precipitation of the polymer;

[0019] wherein the releasable agent is combined with the polymer particle forming stock liquid (a) before the ionic nucleant is combined with the polymer particle forming stock liquid and the releasable agent presents as a suspension within the polymer particle forming stock liquid, and / or (b) at the same time as the ionic nucleant is combined with the polymer particle forming stock liquid and the releasable agent presents as a suspension within the polymer particle forming stock liquid; and

[0020] wherein as the polymer precipitates it coats the releasable agent to form the dispersible polymer particles comprising the releasable agent.

[0021] It has now been found that dispersible polymer particles comprising a releasable agent can be produced efficiently and effectively using a stock liquid comprising (a) polymer in solution, polymer in a colloidal state, or a combination thereof. The releasable agent presents as a suspension in the stock liquid at the same time as promoting precipitation of the polymer in the stock liquid. As the polymer precipitates it coats the releasable agent to form the dispersible polymer particles comprising the releasable agent.

[0022] The process in accordance with the invention advantageously makes use of preformed polymer and therefore avoids problems in the art of trying to coat / encapsulate the releasable agent in a process that involves the polymerisation of monomer to form the polymer.An important feature of the invention is believed to be the use of the stock liquid comprising (a) polymer in solution, polymer in a colloidal state, or a combination thereof, and (b) both a solvent and a non-solvent for the polymer. Unlike conventional techniques that make use of polymer dissolved in solvent so as to produce a bulk polymer mass comprising an agent, use of the stock liquid in accordance with the invention has been found to not only extend control over polymer particle formation through a precipitation process, but the precipitation process can advantageously be used as a means to coat the suspended releasable agent with polymer and produce the dispersible polymer particles comprising the releasable agent.

[0023] Without wishing to be limited by theory, it is believed providing the dissolved polymer and / or colloidal polymer in a stock liquid comprising both a solvent and a non-solvent for the polymer provides a metastable liquid that enables the step of adding the ionic nucleant to impart excellent control over precipitation of the polymer such that it effectively and efficiently coats the suspended releasable agent.

[0024] In one embodiment, the polymer particle forming stock liquid is prepared by (i) providing a polymer stock liquid comprising the polymer dissolved in a solvent and the releasable agent suspended therein, and (ii) combining the polymer stock liquid with the non-solvent for the polymer while maintaining the polymer in solution and / or promoting formation of the polymer in a colloidal state.

[0025] In another embodiment, the polymer particle forming stock liquid comprises: (a) the polymer in solution, polymer in a colloidal state, or a combination thereof, and (b) both the solvent and nonsolvent for the polymer; and the releasable agent and the ionic nucleant are combined with the polymer particle forming stock liquid at the same time.

[0026] In a further embodiment, the releasable agent is selected from a biologically active agent, dyes, fluorescent compounds, radioisotopes, radiographic imaging agents (e.g. diagnostics), paint additives, surface modifying additives, inorganic salt, and combinations thereof.

[0027] In another embodiment, the polymer is selected from one or more of polypeptides, alginates, polyurethanes, polyacrylates, polyacrylamides, polyethers, polyesters, polyolefins, boronic acid functionalised polymers, and inorganic polymers.In further embodiment, the polymer is selected from one or more of polyvinylalcohol, polyallylamine, polyethyleneimine, polyvinyl pyrrolidone, chitosan, starch, collagen, polyacrylic acid, silk fibroin, polylactic acid and polyether sulfone.

[0028] In a further embodiment, the solvent and non-solvent for the polymer are selected from one or more of ketones, alcohols, water, esters, ethers, amides, sulfoxides, hydrocarbons, aromatic hydrocarbons, and halogenated hydrocarbons.

[0029] Further aspects and / or embodiments of the invention are discussed in more detail below.

[0030] BRIEF DESCRIPTION OF DRAWINGS

[0031] The present invention will herein be described with reference to the following non-limiting drawings in which:

[0032] Figure 1 illustrates loading of DNA (herring sperm, sodium salt) into PLA (305 ID) in combination with egg white lysozyme. Fibres were dissolved in 0.5 mL of 5 M sodium hydroxide for evaluation. Loading measured via UV-Vis absorbance measured at 260 nm after subtracting control absorbance (polymer + additives, without DNA); and

[0033] Figure 2 illustrates loading of Type IV RNA (from torula yeast) in 0.1 M, pH 5 citrate buffer when added to PLA-PU3 in acetone (12.5 mg / mL) and precipitated into ethanol with 1% normal saline. Fibres were centrifuged, then washed with 50% ethanol / water. Fibres were dissolved in 0.5 mL of 5 M sodium hydroxide for evaluation. Loading measured via UV-Vis absorbance measured at 260 nm after subtracting control absorbance (polymer + additives, without RNA).

[0034] Figure 3 illustrates the efficiency of loading of bovine serum albumin (BSA) into poly lactide acetate (PLA) fibres with varying concentrations of BSA. Optimal maximum loading was achieved at approximately 1 mg / mL of BSA; and

[0035] Figure 4 illustrates the release of azoalbumin from different types of polymer (PLA and PCL), providing ability to modify the protein release half-life.DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention provides a process for producing dispersible polymer particles comprising a releasable agent.

[0037] By being "dispersible" polymer particles is meant the polymer particles can be readily distributed throughout and suspended in a liquid. That liquid may include one or a combination of solvents described herein. It will be appreciated the liquid in which the polymer particles are dispersed will by default not dissolve the polymer particles. In other words, the liquid in which the polymer particles are dispersed will not act as solvent for the polymer particles. If required, conventional dispersing agents may be used to assist with dispersing the polymer particles in the liquid.

[0038] As is convention in the art and discussed in more detail below, a general reference to "solvent" herein can include both solvents and non-solvents for the polymer. However, it is to be appreciated that reference herein to a "solvent for the polymer" is not intended to include a nonsolvent for the polymer. Similarly, reference herein to a "non-solvent for the polymer" is not intended to include a solvent for the polymer. Provided the liquid in which the polymer particles are dispersed will not act as solvent for the polymer particles, it may nevertheless comprise both solvent and non-solvent for the polymer. As is discussed in more detail below, those skilled in the art will appreciate temperature considerations may sometimes be required when considering solvent and nonsolvent properties for a given polymer.

[0039] Reference in the context of the present invention to "polymer particles" is intended to mean a small mass of polymer, often having a spherical or fibre-like shape, which can be dispersed in a liquid. As the "polymer particles" in the context of the present invention are derived through precipitation of polymer, it will be appreciated they are different from and have a larger mass and size to colloidal polymer from which they may be derived.

[0040] The dispersible polymer particles will generally have an average diameter ranging from about 1pm to about 1mm, or from about 5 pm to about 1 mm.

[0041] The dispersible polymer particles comprise a releasable agent. There is no particular limitation on how the releasable agent forms part of the dispersible polymer particles. The dispersible polymer particles are derived through precipitation of the polymer and the precipitating polymer coats suspended particles of the releasable agent. The precipitating polymer may partially or fully coat(encapsulate) the releasable agent. The releasable agent may be present as primary suspended particles or as an aggregate of suspended particles that become coated by the polymer. The precipitating polymer may coat and join together multiple growing dispersible polymer particles so as to present as an aggregate thereof. No matter how the dispersible polymer particles form, they can end up as a composite of the precipitated polymer and the releasable agent.

[0042] When presented as a suspension in the liquid and before being coated with the precipitating polymer, the releasable agent will generally be in the form of particles having an average diameter ranging from 900 nm to about 900 pm, or from about 2 pm to about 500 pm.

[0043] In one embodiment, the releasable agent presents as a suspension of particles within the polymer particle forming stock liquid, the particles having an average diameter ranging from about 900 nm to about 900 pm, or from about 1 pm to about 900 pm, or from about 1 pm to about 700 pm or from about 1 pm to about 600 pm, or from about 2 pm to about 500 pm.

[0044] If required, conventional dispersing agents may be used to assist with suspending the releasable agent in the liquid.

[0045] By the agent being a "releasable" agent in the context of the present invention is meant the dispersible polymer particles per se, which comprise the releasable agent, are intended to function as a delivery vehicle from which the agent can be separated or removed. The polymer and releasable agent used in preparing the dispersible polymer particles comprising the releasable agent will generally be selected such that the agent is released at an appropriate time and rate upon the dispersible polymer particles being presented at a desired location. For example, upon the dispersible polymer particles comprising the releasable agent being presented at a desired location, the agent will migrate / diffuse / dissolve (i.e. be released) from the polymer particle matrix. The releasable agent may also be released from the dispersible polymer particles by other means, for example as a result of the polymer that forms the particles degrading (e.g. biodegrading).

[0046] By being a releasable "agent" is meant a composition of matter that is to have an active role / function in a biological, chemical or physical process. The agent may therefore also be described herein as a releasable active agent. In the context of the present invention, the agent has the potential for that active role / function in the sense that it forms part of the dispersible polymer particles for the purpose of inevitably being delivered to a desired location at which point it canactively undertake its intended role / function upon being released.

[0047] As will be discussed in more detail below, the releasable agent presents as a suspension within a liquid in the process of preparing the dispersible polymer particles. Provided the releasable agent can be suspended in the liquid and coated with the precipitating polymer, there is no particular limitation on the type of releasable agent that can be used.

[0048] The type of the releasable agent used will typically be dictated by the intended application for the dispersible polymer particles.

[0049] The dispersible polymer particles may comprise one or more different releasable agents. By "one or more" releasable agents is meant 1, 2, 3, 4, 5, 6, 7, and so on, releasable agents.

[0050] The releasable agent may be inorganic or organic.

[0051] In one embodiment, the releasable agent is selected from biologically active agents, dyes, fluorescent compounds, radioisotopes, radiographic imaging agents (e.g. diagnostics), paint additives, surface modifying additives, and inorganic salts.

[0052] As used herein, the expression "biologically active agent(s)" refers to any molecule / compound of synthetic or natural origin that is capable of eliciting a physiological response in a biological system, whether in vitro, ex vivo or in vivo.

[0053] In an embodiment, the dispersible polymer particles comprise at least 1 biologically active agent, at least 2 biologically active agents, at least 3 biologically active agents, at least 4 biologically active agents, at least 5 biologically active agents, at least 6 biologically active agents, at least 7 biologically active agents, at least 8 biologically active agents, at least 9 biologically active agents, or at least 10 biologically active agents.

[0054] Suitable biologically active agents will be known to persons skilled in the art, the choice of which will likely depend on the intended therapeutic, prophylactic and / or diagnostic use of the dispersible polymer particles disclosed herein, such as the nature or type of disease or disorder to be treated. Illustrative examples of suitable biologically active agents include small molecule drugs, hormones, antimicrobial compounds, antimicrobial proteins, antivirals, steroids, chemotherapy drugs, ligands, binding agents (e.g. aptamers, small interfering RNA, antibodiesand antigen-binding fragments thereof, including therapeutic antibodies and antigen-binding fragments thereof), cell lysates, cytokines, growth factors, fusion proteins, immunogens, antigens, viruses, viral proteins, bacteria, bacterial proteins and fragments thereof, bacteria cell lysates, hormones and nucleic acid molecules, including nucleic acid molecules encoding any one or more of the foregoing. It is to be understood that the dispersible polymer particles disclosed herein may comprise one or more biologically active agents selected from one or more classes, including from one or more of the aforementioned classes. Conversely, when the dispersible polymer particles disclosed herein comprise two or more biologically active agents, the biologically active agents may belong to the same class of active agents.

[0055] In an embodiment, the one or more biologically active agents are selected from a hormone, an antimicrobial agent, an antiviral, a steroid, a chemotherapy drug, a therapeutic binding agent (e.g., an aptamer, an antibody or antigen-binding fragments thereof), a cytokine, an immunogen and a nucleic acid molecule.

[0056] In an embodiment, the one or more biologically active agents comprises an immunogen.

[0057] The term “immunogen” as used herein is understood to mean a peptide or protein that is capable of raising an immune response, including a humoral (antibody) response, in vivo. The terms “peptide”, “polypeptide” and "protein" are used interchangeably herein in their broadest sense to refer to a molecule of two or more amino acid residues, or amino acid analogs. The amino acid residues may be linked by peptide bonds, or alternatively by other bonds, e.g. ester, ether etc., but in most cases will be linked by peptide bonds. The terms “amino acid” or “amino acid residue” are used herein to encompass both natural and unnatural or synthetic amino acids, including both the D- or L-forms, and amino acid analogs. An “amino acid analog” is to be understood as a non-naturally occurring amino acid differing from its corresponding naturally occurring amino acid at one or more atoms. For example, an amino acid analog of cysteine may be homocysteine. Suitable immunogens will be familiar to persons skilled in the art, noting that the choice of immunogen will also largely depend on the intended therapeutic or prophylactic use. Illustrative examples of suitable immunogens include a tumour cell, a tumour cell lysate, a virus, a viral antigen, a bacteria, a bacteria cell lysate, a cancer-associated antigen and nucleic acid molecules encoding any one or more of the foregoing. Thus, in an embodiment disclosed herein, the immunogen is selected from the group consisting of a tumour cell, a tumour cell lysate, a virus, a viral antigen, a bacteria, a bacteria cell lysate, a cancer-associated antigen and nucleic acid molecules encoding any one or more of the foregoing.In another embodiment, the one or more biologically active agents comprises a fusion protein.

[0058] The term "fusion protein" as used herein refers to two or more peptide sequences (e.g., immunogens) linked in such a way as to produce a peptide that would not otherwise occur in nature. In an embodiment, the fusion protein comprises two or more peptide sequences linked to one another end-to-end. In an embodiment, the fusion protein comprises two or more peptide sequences linked to one another in a linear configuration via a suitable linking moiety, also referred to herein as a linker. Suitable methods of linking peptide sequences will be familiar to persons skilled in the art, illustrative examples of which include peptide (amide) bonds and linkers. As used herein, the term “linker” refers to a short polypeptide sequence interposed between any two neighboring peptide sequences as herein described. In an embodiment, the linker is a polypeptide linker of 1 to 10 amino acids, or 1, 2, 3, 4 or 5 naturally or non-naturally occurring amino acids. In an embodiment, the linker is a carbohydrate linker. Suitable carbohydrate linkers will be known to persons skilled in the art. In another embodiment disclosed herein, the fusion protein comprises one or more peptidic or polypeptidic linker(s) together with one or more other non-peptidic or non-polypeptidic linker(s). Further, different types of linkers, peptidic or non-peptidic, may be incorporated in the same fusion peptide as deemed appropriate. In the event that a peptidic or polypeptidic linker is used to join two respective peptide sequences, the linker will be advantageously incorporated such that its N-terminal end is bound via a peptide bond to the C-terminal end of the one peptide sequence, and its C-terminal end via a peptide bond to the N-terminal end of the other peptide sequence. The individual peptide sequences within the fusion protein may also have one or more amino acids added to either or both ends, preferably to the C-terminal end. Thus, for example, linker or spacer amino acids may be added to the N- or C-terminus of the peptides or both, to link the peptides and to allow for convenient coupling of the peptides to each other and / or to a delivery system such as a carrier molecule serving as an anchor. An illustrative example of a suitable peptidic linker is LP (leucine-proline). Also contemplated herein are fusion proteins comprising at least two of the peptide sequences concatenated two or more times in tandem repeat. Without being bound by theory or by a particular mode of application, it will be understood that incorporating two or more different peptide sequences into the fusion peptide, as herein described, may generate a more beneficial immune response by eliciting a higher antibody titre as compared to an immunogen comprising a single peptide sequence disclosed herein. Suitable methods of preparing a fusion protein, as herein described, would be familiar to persons skilled in the art. An illustrative example includes peptide synthesis that involves the sequential formation of peptide bonds linking each peptide sequence, as hereindescribed, to its respectively neighbouring peptide sequence, and recovering said fusion peptide. Illustrative examples include the methods described in “Amino Acid and Peptide Synthesis” (Oxford Chemistry Primers; by John Jones, Oxford University Press). Synthetic peptides can also be made by liquid-phase synthesis or solid-phase peptide synthesis (SPPS) on different solid supports (e.g. polystyrene, polyamide, or PEG). SPPS may incorporate the use of F-moc (9H-fluoren-9-ylmethoxy carbonyl) ort-Boc (tert-Butoxy carbonyl). Custom peptides are also available from a number of commercial manufacturers. Alternatively, the fusion protein may be prepared by recombinant methodology. For example, a nucleic acid molecule comprising a nucleic acid sequence encoding the fusion protein can be transfecting into a suitable host cell capable of expressing said nucleic acid sequence, incubating said host cell under conditions suitable for the expression of said nucleic acid sequence, and recovering said fusion protein. Suitable methods for preparing a nucleic acid molecule encoding the fusion protein will also be known to persons skilled in the art, based on knowledge of the genetic code, possibly including optimizing codons based on the nature of the host cell (e.g. microorganism) to be used for expressing and / or secreting the recombinant fusion protein. Suitable host cells will also be known to persons skilled in the art, illustrative examples of which include prokaryotic cells (e.g., E. coli) and eukaryotic cells (e.g., P. pastoris). Reference is made to “Short Protocols in Molecular Biology, 5th Edition, 2 Volume Set: A Compendium of Methods from Current Protocols in Molecular Biology” (by Frederick M. Ausubel (author, editor), Roger Brent (editor), Robert E. Kingston (editor), David D. Moore (editor), J. G. Seidman (editor), John A. Smith (editor), Kevin Struhl (editor), J Wiley & Sons, London).

[0059] In an embodiment, the immunogen is a tumour cell lysate. Persons skilled in the art will understand that the choice of tumour cell lysate will depend on the type of disease or disorder to be treated or prevented. The tumour cell lysate will typically be prepared from a sample of tumour cell derived from the cancer. For instance, where the cancer is a cancer of the liver, the tumour cell lysate may suitably be prepared from one or more cancer cells derived from the tumour in the subject to be treated. In an embodiment, the tumour cell is a glioblastoma tumour cell. In an embodiment, the glioblastoma is glioblastoma multiforme.

[0060] In an embodiment, the one or more biologically active agents comprises a cytokine. Suitable cytokines will be known to persons skilled in the art, illustrative examples of which includes interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Thus, in an embodiment disclosed herein, the cytokine is GM-CSF.In another embodiment, the one or more biologically active agents comprises a hormone. Suitable hormones will be known to persons skilled in the art, illustrative examples of which include insulin and somatotropin, and steroid hormones such as corticosteroids, estrogens, progestogens and androgens. The present disclosure also extends to the use of peptide hormones. A "peptide hormone" is typically understood to be a peptide or protein that has an effect on the endocrine system of a subject. An illustrative example of a suitable peptide hormone is somatotropin. Somatotropin stimulates the growth, cell reproduction and cell regeneration in humans and nonhuman animals and is important in growth and development.

[0061] In another embodiment disclosed herein, the one or more biologically active agents comprises a binding agent, illustrative examples of which will be known to persons skilled in the art and include aptamers, antibodies, and antigen-binding fragments thereof. The binding agent may be a therapeutic antibody to a target antigen of interest, such as a viral protein or a cancer-associated antigen. In other embodiments, the antibody may be used to target the dispersible polymer particles to a biological site of interest (i.e., a targeting antibody or binding fragment thereof).

[0062] In an embodiment, the one or more biologically active agent comprises a cancer-associated antigen. The terms “cancer-associated antigen”, “antigen associated with cancer, “tumour-associated antigen”, “tumour antigen”, “cancer antigen” and the like are used interchangeably herein to mean an antigen that is aberrantly expressed in cancer cells or tissue. In some embodiments, the antigen may be expressed under normal conditions in a limited number of tissues and / or organs or in specific developmental stages. For example, the antigen may be specifically expressed under normal conditions in stomach tissue and is expressed or aberrantly expressed (e.g., overexpressed) in one or more cancer cells. The expression of antigen may be reactivated in cancer cells or tissue irrespective of the origin of the cancer. In some embodiments, the cancer-associated antigen includes differentiation antigens, for example cell type-specific differentiation antigens (i.e., proteins that are specifically expressed under normal conditions in a certain cell type at a certain differentiation stage), cancer / testis antigens (i.e., proteins that are specifically expressed under normal conditions in testis and sometimes in placenta), and germline specific antigens.

[0063] In an embodiment, the cancer-associated antigen is expressed on the cell surface of a cancer cell and is preferably not or only rarely expressed on normal cells and tissues. Preferably, the antigen or the aberrant expression of the antigen identifies cancer cells, preferably tumour cells. In some embodiments, the antigen that is expressed by a cancer cell in a subject (e.g., a patient sufferingfrom cancer) is a self-protein. It will be understood, however, that no autoantibodies directed against the antigen are typically found in a detectable level under normal conditions in a subject carrying the antigen (typically a healthy patient that does not have cancer) or such autoantibodies can only be found in an amount below a threshold concentration that would be necessary to damage the tissue or cells carrying the antigen. Suitable cancer-associated antigens will be known to persons skilled in the art, illustrative examples of which include EGFR (e.g., Her2 / neu, Her-1), BAGE (B melanoma antigen), CEA (carcinoembryonic antigen), Cpg (cytosine-phosphate diesterguanine), GplOO (glycoprotein 100), h-TERT (telomerase transcriptase), MAGE (melanoma antigen-encoding gene), Melan-A (melanoma antigen recognized by T cells) and MUC-1 (mucin-1). Thus, in an embodiment, the cancer-associated antigen is selected from the group consisting of EGFR (e.g., Her2 / neu, Her-1), BAGE (B melanoma antigen), CEA (carcinoembryonic antigen), CpG (cytosine-phosphate diesterguanine), GplOO (glycoprotein 100), h-TERT (telomerase transcriptase), MAGE (melanoma antigen-encoding gene), Melan-A (melanoma antigen recognized by T cells) and MUC-1 (mucin-1). It will also be understood that the choice of antigen that is to be the target of the dispersible polymer particles will typically depend on the intended use of the dispersible polymer particles. For example, if the dispersible polymer particles are intended to treat subjects with breast cancer, then the antigen will typically be an antigen that is associated with (e.g., overexpressed by) the breast cancer. Suitable examples of antigens associated with breast cancer will be familiar to persons skilled in the art, illustrative examples of which include the epidermal growth factor receptors Her2 / neu and Herl. Other illustrative examples of suitable cancer-associated antigens include Wilms tumor- 1 (WT1), surviving, cytomegalovirus (CMV) and alpha-lactalbumin.

[0064] In an embodiment disclosed herein, the one or more biologically active agents comprises a cancer-associated antigen selected from the group consisting of Wilms tumor-1 (WT1), surviving, cytomegalovirus (CMV) and alpha-lactalbumin.

[0065] In another embodiment, the one or more biologically active agents comprise one or more of paclitaxel, cisplatin, and 5 -fluorouracil.

[0066] In a further embodiment, the one or more biologically active agents comprise one or more of, tyrosine kinase inhibitors, ionic substances, peptides, proteins, antibodies, RNA (e.g mRNA, siRNA, circRNA, IncRNA, miRNA) and DNA.

[0067] Provided the dispersible polymer particles can be prepared there is no particular limitation on theamount of releasable agent they may contain.

[0068] The amount of the releasable agent in the dispersible polymer particles will vary, depending on, for example, the type of polymer that is precipitated and the characteristics of the releasable agent (e.g., size, net charge, molecular weight).

[0069] The amount of releasable agent contained within the dispersible polymer particles may be referred to herein as the "loading rate"; that is, the amount of releasable agent loaded into the dispersible polymer particles as a proportion of the total weight of the loaded dispersible polymer particles.

[0070] In an embodiment, the dispersible polymer particles comprise the releasable agent in an amount from about 2% to about 95%, or about 10% to about 60%, or about 20% to about 50%, or about 30% to about 50% by weight, relative to the weight of the dispersible polymer particles comprising the releasable agent.

[0071] In other embodiments, the dispersible polymer particles comprise the releasable agent in an amount from about 2% to about 50%, or about 2% to about 40%, or about 2% to about 30%, or about 2% to about 20%, or about 2% to about 10% by weight, relative to the weight of the dispersible polymer particles comprising the releasable agent.

[0072] The process according to the invention comprises providing a polymer particle forming stock liquid comprising (a) polymer in solution, polymer in a colloidal state, or a combination thereof.

[0073] The polymer particle forming stock liquid is a liquid in which the dispersible polymer particles are produced. That stock liquid initially comprises polymer in solution, polymer in a colloidal state, or a combination thereof.

[0074] By the polymer being in "solution" is meant the polymer is solvated in the stock liquid.

[0075] By the polymer being in a "colloidal state" is meant the polymer presents as ultramicroscopic dispersed polymer particles throughout the stock liquid that will not settle on standing and cannot be readily separated from the stock liquid by conventional filtering or centrifugation. For convenience, the polymer being in a colloidal state may also herein be referred to as "colloidal polymer".As the dispersible polymer particles comprising the releasable agent can be produced through precipitation of the colloidal polymer, it will be appreciated the dispersible polymer particles are consequently not in a colloidal state (i.e. the so formed dispersible polymer particles are not colloidal polymer particles).

[0076] The colloidal polymer particles will typically have an average diameter ranging from 1 nm to about 5 pm, or about 2 nm to about 1 pm.

[0077] There is no particular limitation on the type of polymer that may be used in preparing the stock liquid, provided it can (i) be dissolved and / or presented in a colloidal state, and (ii) undergoes precipitation according to the process of the invention.

[0078] In one embodiment, the polymer is a biocompatible polymer.

[0079] As used herein, the term “biocompatible polymer” refers to a polymer material that, when introduced into or placed in contact with a biological system (e.g. in vitro, ex vivo or in vivo), does not cause any unacceptable toxic, immunogenic, inflammatory, or otherwise adverse biological response, and is tolerated by the biological system for the duration of its intended use. The term encompasses polymers that are naturally occurring, synthetic, biodegradable, non-biodegradable, or substantially non-biodegradable, provided they are suitable for use in a biological environment without producing deleterious effects that would materially impair their function or the function of the biological system.

[0080] The polymer can be a synthetic or a natural (i.e., naturally-occurring) polymer. Illustrative examples of suitable natural polymers include proteins such as albumin, collagen, gelatin and prolamins, for example, zein, and polysaccharides such as alginate, cellulose derivatives and polyhydroxyalkanoates, for example, polyhydroxybutyrate.

[0081] The polymer may be a biodegradable polymer, a non-biodegradable polymer, or substantially non-biodegradable polymer. It would be understood, however, that it is generally desirable that the biocompatible polymer is biodegradable, or substantially biodegradable, so as to avoid or minimise the impact the polymer may otherwise have on a biological system over time.

[0082] In an embodiment, the polymer is a biodegradable polymer.Examples of suitable polymers include, but are not limited to, polypeptides, alginates, chitosan, starch, collagen, silk fibroin, polyurethanes, polyacrylic acid, polyacrylates, polyacrylamides, polyesters, polyolefins, polyethers, boronic acid functionalised polymers, polyvinylalcohol, polyallylamine, polyethyleneimine, polyvinyl pyrrolidone, polylactic acid, polyhydroxyalkanoates, polyether sulfone, inorganic polymers, and a combination of any of foregoing.

[0083] In one embodiment, the polymer is selected from one or more of polypeptides, alginates, polyurethanes, polyacrylates, polyacrylamides, polyethers, polyesters, polyolefins, boronic acid functionalised polymers, and inorganic polymers.

[0084] In further embodiment, the polymer is selected from one or more of polyvinylalcohol, polyallylamine, polyethyleneimine, polyvinyl pyrrolidone, chitosan, starch, collagen, polyacrylic acid, silk fibroin, polylactic acid and polyether sulfone.

[0085] The polymer can be selected to degrade over a time period ranging from one day to more than one year, or from seven days to 26 weeks, or from seven days to 20 weeks, or from seven days to 16 weeks.

[0086] It will be understood that the choice of polymer may depend on the intended use of the dispersible polymer particles. In some embodiments, a synthetic polymer may be preferred. In other embodiments, a natural polymer may be preferred.

[0087] Other illustrative examples of suitable polymers include poly(lactic acid), poly(glycolic acid), poly(lactic acid-co-glycolic acids), polyhydroxyalkanoates such as poly 3 -hydroxybutyrate or poly4-hydroxybutyrate; polycaprolactones; poly(orthoesters); polyanhydrides; poly(phosphazenes); poly(lactide-co-caprolactones); poly(glycolide-co-caprolactones); polycarbonates such as tyrosine polycarbonates; polyamides (including synthetic and natural polyamides), polypeptides, and poly(amino acids); polyesteramides; other biocompatible polyesters; poly(dioxanones); poly(alkylene alkylates); hydrophilic polyethers; polyurethanes; polyetheresters; polyacetals; polycyanoacrylates; polysiloxanes; poly(oxyethylene) / poly(oxypropylene) copolymers; polyketals; polyphosphates; polyhydroxyvalerates; polyalkylene oxalates; polyalkylene succinates; poly(maleic acids), polyvinyl alcohols, polyvinylpyrrolidone; poly(alkylene oxides) such as polyethylene glycol (PEG); derivativized celluloses such as alkyl celluloses (e.g., methyl cellulose), hydroxyalkylcelluloses (e.g., hydroxypropyl cellulose), cellulose ethers, cellulose esters, nitrocelluloses, polymers of acrylic acid, methacrylic acid or copolymers or derivatives thereof including esters, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly (isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate) (jointly referred to herein as "polyacrylic acids"), as well as derivatives, copolymers, and blends thereof. As used herein, "derivatives" include polymers having substitutions, additions of chemical groups and other modifications to the polymeric backbones described above routinely made by those skilled in the art.

[0088] Polylactic acid (PLA) is classified under the family of aliphatic polyesters. PLA is mainly derived from renewable resources, particularly sugar and starch. The PLA family includes poly(l-lactide) (PLLA), poly(d-lactide) (PDLA), poly(dl-lactide) (PDLLA), poly(meso-lactide), and copolymers obtained from the monomers. Polymerization of 1-lactide yields poly(l-lactide) while poly(d-lactide) is produced by polymerization of d-lactide

[0089] In an embodiment, the polymer comprises poly(lactic acid).

[0090] In another embodiment, the poly(lactic acid) is poly(lactic-co-glycolic acid) (PLGA) or poly(lactide-co-caprolactone) (PLCL). In some embodiments, the poly(lactic-co-glycolic acid) is poly(D,L-lactide-co-glycolide) and the poly(lactide-co-caprolactone) is poly(L-lactide-co-caprolactone) copolymer.

[0091] In an embodiment, the poly(lactic-co-glycolic acid) has a lactide:glycolide ratio of about 85:15.

[0092] In an embodiment, the poly(lactide-co-caprolactone) has a lactide:caprolactone ratio of about 50:50 to about 90:10.

[0093] In an embodiment, the poly(lactic-co-glycolic acid), for example poly(D,L-lactide-co-glycolide), or poly(lactide-co-caprolactone), for example poly(L-lactide-co-caprolactone) has an Mw from about 50 kDa to about 75 kDa. In another embodiment, the poly(lactic-co-glycolic acid) or poly(lactide-co-caprolactone) has an Mw from about 190 kDa to about 240 kDa.

[0094] In one embodiment, the biocompatible polymer comprises a combination of a first poly(lactic-co-glycolic acid) polymer component having an Mw from about 50 kDa to about 75 kDa and a secondpoly(lactic-co-gly colic acid) polymer component having an Mw from about 190 kDa to about 240 kDa. Mw is the weight average molecular weight of the polymer. In an embodiment, the PLGA may suitably comprise a combination of different forms of PLGA, including those described herein.

[0095] Different forms of PLGA may be combined in proportions or absolute amounts suitable to produce the dispersible polymer particles with the desired properties as herein described. Suitable combinations can be ascertained using methods known to persons skilled in the art, illustrative examples of which include combinations of poly(D,L-lactide-co-glycolide) having an Mw from about 50 kDa to about 75 kDa and poly(D,L-lactide-co-glycolide) having an Mw from about 190 kDa to about 240 kDa. Thus, in an embodiment, the PLGA comprises poly(D,L-lactide-co-glycolide) having an Mw from about 50 kDa to about 75 kDa and poly(D,L-lactide-co-glycolide) having an Mw from about 190 kDa to about 240 kDa. In another embodiment, the PLGA comprises from about 5% to about 50% poly(D,L-lactide-co-glycolide) having an Mw from about 50 kDa to about 75 kDa and from about 50% to about 95% poly(D,L-lactide-co-glycolide) having an Mw from about 190 kDa to about 240 kDa. In another embodiment, the PLGA comprises from about 5% to about 20% poly(D,L-lactide-co-glycolide) having an Mw from about 50 kDa to about 75 kDa and from about 80% to about 95% poly(D,L-lactide-co-glycolide) having an Mw from about 190 kDa to about 240 kDa. In yet another embodiment, the PLGA comprises from about 10% poly(D,L-lactide-co-glycolide) having an Mw from about 50 kDa to about 75 kDa and about 90% poly(D,L-lactide-co-glycolide) having an Mw from about 190 kDa to about 240 kDa.

[0096] Polyhydroxyalkanoates (PHAs) are a class of polyester that is typically biodegradable and biocompatible and are produced naturally by various microorganisms as intracellular energy and carbon storage materials. PHAs are characterised by hydroxyalkanoate repeat units and can vary widely in their mechanical and physical properties depending on the monomer composition, which may include short-chain-length (SCL) or medium-chain-length (MCL) hydroxyalkanoates. PHAs may be used alone or in copolymer form.

[0097] In one embodiment, the polymer is a polyhydroxyalkanoate selected from one or more of poly (3 -hydroxybutyrate) (PHB), poly(3 -hydroxy valerate) (PHV), poly(3-hydroxybutyrate-co-3 -hydroxy valerate) (PHBV), poly(4-hydroxybutyrate) (P4HB), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB-CO-4HB), poly (3 -hy droxybutyrate-co-3 -hy droxyhexanoate) (PHBHHx), poly (3 -hy droxyhexanoate) (P3HHx), poly (3 -hydroxy octanoate) (P3HO), poly (3 -hydroxy decanoate) (P3HD),poly (3 -hydroxy dodecanoate) (P3HDD), poly(3-hydroxyhexanoate-co-3 -hydroxy octanoate) (P3HHx-co-P3HO), poly(3 -hydroxy octanoate-co-3 -hydroxy decanoate) (P3HO-CO-P3HD), poly(3 -hydroxy valerate-co-3 -hydroxyhexanoate) (PHV-co-PHHx), poly(4-hydroxyvalerate) (P4HV), poly(5-hydroxyvalerate) (P5HV), poly(3-hydroxyheptanoate) (P3HHp), and poly(3-hydroxynonanoate) (P3HN).

[0098] In one embodiment, the polymer is a biodegradable polyester.

[0099] In a further embodiment, the biodegradable polyester is selected from poly lactic acid, poly glycolic acid, poly(lactic-co-glycolic acid), polycaprolactone, and copolymers thereof.

[0100] In another embodiment, the polymer is a polysaccharide selected from chitosan, alginate, starch, cellulose derivatives, and combinations thereof.

[0101] In a further embodiment, the polymer is polylactic acid having a molecular weight in the range of 10,000 to 200,000 Da.

[0102] In an embodiment, the polymer comprises one or more photo-polymerizable groups, allowing for the crosslinking of the so formed dispersible polymer particles. Illustrative examples of suitable photo-polymerizable groups include vinyl groups, acrylate groups, methacrylate groups, and acrylamide groups. Photo-polymerizable groups, when present, may be incorporated within the backbone of the polymer, within one or more of the sidechains of the polymer, at one or more of the ends of the polymer, or combinations thereof.

[0103] The polymer, which may be a single polymer or two or more different polymers, may be present in the stock liquid (either dissolved and / or in a colloidal state) at a concentration ranging from about 0.05 mg / ml to about 50 mg / ml, or about 0.1 mg / ml to about 30 mg / ml, or from about 0.15 mg / ml to about 15 mg / ml, or about 0.5 mg / ml to about 10 mg / ml, or about 0.5 mg / ml to about 5 mg / ml, or about 0.5 mg / ml to about 2 mg / ml.

[0104] In addition to the polymer (in solution and / or in a colloidal state), the polymer particle forming stock liquid comprises both a solvent and a non-solvent for the polymer.

[0105] By being a "solvent for the polymer" is meant a solvent that can dissolve or solubilise the polymer. By being a "non-solvent for the polymer" is meant a solvent that cannot dissolve or solubilise thepolymer. The "solvent" or "non-solvent" character of a given solvent for the polymer is that determined at a temperature used in one or more steps of the process of the invention. As those skilled in the art will appreciate, the solvent characteristics of a given solvent for a polymer can vary depending upon the molecular weight of the polymer.

[0106] As is convention in the art, a general reference to "solvent" herein can include both solvents and non-solvents for the polymer. However, it is to be appreciated that reference herein to a "solvent for the polymer" is not intended to include a non-solvent for the polymer. Similarly, reference herein to a "non-solvent for the polymer" is not intended to include a solvent for the polymer.

[0107] Those skilled in the art will appreciate that a solvent for one polymer may in fact be a non-solvent for another polymer. The solvent or non-solvent characteristics of a particular solvent for a particular polymer can be readily determined by those skilled in the art. In other words, those skilled in the art can readily look up or determine if a particular solvent is a solvent or non-solvent for a particular polymer.

[0108] With that in mind, it appropriate to list herein a common list of solvents from which both the solvent and non-solvent for the polymer may be selected.

[0109] Examples of suitable solvents and non-solvents for the polymer include, but are not limited to, one or more selected from ketones, carboxylic acids, alcohols, water, esters, ethers, amides, nitriles, amines, nitro compounds, sulfoxides, hydrocarbons, aromatic hydrocarbons, and halogenated hydrocarbons.

[0110] Both the solvent and non-solvent for the polymer may comprise a single solvent or a mixture of solvents.

[0111] Examples of suitable ketones include, but are not limited to, acetone, butanone and pentanone.

[0112] Examples of suitable carboxylic acids include, but are not limited to, acetic acid, propanoic acid, and butanoic acid.

[0113] Examples of suitable alcohols include, but are not limited to, methanol, ethanol, isopropanol, butanol, tert-butanol, pentanol, cyclohexanol, ethylene glycol, glycerol, and propargyl alcohol.Examples of suitable esters include, but are not limited to, ethyl actetate, butyl acetate, tert-butyl acetate, ethyl butanoate, and cyclohexyl acetate.

[0114] Examples of suitable ethers include, but are not limited to, diethylether, tetrahydrofuran, dioxane, trioxane, dimethoxyethane (glyme), diglyme, and tert-butyl methyl ether.

[0115] Examples of suitable amides include, but are not limited to, dimethylformamide, dimethylacetamide, n-methylpyrrolidone, hexamethylphosphoramide, and hexamethylphosphorous triamide.

[0116] Examples of suitable nitriles include, but are not limited to, acetonitrile, benzonitrile, and phenyl acetonitrile.

[0117] Examples of suitable amines include, but are not limited to, triethylamine, diisopropylamine, and ethylenediamine.

[0118] Examples of suitable nitro compounds include, but are not limited to, nitromethane, nitroethane, and nitrobenzene.

[0119] Examples of suitable sulfoxides include, but are not limited to, dimethyl sulfoxide, methyl phenyl sulfoxide, and methyl propyl sulfoxide.

[0120] Examples of suitable hydrocarbons include, but are not limited to, pentane, hexane, heptane, octane, nonane, decane, cyclohexane, and cycloheptane.

[0121] Examples of suitable aromatic hydrocarbons, include, but are not limited to, benzene, toluene, xylene, and ethylbenzene.

[0122] Examples of suitable halogenated hydrocarbons, include, but are not limited to, dichloromethane, chloroform, trichloromethane, carbon tetrachloride, dichloroethane, and chlorobenzene.

[0123] As an example of using such solvents, where the polymer is polylactic acid, a solvent for that polymer may be a ketone such as acetone and a non-solvent for that polymer may be water or ethanol.In one embodiment, both the solvent and non-solvent for the polymer are miscible.

[0124] An important feature of the present invention is that the stock liquid comprise both a solvent and a non-solvent for the polymer.

[0125] Where the polymer in the stock liquid is dissolved / solvated therein, the amount of non-solvent for the polymer present will not be sufficient to promote precipitation of the polymer.

[0126] Similarly, where the polymer in the stock liquid is in a colloidal state, the amount of non-solvent for the polymer present will not be sufficient to promote precipitation of the polymer.

[0127] The polymer (which may be a single polymer of two or more different polymers) may be also be present in the stock liquid such that some of it is dissolved therein and some of it is in a colloidal state. Depending on temperature, the polymer may transition in the stock liquid from being solvated to being in a colloidal state.

[0128] Accordingly, the amount of solvent and non-solvent for the polymer used in the polymer particle forming stock liquid is suitably balanced to retain the polymer in solution and / or a colloidal state. The respective amounts of solvent and non-solvent to use for a given polymer may vary depending upon the type of polymer, its molecular weight, the type of solvents being used and the temperature of the stock liquid. Those skilled in the art can readily determine the appropriate amount of solvent and non-solvent to use for a given polymer.

[0129] In some embodiments, in the stock liquid the amount of non-solvent for the polymer is greater than the amount of solvent for the polymer.

[0130] In a further embodiment, in the stock liquid the ratio of non-solvent for the polymer to solvent for the polymer ranges from about 2:1 to about 25:1, or about 3:1 to about 22:1, about 4:1 to about 20:1, about 5:1 to about 20:1.

[0131] There is no particular limitation on the way in which the polymer particle forming stock liquid comprising the polymer is prepared.

[0132] For example, in one embodiment the polymer particle forming stock liquid comprises: the polymer in solution, polymer in a colloidal state, or a combination thereof, both the solvent andnon-solvent for the polymer and the releasable agent; and the ionic nucleant is then combined with the polymer particle forming stock liquid.

[0133] In that case, the polymer particle forming stock liquid may be prepared by (i) providing a polymer stock liquid comprising the polymer dissolved in a solvent and the releasable agent suspended therein, and (ii) combining the polymer stock liquid with the non-solvent for the polymer such that the polymer stays in solution and / or transitions into a colloidal state.

[0134] Reference to the "polymer dissolved in a solvent" is of course intended to mean the polymer is dissolved / solvated in a solvent for the polymer.

[0135] In such an embodiment, the polymer colloid can be formed in the presence of the suspended releasable agent.

[0136] Alternatively, the polymer particle forming stock liquid may be prepared by (i) providing a polymer stock liquid comprising the polymer dissolved in a solvent, (ii) combining the polymer stock liquid with the non-solvent for the polymer such that the polymer stays in solution and / or transitions into a colloidal state, and then (iii) combing that resulting liquid with the releasable agent.

[0137] In such an embodiment, the polymer colloid can be formed first and then it is combined with releasable agent, which itself is suspended in the so formed polymer particle forming stock liquid.

[0138] As another example, in one embodiment the polymer particle forming stock liquid comprises: the polymer in solution, polymer in a colloidal state, or a combination thereof and both the solvent and non-solvent for the polymer; and the releasable agent and the ionic nucleant are then combined with the polymer particle forming stock liquid at the same time.

[0139] In such an embodiment, the polymer in solution and / or polymer in a colloidal state can be prepared first and then combined with releasable agent, which itself becomes suspended in the so formed polymer particle forming stock liquid. However, unlike other embodiments, the releasable agent is combined with the polymer particle forming stock liquid at the same time as the ionic nucleant.

[0140] In that case, the polymer particle forming stock liquid may be prepared by (i) providing a polymer stock liquid comprising the polymer dissolved in a solvent, and (ii) combining the polymer stockliquid with the non-solvent for the polymer such that the polymer stays in solution and / or transitions into a colloidal state.

[0141] Those skilled will be able to readily prepare a polymer stock liquid comprising the polymer dissolved in a solvent for the polymer. Depending upon the polymer and solvent for the polymer, the polymer may dissolve in the solvent at room temperature. Alternatively, it may be necessary to heat the polymer and the solvent to facilitate the polymer dissolving in the solvent. It will be appreciated that dissolving the polymer in the solvent is undertaken so as to ultimately precipitate it form the stock liquid either directly from solution and / or via its colloidal state.

[0142] The so formed polymer particle forming stock liquid will comprise (a) the polymer in solution, the polymer in a colloidal state, or a combination thereof, and (b) both a solvent and a non-solvent for the polymer, optionally together with the releasable agent. Without wishing to be limited by theory, it is believed the dissolved polymer and / or colloidal polymer in the stock liquid comprising both a solvent and a non-solvent for the polymer presents as a metastable liquid. That metastable liquid is poised to transition into a lower energy state, through precipitation of the polymer, upon being triggered by external stimuli. Addition of the ionic nucleant is believed to function as that external stimuli by disrupting the solvation layer associated with the dissolved polymer / polymer colloid and in turn provide a means to impart excellent control over precipitation of the polymer such that it effectively and efficiently coats the suspended releasable agent that is present at the time perception is induced.

[0143] In performing the process of the invention the releasable agent presents as a suspension within the polymer particle forming stock liquid. The releasable agent can be combined with preformed polymer particle forming stock liquid or combined with one or more liquids in the process of producing the polymer particle forming stock liquid. The releasable agent may combined with a relevant liquid in its native form, for example as solid particulate releasable agent per se, or in the form of a preformed suspension within a liquid. When used as a preformed suspension in a liquid, the releasable agent will simply be combined with a liquid, for example combined with one or more solvents described herein. In that case, the releasable agent will generally present as a suspension within that liquid, the likes of which may then be combined with preformed polymer particle forming stock liquid or combined with one or more liquids in the process of producing polymer particle forming stock liquid (e.g. the polymer stock liquid).

[0144] In one embodiment, the releasable agent is provided in the form of a suspension within a solventas described herein.

[0145] By being provided in the form of a suspension, it will be appreciated the releasable agent will not be soluble in the solvent system within which it presents.

[0146] There is no particular limitation on the amount of releasable agent that can be used in the polymer particle forming stock liquid. The amount used can influence the amount of releasable agent that ultimately forms part of the dispersible polymer particles.

[0147] Generally, it will be desirable for the dispersible polymer particles to comprise the releasable agent in an amount from about 2% to about 95%, or about 10% to about 60%, or about 20% to about 50%, or about 30% to about 50% by weight, relative to the weight of the dispersible polymer particles comprising the releasable agent.

[0148] In some embodiments, the dispersible polymer particles comprise the releasable agent in an amount from about 2% to about 50%, or about 2% to about 40%, or about 2% to about 30%, or about 2% to about 20%, or about 2% to about 10% by weight, relative to the weight of the dispersible polymer particles comprising the releasable agent.

[0149] Releasable agent that becomes incorporated within the so formed dispersible polymer particles will need to be suspended within the polymer particle forming stock liquid at the time when precipitation of the polymer occurs. If required, suspending the releasable agent in the stock liquid may be assisted with stirring. It is possible that releasable agent may be present in the stock liquid that is no longer suspended and consequently may not become coated with the polymer as it precipitates. The overall amount of releasable agent in the stock liquid may therefore not reflect how much releasable agent ultimately becomes incorporated within the dispersible polymer particles.

[0150] The conversion of suspended releasable agent in the polymer particle forming stock liquid to become incorporated into the dispersible polymer particles can vary depending upon the nature of at least the releasable agent, solvent system and polymer being precipitated.

[0151] In some embodiments, it may be desirable to have an excess of suspended releasable agent present in the polymer particle forming stock liquid (i.e. more releasable agent than is intended to be incorporated in the dispersible polymer particles) to maximise the amount of releasable agent thatbecomes incorporated within the dispersible polymer particles.

[0152] Those skilled in the art can readily determine the amount of releasing agent to suspend within a given polymer particle forming stock liquid to arrive at a desired amount of that releasing agent being incorporated within the so formed dispersible polymer particles.

[0153] Some releasable agents may have a lower propensity than desired to be coated by the polymer as it is precipitated from the polymer particle forming stock liquid. In that case, the releasable agent may be combined with an additive in the stock liquid that enhances its ability to be coated with the precipitating polymer and thereby become incorporated within the so formed dispersible polymer particles. Such additives can serve to modify the surface properties of the suspended releasing agent to make them more compatible with the polymer that is being precipitated. There is no particular limitation on the nature of such additives that can be used, the likes of which would be well known to those skilled in the art. For example, egg white lysozyme may be combined with DNA sodium salt to enhance the propensity for the DNA sodium salt to be coated with PLA polymer.

[0154] Having provided the polymer particle forming stock liquid, according to the invention it is then combined with an ionic nucleant that promotes precipitation of the polymer.

[0155] Reference herein to an "ionic nucleant" is intended to mean a compound that (i) comprises cationic and anionic species, and (ii) promotes or initiates (i.e. nucleates) precipitation of the polymer residing (either solvated or as a colloid) in the polymer particle forming stock liquid.

[0156] The ionic nucleant used in accordance with the invention may be provided / used in the form of a solid or liquid.

[0157] If not already in a liquid form, upon being combined with the polymer particle forming stock liquid the ionic nucleant will generally dissolve in the polymer particle forming stock liquid.

[0158] Without wishing to be limited by theory, it is believed ionic species derived from the ionic nucleant destabilises the solvation shell of the dissolved polymer and / or colloidal polymer to promote its precipitation from the polymer particle forming stock liquid.

[0159] Addition of the ionic nucleant to the polymer particle forming stock liquid is believed tomodify / destabilise the solvation shell of the dissolved polymer and / or colloidal polymer to promote its precipitation without adversely interfering with the suspended releasable agent. Accordingly, the suspended releasable agent can remain suspended the time when the polymer precipitates.

[0160] By "precipitation of the polymer" is meant the polymer present in the polymer particle forming stock liquid, be it dissolved and / or in a colloidal state, forms through action of the ionic nucleant particles of polymer that are of a size that precipitate or crash / drop out from the liquid.

[0161] Those skilled in the art will appreciate that such precipitated polymer is not intended to be a reference to colloidal polymer and vice versa.

[0162] In one embodiment, the ionic nucleant comprises a cation selected from ammonium, lithium, sodium, potassium, calcium, aluminium, magnesium, zinc and combinations thereof.

[0163] In another embodiment, the ionic nucleant comprises an anion selected from chloride, bromide, iodide, sulfate, hydrogen sulfate, carbonate, bicarbonate, citrate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, nitrate and combinations thereof.

[0164] Those skilled in the art appreciate the combined cation and anion species of the ionic nucleant will be provided in a suitable ratio to afford a neutral salt.

[0165] In one embodiment, the ionic nucleant is selected from sodium chloride, lithium chloride, potassium chloride, sodium citrate, sodium hydrogen phosphate, potassium citrate, potassium hydrogen phosphate and combinations thereof.

[0166] In another embodiment, the ionic nucleant is provided in the form of a liquid.

[0167] In the form of a liquid, the ionic nucleant may present as solubilised cations and anions.

[0168] In a further embodiment, the ionic nucleant is provided as an aqueous solution of anions and cations.

[0169] The ionic nucleant is used in an amount sufficient to promote precipitation of the polymer present in the polymer particle forming stock liquid. The amount of ionic nucleant required for that tooccur can vary depending upon parameters such as the amount of polymer present in the stock liquid and the solvent / non-solvent composition of the stock liquid. Those skilled in the art can readily determine the amount of ionic nucleant required to perform precipitation having regard to at least those parameters.

[0170] In one embodiment, the ionic nucleant is added at a concentration sufficient to shift the polymer from a soluble or colloidal state to a precipitated state within the stock liquid.

[0171] Generally, the amount of ionic nucleant combined with the polymer particle forming stock liquid will provide for a concentration in the polymer particle forming stock liquid ranging from about 0.001 w / v to about 1 % w / v, 0.001 w / v to about 0.9% w / v, 0.001 w / v to about 0.8% w / v, 0.001 w / v to about 0.7% w / v, 0.001 w / v to about 0.6% w / v, 0.001 w / v to about 0.5% w / v, 0.001 w / v to about 0.4% w / v, 0.001 w / v to about 0.3% w / v, 0.001 w / v to about 0.2% w / v, 0.001 w / v to about 0.1% w / v, or from about 0.003 w / v to about 0.05 w / v.

[0172] In one embodiment, the amount of ionic nucleant combined with the polymer particle forming stock liquid will provide for a concentration in the polymer particle forming stock liquid ranging from about 0.001 w / v to about 1 % w / v, 0.001 w / v to about 0.9% w / v, 0.001 w / v to about 0.8% w / v, 0.001 w / v to about 0.7% w / v, 0.001 w / v to about 0.6% w / v, 0.001 w / v to about 0.5% w / v, 0.001 w / v to about 0.4% w / v, 0.001 w / v to about 0.3% w / v, 0.001 w / v to about 0.2% w / v, 0.001 w / v to about 0.1% w / v, or from about 0.003 w / v to about 0.05 w / v.

[0173] When the ionic nucleant is provided in the form of a liquid, that liquid will generally be miscible with polymer particle stock liquid with which it is combined.

[0174] The ionic nucleant plays an important role in the process of the invention that it promotes precipitation of the polymer that is in solution and / or in a colloidal state that resides in the polymer particle forming stock liquid. That precipitating polymer in turn coats the releasable agent to form the dispersible polymer particles comprising the releasable agent.

[0175] To achieve the required coating of the precipitating polymer the releasable agent must be present in the form of a suspension at the time when precipitation of the colloidal polymer occurs.

[0176] In accordance with the invention, the releasable agent is combined with the polymer particle forming stock liquid (a) before the ionic nucleant is combined with the polymer particle formingstock liquid, and / or (b) at the same time as the ionic nucleant is combined with the polymer particle forming stock liquid. Either way, it will be appreciated the releasable agent will be present in the polymer particle forming stock liquid at the time when precipitation of the colloidal polymer occurs.

[0177] There is no particular limitation on the matter in which the ionic nucleant is combined with the polymer particle forming stock liquid. For example, the ionic nucleant, as a solid or in solution, may be combined with the polymer particle forming stock liquid gradually or rapidly, in parts or as one batch. Combining the only nucleant and the polymer particle forming stock liquid may be facilitated with stirring.

[0178] Producing dispersible polymer particles in accordance with the invention advantageously can be undertaken at room temperature and avoids the need for using harsh chemicals such as acids and bases, the likes of which can adversely affect sensitive agents (e.g. denature proteins) such as many of the bioactive agents described herein. Accordingly, the process in accordance with the invention advantageously provides for a relatively inert means for coating agents with polymer to produce dispersible polymer particles comprising an agent from which the agent can subsequently be released.

[0179] Production of the dispersible polymer particles comprising the releasable agent can be readily confirmed by conventional techniques that can detect (i) the presence of polymer associated with the agent, or (ii) the presence of the agent associated with the polymer. For example, UV-VIS spectrometry and biuret testing may be performed.

[0180] The dispersible polymer particles may be used in various applications to deliver the releasable agent over select periods of time, ranging from days to months. The polymer particles can also be tailored to provide an initial “burst” of releasable agent that is then followed by a longer period of continuous or sustained release. The amount of releasable agent made available by such a "burst" can be adjusted by including a washing step in the final stage of polymer particle production to reduce the amount of surface based releasable agent available for immediate release, thus reducing the size of the burst from its maximum level prior to washing.

[0181] There is no particular limitation on the applications in which the dispersible polymer particles in accordance with the invention can be used. Examples of suitable applications include but are not limited to, delivery of drugs in medical applications (e.g. injectables for vaccines, drug delivery,and theranostics), the delivery of a pesticide or herbicide to a crop for combatting pest or weed infestation, through to the delivery of a fragrance on a substrate to alter the odour of or around the substrate.

[0182] EXAMPLES

[0183] Example 1

[0184] A 1.6 mg / mL solution of DNA sodium salt (from herring sperm) was prepared in an 80% v / v DMSO / water stock solution. This solution was added to a 12.5 mg / mL solution of PLA (Natureworks 305 ID) in acetone containing 0.15 mL of 20 mg / mL solution (80% acetone / water) of egg white lysozyme. The solution was added to ethanol and then a 1% normal saline was added, which precipitated the PLA in the form particles having a fibre-like shape. These fibres are centrifuged and washed with 50% ethanol / water. Figure 1 illustrates the efficiency of loading of DNA as a factor of DNA concentration in the combined solution prior to its introduction into the ethanol / saline solution.

[0185] Example 2

[0186] Type IV RNA (from Torula yeast) was made up to 20 mg / mL in 0.1 M Citrate buffer and added to PLA-PU3 in acetone (12.5 mg / mL). The solution was added to ethanol and then a 1% normal saline was added, which precipitated the PLA-PU3 in the form particles having a fibre-like shape. The Fibres were centrifuged and washed with 50% ethanol / water. Figure 2 illustrates the efficiency of loading of RNA as a factor of RNA concentration in the combined solution prior to its introduction into the ethanol / saline solution.

[0187] Example 3

[0188] For preparation of protein containing dispersible particles, a solution of 80 mg of protein (e.g., Bovine serum albumin, BSA) in 2.4 mL of deionized water was prepared. This solution was added to 9.6 mL of acetone and mixed at room temperature. The polymer-protein solution was then prepared by dissolving 25 mg of polymer (see Table 1) in 1.7 mL of acetone and then adding 0.3 mL of the protein (BSA) solution with mixing. The combined solution was drawn into a syringe, the outlet of which is placed into a Falcon tube containing 50 mL of ethanol and 0.2 mL of 0.9% saline. The polymer-protein solution was delivered via the syringe using a syringe pump set at a rate of 1 mL / min. After the polymer-protein solution has been delivered the Falcon tubes are allowed to settle for 20 minutes, after which the mix was centrifuged at 3,000 RPM for 1 minute.The liquid was then drawn from the top and disposed of, leaving the precipitated material in the form particles having a fibre-like shape.

[0189] Table 1 presents details of the polymers used and Table 1 A presents the loading efficiencies for BSA with various polymer preparations and types of polymers. Figure 3 illustrates the efficiency of loading of BSA as a factor of BSA concentration in the combined solution prior to its introduction into the ethanol-saline solution. Figure 4 illustrates the release over time of BSA from the final polymer particles into normal saline.

[0190] The polymers used were polylactic acid, poly(lactic acid-co-caprolactone) copolymer and polycaprolactone.

[0191] For all preparations, the amount of water used to dissolve any materials (e.g., proteins) should be kept to a minimum, as increasing amounts of water used in protein stock solutions tends to decrease the loading efficiency.

[0192] If necessary, to ensure complete dissolution of the polymer in acetone, the polymer-acetone mixture can be heated until it is all dissolved (e.g., via a heat gun with the solution in a round bottom flask for several seconds), but then must be cooled before adding the protein solution to avoid denaturing the protein. Alternatively, the mixture could be sonicated for several minutes. After mixing the protein solution with the polymer, if the solution appears cloudy or some precipitate is seen the solution should be sonicated for several minutes. If very little polymer is observed after being centrifuged, 0.5 mL of saline, or several drops of brine, should be added and mixed, and the mixture centrifuged again.

[0193] Table 1

[0194]

[0195]

[0196] Table 1A

[0197]

[0198]

[0199] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0200] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS1. A process for producing dispersible polymer particles comprising a releasable agent, the process comprising:(i) providing a polymer particle forming stock liquid comprising (a) polymer in solution, polymer in a colloidal state, or a combination thereof, and (b) both a solvent and a non-solvent for the polymer, and(ii) combining with the polymer particle forming stock liquid an ionic nucleant that promotes precipitation of the polymer;wherein the releasable agent is combined with the polymer particle forming stock liquid (a) before the ionic nucleant is combined with the polymer particle forming stock liquid and the releasable agent presents as a suspension within the polymer particle forming stock liquid, and / or (b) at the same time as the ionic nucleant is combined with the polymer particle forming stock liquid and the releasable agent presents as a suspension within the polymer particle forming stock liquid; andwherein as the polymer precipitates it coats the releasable agent to form the dispersible polymer particles comprising the releasable agent.

2. The process according to claim 1, wherein the polymer particle forming stock liquid comprises: the polymer in solution, polymer in a colloidal state, or a combination thereof, both the solvent and non-solvent for the polymer and the releasable agent, and the ionic nucleant is then combined with the polymer particle forming stock liquid.

3. The process according to claim 2, wherein the polymer particle forming stock liquid is prepared by (i) providing a polymer stock liquid comprising the polymer dissolved in a solvent and the releasable agent suspended therein, and (ii) combining the polymer stock liquid with the non-solvent for the polymer such that the polymer stays in solution and / or transitions into a colloidal state.

4. The process according to claim 2, wherein the polymer particle forming stock liquid is prepared by (i) providing a polymer stock liquid comprising the polymer dissolved in a solvent, (ii) combining the polymer stock liquid with the non-solvent for the polymer such that the polymer stays in solution and / or transitions into a colloidal state, and then (iii) combing that resulting liquid with the releasable agent.

5. The process according to claim 1, wherein the polymer particle forming stock liquid comprises: the polymer in solution, polymer in a colloidal state, or a combination thereof and both the solvent and non-solvent for the polymer, and the releasable agent and the ionic nucleant are then combined with the polymer particle forming stock liquid at the same time.

6. The process according to claim 5, wherein the polymer particle forming stock liquid is prepared by (i) providing a polymer stock liquid comprising the polymer dissolved in a solvent, and (ii) combining the polymer stock liquid with the non-solvent for the polymer such that the polymer stays in solution and / or transitions into a colloidal state.

7. The process according to any one of claims 1 to 6, wherein the releasable agent is selected from a biologically active agent, dye, fluorescent compound, radioisotope, radiographic imaging agent, paint additive, surface modifying additive, inorganic salt, and combinations thereof.

8. The process according to any one of claims 1 to 7, wherein the polymer is selected from one or more of polypeptides, alginates, polyurethanes, polyacrylates, polyacrylamides, polyethers, polyesters, polyolefins, boronic acid functionalised polymers and inorganic polymers.

9. The process according to any one of claims 1 to 8, wherein the solvent and non-solvent for the polymer are selected from one or more of ketones, alcohols, water, esters, ethers, amides, sulfoxides, hydrocarbons, aromatic hydrocarbons, and halogenated hydrocarbons.

10. The process according to any one of claims 1 to 9, wherein the releasable agent is a biologically active agent selected from small molecule drugs, hormones, antimicrobial compounds, antimicrobial proteins, antivirals, steroids, chemotherapy drugs, ligands, binding agents, cell lysates, cytokines, growth factors, fusion proteins, immunogens, antigens, viruses, viral proteins, bacteria, bacterial proteins and fragments thereof, bacteria cell lysates, hormones and nucleic acid molecules.

11. The process according to any one of claims 1 to 10, wherein dispersible polymer particles comprise the releasable agent in an amount from about 10% to about 60% by weight, relative to the weight of the dispersible polymer particles comprising the releasable agent.

12. The process according to any one of claims 1 to 11, wherein the ratio of non-solvent for the polymer to solvent for the polymer in the stock liquid ranges from about 3 : 1 to about 22: 1.

13. The process according to any one of claims 1 to 12, wherein the ionic nucleant comprises a cation selected from ammonium, lithium, sodium, potassium, calcium, aluminium, magnesium, zinc and combinations thereof.

14. The process according to any one of claims 1 to 12, wherein the ionic nucleant comprises an anion selected from chloride, bromide, iodide, sulfate, hydrogen sulfate, carbonate, bicarbonate, phosphate, hydrogen phosphate, dihydrogen phosphate, citrate, acetate, nitrate and combinations thereof.

15. The process according to any one of claims 1 to 12, wherein the ionic nucleant is selected from sodium chloride, lithium chloride, potassium chloride, sodium citrate, sodium hydrogen phosphate, potassium citrate, potassium hydrogen phosphate and combinations thereof.

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