Protein-based nanoparticles suitable for tumour targeting of Anti-cancer drugs and agents

Abstract

The invention relates to a self-assembling conjugate molecule, the conjugate molecule comprising a hydrophobic polymer covalently bound to human serum albumin (HSA) only at residue Cys34; or a hydrophobic polymer covalently bound to a non-human serum albumin protein only at an equivalent residue to Cys34 of HSA; and associated compositions, uses and methods of treatment.

Description

[0001] Protein-based nanoparticles suitable for tumour targeting of anti-cancer drugs and agents

[0002] The present invention relates to a self-assembling conjugate molecule for use as a drug carrier, the resulting nanoparticles and compositions, and methods of manufacture and methods of treatment, for example for cancer.

[0003] Cancer remains one of the leading causes of mortality worldwide. Traditional chemotherapy, while effective to some extent, is often associated with significant drawbacks such as limited drug solubility, poor tumour targeting, and severe systemic toxicity. These challenges have prompted the scientific community to explore alternative strategies to improve cancer treatment outcomes.

[0004] Nanoparticle-based drug delivery systems have emerged as a promising solution for overcoming the limitations of conventional chemotherapy. The use of nanoparticles allows for precise control over drug release, improved bioavailability, enhanced tumour penetration, and reduced adverse effects. Among the various nanoparticle formulations developed, Abraxane® nanoparticles have shown some potential in cancer therapy.

[0005] Abraxane® is a nanoparticle formulation comprising paclitaxel, an established chemotherapeutic agent, bound to albumin nanoparticles. This approach exploits the benefits of nanoparticle technology and harnesses the properties of albumin to enhance drug delivery and therapeutic efficacy.

[0006] The nanoparticles have a mean hydrodynamic diameter of approximately 130 nm, allowing them to passively accumulate within tumour tissues due to the enhanced permeability and retention (EPR) effect. The EPR effect takes advantage of the leaky vasculature and impaired lymphatic drainage commonly found in solid tumours, leading to preferential accumulation of nanoparticles at tumour sites.

[0007] Moreover, the albumin acts as a natural carrier for paclitaxel, protecting it from enzymatic degradation and promoting its solubility.

[0008] Clinical studies have demonstrated the effectiveness of Abraxane® nanoparticles in treating several types of cancer, including breast, lung, and pancreatic cancer. Although Abraxane® has shown lower systematic toxicity than the chemotherapy Taxol®, the overall survival of metastatic breast cancer patients is not significantly improved. Because the therapeutic efficacy of PTX from Abraxane® in patients is not improved due to the immunosuppression, poor colloidal stability at low concentrations and passive targeting of Abraxane® nanoparticles.

[0009] Therefore, there is a need to improve such nanoparticle-based therapies. This patent application aims to protect the novel composition and methods of using nanoparticles in treatments, such as cancer.

[0010] According to a first aspect of the invention there is provided a self-assembling conjugate molecule, for example for use as an agent carrier, the conjugate molecule comprising a hydrophobic polymer, such as polylactic acid (PLA), covalently bound to human serum albumin (HSA) only at residue Cys34; or a hydrophobic polymer, such as PLA, covalently bound to a non-human serum albumin protein only at an equivalent residue to Cys34 of HSA.

[0011] The self-assembling conjugate molecule (also termed herein as HSA-PLA) advantageously provides a new nanocarrier for the parenteral administration of agents, such as therapeutic molecules (e.g. taxanes such as paclitaxel (PTX)). Acetylated embodiments are also provided (termed herein as Ac-HSA-PLA). These amphiphilic polymers, HSA-PLA and Ac-HSA-PLA can then self-assembly into polymeric nanoparticles, which may be about 100 nm in diameter. Advantageously, many hydrophobic small molecules, such as paclitaxel, or other agents such as nanoparticles, can be incorporated into the hydrophobic part of HSA-PLA and Ac-HSA-PLA nanocarriers.

[0012] The cysteine conjugation of the hydrophobic polymer, such as PLA, to a single Cys34 site of HSA is beneficial, as this conjugation leaves many other lysine groups available for acetylation and allows both conjugations to take place on one molecule in a controlled fashion.

[0013] By conjugating acetyl groups to HSA, an additional hydrophobic focus is provided for agent loading (such as drug loading) and colloidal stability, which is different and advantageous over alternative systems that may surface-acetylate nanoparticles. HSA- PLA nanoparticles have a small amount of agglomeration in aqueous media, which may lead to inhomogeneous surface acetylation. Ac-PLA-HSA also provides for more monodisperse nanoparticles, which are advantageous in pharmaceutical development as they are easier to characterise and their biological activity easier to predict.

[0014] The present invention improves on the commercial PTX injection Abraxane® with enhanced colloidal stability of both nanocarriers (HSA-PLA nanoparticle and Ac-HSA- PLA nanoparticle) for carrying paclitaxel and targeted tumour delivery of PTX by Ac- HSA-PLA nanoparticles. This results in improved pharmacokinetics and a better tumoricidal effect in 4T1 and MDA-MB-231 tumour-bearing models without additional toxicity when compared with Abraxane®. Thus, the formulations have potential to prolong the life of metastatic breast cancer patients.

[0015] Based on the colloidally stable nanoplatform HSA-PLA polymeric micelle, we further acetylated human serum albumin and then produced the active targeting nanoparticle with the higher colloidal stability, acetylated HSA-PLA (Ac-HSA-PLA). According to our in vitro studies, the Ac-HSA-PLA nanoparticles are able to bind with folate receptor and CD44 receptor of MDA-MB-231 human triple negative breast cancer cells. And the cell-association efficiency of Ac-HSA-PLA nanoparticles was increased around 10-fold when compared with human serum albumin standard and HSA-PLA nanoparticles. Meanwhile, the excellent active targeting of Ac-HSA-PLA nanoparticle to tumours has been demonstrated, and tumour bearing mice injected with Ac-HSA-PLA (PTX) have been cured with no recurrent tumour.

[0016] It's worth noting that HSA-PLA nanoparticles are unable to actively target the tumour as the Ac-HSA-PLA nanoparticle did, but HSA-PLA nanoparticles also have their own unique advantages such as easy surface functionalization. During the synthesis of HSA- PLA polymer, we utilized the residue of cysteine to react with maleimide-PLA. So, HSA-PLA polymer still has 59 free lysine amine groups for modification for diverse purposes, such as conjugation of stealth polymer to evade immune system, addition of various ligands to target different sites.

[0017] Further advantageously, it has been demonstrated that Irinotecan, artemisinin and imatinib have been successfully encapsulated in Ac-HSA-PLA nanocarrier. SN-38, other taxanes, such as cabazitaxel (CTX), and gold nanoparticles have also been demonstrated to be successfully encapsulated.

[0018] Acetylation of the conjugate molecule

[0019] In one embodiment, the conjugate molecule is acetylated. The conjugate molecule may comprise multiple sites of acetylation. Alternatively, the conjugate molecule may not be acetylated.

[0020] In an embodiment wherein the conjugate molecule is acetylated, preferably acetylation occurs prior to any self-assembly of the conjugate molecule into a complex, such as a nanoparticle.

[0021] The Serum Albumin

[0022] The non-human serum albumin protein may comprise or consist of a mammalian serum albumin. The non-human serum albumin protein may comprise or consist of simian, bovine, horse, pig, rat, mouse or goat serum albumin. In one embodiment, the non- human serum albumin protein comprises Bovine Serum Albumin (BSA). In a preferred embodiment, the single polylactic acid (PLA) is covalently bound to human serum albumin (HSA) at Cys34.

[0023] The HSA may comprise or consist of the amino acid sequence of SEQ ID NO: 1, or a variant thereof. The BSA may comprise or consist of the amino acid sequence of SEQ ID NO: 2, or a variant thereof.

[0024] The HSA variant or the non-human serum albumin may comprise or consist of an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1. In another embodiment, the HSA variant or the non-human serum albumin may comprise or consist of an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 1. In another embodiment, the HSA variant or the non-human serum albumin may comprise or consist of an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1. In another embodiment, the HSA variant or the non-human serum albumin may comprise or consist of an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1. In another embodiment, the HSA variant or the non-human serum albumin may comprise or consist of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. In another embodiment, the HSA variant or the non-human serum albumin may comprise or consist of an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1. In another embodiment, the HSA variant or the non-human serum albumin may comprise or consist of an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 1. In another embodiment, the HSA variant or the non-human serum albumin may comprise or consist of an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 1. The variant of HSA may be a functional variant (i.e. has substantially the same function, such as the same natural ligand binding ability, as HSA).

[0025] The skilled person will recognise that there are several genetic variants of HSA that have been identified. These variants are mainly due to genetic polymorphisms in the HSA gene (ALB) that lead to differences in the amino acid sequence of the protein. Some of the well-known HSA variants include: HSAF1 and HSAF2: These are the two most common genetic variants of HSA. They are also known as HSA allele 1 and HSA allele 2, respectively. They differ in their amino acid sequences at position 428, with HSAF1 having an arginine (Arg) residue and HSAF2 having a tryptophan (Trp) residue. HSA*S: This variant has a substitution of Arg at position 410 with cysteine (Cys). HSA*E: This variant has a substitution of Asp at position 469 with asparagine (Asn). HSA*H: This variant has a substitution of His at position 146 with arginine (Arg). HSA*Z: This variant has a substitution of Lys at position 281 with glutamine (Gin). HSA*V: This variant has a substitution of Thr at position 436 with isoleucine (He). HSA*Afinsa: This is a rare variant that is characterized by a deletion of a single amino acid (Asp) at position 48. In one embodiment, the HSA variant may comprise or consist of any of the HSA variants selected from the group consisting of HSAF1; HSAF2; HSA*S; HSA*E; HSA*H; HSA*Z; HSA*V and HSA*Afinsa; or combinations thereof.

[0026] The variant of the HSA or non-human serum albumin protein may be a truncated variant. The truncated HSA or non-human serum albumin protein may still function substantially the same as full size / wild-type HSA or non-human serum albumin protein, for example having substantially the same binding characteristics.

[0027] The hydrophobic polymer In a preferred embodiment, the hydrophobic polymer comprises or consists of PLA. The skilled person will recognise that alternative hydrophobic polymers to PLA may provide the same effect as PLA. Preferably the hydrophobic polymer is a pharmaceutically acceptable hydrophobic polymer.

[0028] The hydrophobic polymer may be selected from the group consisting of PLA, such as poly(l-lactic acid); a poly(l-lactic acid)-containing co-polymer such as poly(L-lactic acid)-co-(L-glycolic acid); a polycaprolactone; a poly(orthoester); a poly(phosphoester); a polystyrene; and maleimide PCL; or combinations thereof.

[0029] The hydrophobic polymer may be selected from the group consisting of poly(methyl methacrylate) (PMMA); polycaprolactone (PCL); Poly(lactic acid) (PLA); poly (lactic- co-glycolic acid) (PLGA); PEG-polycaprolactone (PEG-PCL); PEG-poly(lactic acid) (PEG-PLA); and PEG-Poly (lactic-co-glycolic acid) (PEG-PLGA); or combinations thereof.

[0030] The hydrophobic polymer may comprise a maleimide group, for example for conjugation to the HSA or non-human serum albumin protein. The hydrophobic polymer may be maleimide-polymers selected from the group consisting of maleimide- Poly(methyl methacrylate) (MAL-PMMA); maleimide-Polycaprolactone (MAL-PCL); maleimide-Poly(lactic acid) (MAL-PLA); maleimide-Poly (lactic-co-glycolic acid) (MAL-PLGA); maleimide-PEG-Polycaprolactone (MAL-PEG-PCL); maleimide-PEG- Poly(lactic acid) (MAL-PEG-PLA); and maleimide-PEG-Poly (lactic-co-glycolic acid) (MAL-PEG-PLGA); or combinations thereof.

[0031] The PLA may comprise the formula: or [-C(CH3)HC(=O)O-]„, wherein n is a number of repeat units.

[0032] Other hydrophobic polymers may comprise repeating units, with an integer of n. The integer n may be any suitable number, such as to provide the molecular weights herein described. The hydrophobic polymer, such as PLA, may have a MW of up to about 30 kDa. In another embodiment, the hydrophobic polymer, such as PLA, may have a MW of from 1 to 30 kDa. In another embodiment, the hydrophobic polymer, such as PLA, may have a MW of up to about 10,000 Da. In another embodiment, the hydrophobic polymer, such as PLA, may have a MW of up to 5000 Da. In another embodiment, the hydrophobic polymer, such as PLA, may have a MW of from about 1000 to 10,000 Da. In another embodiment, the hydrophobic polymer, such as PLA, may have a MW of from about 1500 to 10,000 Da. In another embodiment, the hydrophobic polymer, such as PLA, may have a MW of from about 2000 to 10,000 Da. In another embodiment, the hydrophobic polymer, such as PLA, may have a MW of from about 1500 to 4500 Da. In another embodiment, the hydrophobic polymer, such as PLA, may have a MW of from about 1500 to 5000 Da. In another embodiment, the hydrophobic polymer, such as PLA, may have a MW of from about 1500 to 2500 Da. In one embodiment, the hydrophobic polymer, such as PLA, has a MW of about 2000 Da. In a preferred embodiment, the hydrophobic polymer, such as PLA, may have a MW of from 1 to 5 kDa.

[0033] In a preferred embodiment, the PLA is poly(l-lactic acid).

[0034] The hydrophobic polymer binding

[0035] In one embodiment, there is a single hydrophobic polymer molecule covalently bound to human serum albumin, wherein the hydrophobic polymer is covalently bound at Cys34 of HSA or an equivalent residue of a non-human serum albumin.

[0036] The hydrophobic polymer may be covalently bound to the Cys34 side chain of serum albumin via a maleimide group. The hydrophobic polymer may be covalently bound to the serum albumin by reducing the Cys34 side chain to a free sulfhydryl group by using a reductant, such as TCEP, then 4-azidobenzoic acid may be added to neutralize the excess TCEP molecules and release acidic by-products. Functionalised maleimide- hydrophobic polymer, such as maleimide-polylactic acid, may be conjugated to the reduced serum albumin to create the conjugate molecule. In one embodiment, the hydrophobic polymer may comprise an alkyl halide, which can undergo a nucleophilic substitution reaction with thiol (e.g. of Cys34) to form a thioether (e.g. alkyl thioether or alkyl sulfide).

[0037] In another embodiment, the hydrophobic polymer may comprise an acyl halide (such as acetyl chloride), which can react with thiol (e.g. of Cys34) through nucleophilic acyl substitution to form a thioester.

[0038] In another embodiment, the hydrophobic polymer may comprise a sulfonyl halide (e.g., methyl sulfonyl chloride), which can react with thiol (e.g. of Cys34) through nucleophilic substitution to form sulfone.

[0039] In another embodiment, the hydrophobic polymer may comprise a disulfide, which can undergo thiol-disulfide exchange reactions with other thiols (e.g. Cys34) or disulfide compounds to form a new disulfide linkage.

[0040] In another embodiment, the hydrophobic polymer may comprise an epoxides, which can react with thiol (e.g. Cys34) through ring-opening reactions to form a thioether.

[0041] In another embodiment, the hydrophobic polymer may comprise an isocyanate, which can react with thiol (e.g. Cys34) to form thiourea.

[0042] In another embodiment, the hydrophobic polymer may comprise maleimide, which can react with thiol (e.g. Cys34) through a Michael addition reaction to form a thioether linkage.

[0043] In another embodiment, the hydrophobic polymer may comprise a vinyl sulfone, which can react with thiol (e.g. Cys34) to form a thioether linkage.

[0044] Polymeric Nanoparticles

[0045] A plurality of self-assembling conjugate molecules may be provided. The selfassembling conjugate molecule(s) may be in an assembled form. For example, a population of the self-assembling conjugate molecules may be provided in the form of a complex, such as a nanoparticle (also known as a polymeric nanoparticle). The conjugate molecule may be self-assembled into micelles during its formation (e.g. during the reaction of the hydrophobic polymer to the HSA or non-human serum binding protein. The nanoparticle(s) may be from 50nm to 500nm in size. In another embodiment, the nanoparticle(s) may be from 50nm to 300nm in size. In another embodiment, the nanoparticle(s) may be from 80nm to 200nm in size. In another embodiment, the nanoparticle(s) may be from 90nm to 150nm in size. In one embodiment, the nanoparticle(s) is about 100 nm in size (i.e. diameter). Reference to the size / diameter of the nanoparticles may refer to the average size / diameter in a population of nanoparticles.

[0046] In a preferred embodiment, the nanoparticle size distributions in a population of nanoparticles have a polydispersity index of less than 1 (<1).

[0047] The size of the nanoparticles may be determined by dynamic light scattering (DLS), for example as measured by a Malvern Nano-ZS instrument or an equivalent thereof. Additionally or alternatively, zeta potential of the nanoparticles may be determined by electrophoresis, for example as measured by a Malvern Nano-ZS instrument or an equivalent thereof.

[0048] The nanoparticle(s) may have a zeta potential of from about -5 to about -100 mV, for example for nanoparticles that are non-acetylated, such as HSA-PLA (PTX). In one embodiment, the nanoparticle(s) may have a zeta potential of about -14 to about -50 mV, for example for nanoparticles that are non-acetylated, such as HSA-PLA (PTX). In another embodiment, the nanoparticle(s) may have a zeta potential of about -15 to about -60 mV, for example for nanoparticles that are acetylated, such as Ac-HSA-PLA (PTX).

[0049] Acetylation and other modifications

[0050] The nanoparticle may be acetylated, for example by incorporation of the acetylated conjugate molecules described herein. Preferably, the conjugate molecule is acetylated prior to assembly into the nanoparticle.

[0051] The serum albumin of the conjugate molecule may be acetylated, for example by acetylating the amine groups of lysine residues of the serum albumin, such as HSA. The acetate may be bound to the lysine residues, or other residues, via a covalent amide bond. The acetylation may be by reacting the conjugate molecule with sulfo-NHS- acetate, or another acetylating agent. Other acetylating agents that may be used include NHS-acetate (N-hydroxysuccinimide acetate), acetic anhydride, aspirin (acetylsalicylic acid), and acetate esters.

[0052] Preferably lysine residues are acetylated. Additionally or alternatively, other residues may be acetylated, such as those residues comprising carboxylic acid groups. For example, one or more of glutamic acids, serine, and aspartic acid residues may be acetylated.

[0053] The nanoparticle may be acetylated throughout the nanoparticle, such as in the core of the nanoparticle. The nanoparticle may be acetylated in the core and on the surface. The acetylation may not be only surface acetylation of the nanoparticle.

[0054] The HSA or non-human serum albumin may comprise acetylated residues, such as lysine residues. The level of acetylation of the lysine and / or other residues in the HSA or non- human serum albumin may be at least 25%. In one embodiment, the level of acetylation of the lysine and / or other residues in the HSA or non-human serum albumin may be at least 50%. In another embodiment, the level of acetylation of the lysine and / or other residues in the HSA or non-human serum albumin may be at least 80%. In another embodiment, the level of acetylation of the lysine and / or other residues in the HSA or non-human serum albumin may be at least 90%. In another embodiment, the level of acetylation of the lysine and / or other residues in the HSA or non-human serum albumin may be from 25% to 100%. In another embodiment, the level of acetylation of the lysine and / or other residues in the HSA or non-human serum albumin may be from 90% to 100%. In one embodiment, about 100% of the lysine and / or other residues in the HSA or non-human serum albumin are acetylated.

[0055] The nanoparticle may be further modified with one or more functional groups. For example, the nanoparticle may be covalently bound to a functional molecule via one or more of the free lysine amine groups provided in the nanoparticle.

[0056] Advantageously, there would be 59 free lysine amine groups for further functional modification in a HSA that may be conjugated in accordance with the present invention. The functional groups may comprise a chemical moiety. The functional groups may comprise an amino acid, or a polymer, such as a peptide, a polypeptide, a nucleic acid, or a synthetic polymer; or combinations thereof. The functional groups may comprise a ligand or receptor molecule. The functional groups may comprise an antigen binding molecule, such as an antibody, antibody fragment, or antibody mimetic.

[0057] In one embodiment, the functional groups are selected from the group consisting of carboxylic acid groups, such as Asp and Glu; hydroxyl groups, such as Ser and Thr; or combinations thereof.

[0058] Encapsulation of agents, such as pharmaceutically active agents (e.g. drug)

[0059] The nanoparticle may encapsulate one or more agents, which may be pharmaceutically active agents or diagnostic agents or contrast agents.

[0060] The nanoparticle may encapsulate one or more small molecules (e.g. molecules of 2500Da or less). Preferably the small molecules are hydrophobic small molecules. The small molecule may be a peptide, such as a hydrophobic peptide. The encapsulated small molecule may comprise or consist of a hydrophobic peptide of 2500 Da or less, such as 2000 Da or less.

[0061] The nanoparticle may encapsulate one or more pharmaceutically active agents, such as paclitaxel (PTX), irinotecan (IRI, or CPT-11, or CPT), SN-38, artemisinin (ART) or imatinib (IMA); or combinations thereof. In one embodiment, the nanoparticle encapsulates SN-38. The pharmaceutically active agent may comprise or consist of an agent selected from docetaxel (DTX), imatinib, artemisinin, irinotecan and cyclosporin A (CsA); or combinations thereof. The nanoparticle may encapsulate one or more chemotherapeutic drugs, which may be cytotoxic. The pharmaceutically active agent may comprise or consist of a taxane. The pharmaceutically active agent may comprise or consist of an agent selected from PTX, DTX, and cabazitaxel (CTX); or combinations thereof. In one embodiment, the nanoparticle encapsulates PTX. In one embodiment, the nanoparticle encapsulates CTX.

[0062] The encapsulated small molecules and / or pharmaceutically active agents may otherwise be termed herein as “drugs”. In an embodiment wherein the conjugate of the nanoparticle is an acetylated form, the encapsulated pharmaceutically active agent may comprise paclitaxel.

[0063] The pharmaceutically active agent may be a hydrophobic molecule, such as a small molecule of 2500 Da or less. The pharmaceutically active agent may be a small molecule with a positive Log P.

[0064] The nanoparticle may encapsulate one or more of the pharmaceutically active agents selected from curcumin, griseofulvin, ritonavir, and lomustine.

[0065] The nanoparticle may encapsulate one or more of a type II topoisomerase inhibitor (e.g. Mitoxantrone); an antineoplastic enzyme inhibitor (e.g. Irinotecan, or its active metabolite SN-38); an antimitotic anticancer agent (e.g. Vinorelbine); an anthracycline topoisomerase II inhibitor (e.g. Epirubicin and / or Etoposide); an antineoplastic agent (e.g. Methotrexate); a microtubules stabilizer (e.g. Docetaxel and / or Paclitaxel and / or cabazitaxel); and a STAT3 and / or NF-KB signaling pathways inhibitor (e.g. Curcumin).

[0066] The agent, such as a pharmaceutically active agent, may be loaded into the nanoparticle in an amount up to 30% w / w, optionally wherein the nanoparticle comprises the acetylated form of the conjugate. In another embodiment, the agent, such as a pharmaceutically active agent, may be loaded into the nanoparticle in an amount up to 20% w / w, optionally wherein the nanoparticle comprises the acetylated form of the conjugate. In another embodiment, the agent, such as a pharmaceutically active agent, may be loaded into the nanoparticle in an amount up to about 15% w / w, optionally wherein the nanoparticle comprises the acetylated form of the conjugate. The agent, such as a pharmaceutically active agent, may be loaded into the nanoparticle in an amount greater than 12%. The agent, such as a pharmaceutically active agent, may be loaded into the nanoparticle in an amount from about 12% to 20%.

[0067] In an embodiment wherein the pharmaceutically active agent comprises PTX, the PTX may be loaded into the nanoparticle in an amount up to 16% w / w. In one embodiment wherein the pharmaceutically active agent comprises PTX, the PTX may be loaded into the nanoparticle in an amount greater than 12% w / w. In one embodiment wherein the pharmaceutically active agent comprises PTX, the PTX may be loaded into the nanoparticle in an amount from 12% to 16% w / w.

[0068] In an embodiment wherein the pharmaceutically active agent comprises CTX, the CTX may be loaded into the nanoparticle in an amount up to 15% w / w. In one embodiment wherein the pharmaceutically active agent comprises CTX, the CTX may be loaded into the nanoparticle in an amount greater than 12% w / w. In one embodiment wherein the pharmaceutically active agent comprises CTX, the CTX may be loaded into the nanoparticle in an amount from 12% to 15% w / w.

[0069] In an embodiment wherein the pharmaceutically active agent comprises imatinib, the imatinib may be loaded into the nanoparticle in an amount up to 13% w / w. In an embodiment wherein the pharmaceutically active agent comprises irinotecan, the irinotecan may be loaded into the nanoparticle in an amount up to 20% w / w. In an embodiment wherein the pharmaceutically active agent comprises cyclosporin A, the cyclosporin A may be loaded into the nanoparticle in an amount up to 17% w / w.

[0070] The nanoparticle may be loaded with one or more small molecules and / or pharmaceutically active agents by the process of adding the hydrophobic polymer, such as PLA, to aqueous medium followed by the addition of the pharmaceutically active agent dissolved in a lower volume of solvent and mixing by probe sonication, followed by lyophilisation to produce a powder that is readily reconstituted into drug loaded nanoparticles.

[0071] The nanoparticle may alternatively or additionally be loaded with other molecules, which may have a therapeutic or diagnostic effect, such as metal particles. These molecules, such as metal particles, may only have therapeutic effect under specific conditions e.g. when first irradiated with a suitable excitation laser. For example, the nanoparticle may be loaded with gold (Au) particles, or iron oxide particles, or both. The gold particles may comprise or consist of ultrasmall gold nanoclusters (AuNCs), for example less than about 3 nm. The gold nanoclusters may comprise from about 10 to about 100 gold atoms. The iron oxide particles may comprise magnetite (FesCh) and / or maghemite (y-Fe2C>3) particles. In some preferred embodiments, the nanoparticle of the invention comprises a Ac-HSA nanoparticle further comprising PLGA and AuNC particles; these embodiments may be referred to as Ac-HSA-PLGA-AuNC. The excitation laser may be of sufficient power to excite the additional molecules, such as metal particles, through the skin of the subject. The person skilled in the art will be familiar with the typical excitation wavelengths of commonly used metal particles, and will be able to match a suitable laser to a given type of metal particle, e.g. an 808 nm laser may be used to excite nanoparticles comprising AuNC particles. In some embodiments, nanoparticles comprising i) metal particles, such as AuNC particles, and ii) no other therapeutic molecule, are delivered to the same subject alongside (concurrently with, prior to, or after) the delivery of nanoparticles comprising i) a therapeutic molecule, such as PTX, and ii) no metal particles, e.g. by administering a mixture of the two types of nanoparticles. The metal particles may be nano-scale, such as less than 500 nm. In one embodiment, the metal particles are less than 100 nm. In one embodiment, the metal particles are less than 50 nm. In one embodiment, the metal particles are less than 10 nm. In another embodiment, the metal particles are less than 5 nm. In another embodiment, the metal particles are less than 3 nm. In another embodiment, the metal particles are 2 nm or less. In another embodiment, the metal particles are 1-3 nm on average.

[0072] Other Aspects

[0073] According to another aspect of the invention there is provided a nanoparticle as described herein.

[0074] The nanoparticle may comprise a plurality of the conjugate molecules according to the invention herein.

[0075] In one embodiment, the nanoparticles encapsulate one or more pharmaceutically active agents, for example as described herein. In another embodiment, the nanoparticles encapsulate metal nanoparticles, such as gold or iron oxide.

[0076] In one embodiment, the nanoparticle comprises the PLA-HSA or Ac-PLA-HSA as described herein. In another embodiment, the nanoparticle comprises the PLA-HSA- PTX or Ac-PLA-HSA-PTX as described herein.

[0077] Compositions According to another aspect of the invention there is provided a composition comprising conjugate molecules according to the invention herein.

[0078] According to another aspect of the invention there is provided a composition comprising the nanoparticles of the invention.

[0079] The composition may comprise two or more types of nanoparticle according to the invention, for example the composition may comprise a nanoparticle encapsulating a metal particle, such as gold, and a nanoparticle encapsulating a therapeutic drug, such as a taxane. In one embodiment, the composition comprises a nanoparticle encapsulating a gold nanoparticle, and a nanoparticle encapsulating a taxane, such as PTX.

[0080] The composition may be a pharmaceutically acceptable composition. The composition may comprise one or more pharmaceutically acceptable excipients. In one embodiment, the nanoparticles are suspended in a carrier, such as an aqueous carrier.

[0081] In one embodiment, the carrier is a pharmaceutically acceptable carrier. The carrier may comprise or consist of an aqueous medium, such as a saline solution or buffer. The carrier may comprise sodium chloride and / or a sugar such as dextrose. In one embodiment, the carrier comprises sodium chloride (e.g. about 0.9%), dextrose (e.g. about 5%) or water. The carrier may be suitable for delivery by injection, such as s.c. or i.v. injection.

[0082] The nanoparticles are provided in the composition at a concentration of from 10 to 200 mg / ml. In another embodiment, the nanoparticles are provided in the composition at a concentration of up to 80 mg / mL.

[0083] Method of Manufacture

[0084] According to another aspect of the invention there is provided a method of producing a conjugate molecule according to the invention, the method comprising covalently binding a hydrophobic polymer, such as PLA, to human serum albumin (HSA) only at residue Cys34; or covalently binding a hydrophobic polymer, such as PLA, to a nonhuman serum albumin protein only at an equivalent residue to Cys34 of HSA. The hydrophobic polymer may be covalently bound to the Cys34 side chain of serum albumin via a Maleimide group. The hydrophobic polymer may be covalently bound to the serum albumin by reducing the Cys34 side chain to a free sulfhydryl group by using a reductant, such as TCEP, then 4-azidobenzoic acid may be added to neutralize the excess TCEP molecules and release acidic by-products. Functionalised maleimide- hydrophobic polymer, such as maleimide-polylactic acid, may be conjugated to the reduced serum albumin to create the conjugate molecule.

[0085] The method may further comprise acetylating the human or non-human serum albumin. Preferably the acetylation of the human or non-human serum albumin is before the hydrophobic polymer, such as PLA, is covalently bound to the Cys34.

[0086] According to another aspect of the invention there is provided a method of producing a nanoparticle according to the invention, the method comprising assembling a plurality of the conjugate molecules according to the invention into a nanoparticle.

[0087] Preferably, the nanoparticle is a population of nanoparticles.

[0088] The assembly may be self-assembly of the conjugate molecules in aqueous media. The method may comprise mixing a suspension of conjugate molecules according to the invention in an aqueous solution. The method may further comprise the addition of one or more agents for encapsulation into the nanoparticles. The method may further comprise the addition of one or more small molecules and / or pharmaceutically active agents for encapsulation into the nanoparticles. The addition of one or more agents for encapsulation into the nanoparticles may be before the hydrophobic polymer is covalently bound to the human or non-human serum albumin. The addition of one or more small molecules and / or pharmaceutically active agents for encapsulation into the nanoparticles may be before the hydrophobic polymer is covalently bound to the human or non-human serum albumin.

[0089] According to another aspect of the invention there is provided a method of producing a composition according to the invention, The assembly may comprise emulsification. The method may further comprise the addition of one or more agents for encapsulation into the nanoparticles. The method may further comprise the addition of one or more small molecules and / or pharmaceutically active agents for encapsulation into the nanoparticles.

[0090] Methods of Treatment

[0091] The nanoparticles and / or composition according to the invention may be a medicament. The nanoparticles and / or composition according to the invention may be suitable for treatment or prevention of a disease or condition of a subject.

[0092] The disease may be cancer. The cancer may be metastatic, such as metastatic breast cancer. The cancer may be neuroblastoma cancer, pancreatic cancer, ovarian cancer, or prostate cancer. The cancer may be neuroblastoma cancer, pancreatic cancer, ovarian cancer, bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, non-melanoma skin cancer, thyroid cancer or prostate cancer.

[0093] In one embodiment, the cancer is selected from any of the list comprising Adamantinoma, Chondrosarcoma, Chordoma, Ewing's sarcoma, Fibrocartilaginous mesenchymoma of bone, Leiomyosarcoma, Malignant fibrous histiocytoma of bone / osteosarcoma, Myxosarcoma, Osteosarcoma, Rhabdomyosarcoma, Astrocytoma, Brainstem glioma, Choroid plexus carcinoma, Cerebellar astrocytoma, Cerebral astrocytoma, Craniopharyngioma, Ependymoma, Ganglioneuroma, Glioblastoma, Glioma, Hemangioblastoma, Medulloblastoma, Meningioma, Neuroblastoma, Neurofibroma, Oligodendroglioma, Paraganglioma, Pineal astrocytoma, Pineocytoma, Pineoblastoma, Pituitary adenoma, Pilocytic astrocytoma, Primary central nervous system lymphoma, Primitive neuroectodermal tumor, Schwannoma, Visual pathway and hypothalamic glioma, Breast cancer, Ductal carcinoma in situ, Inflammatory breast cancer, Invasive ductal carcinoma, Invasive lobular carcinoma, Tubular carcinoma, Invasive cribriform carcinoma of the breast (also termed invasive cribriform carcinoma), Medullary carcinoma, Male breast cancer, Phyllodes tumor, Mammary secretory carcinoma, Mucinous carcinoma of the breast, Papillary carcinomas of the breast, Adrenocortical adenoma, Adrenocortical carcinoma, Carcinoid, Gastrinoma, Glucagonoma, Insulioma, Islet cell carcinoma (endocrine pancreas), Merkel cell carcinoma, Multiple endocrine neoplasia syndrome, Pancreatic Cancer, Parathyroid cancer, Pheochromocytoma, Somatostatinoma, Thyroid cancer, VIPoma, Conjunctival melanoma, Optic nerve glioma, Orbital lymphoma, Retinoblastoma, Uveal melanoma, Gastrointestinal, Anal cancer, Appendix cancer, Cholangiocarcinoma, Carcinoid tumor, gastrointestinal, Colon cancer, Duodenal cancer, Extrahepatic bile duct cancer, Gallbladder cancer, Gastric (stomach) cancer, Gastrointestinal carcinoid tumor, Gastrointestinal stromal tumor (GIST), Hepatoblasoma, Hepatocellular cancer, Pancreatic cancer, islet cell, Rectal cancer, Small intestine cancer, Bladder cancer, Cervical cancer, Choriocarcinoma, Embryonal carcinoma, Endometrial cancer, Endodermal sinus tumor, Extragonadal germ cell tumor, Fallopian tube cancer, Gestational trophoblastic tumor, Kidney cancer, Leydig cell tumour, Ovarian cancer, Ovarian epithelial cancer (surface epithelial-stromal tumor), Ovarian germ cell tumor, Penile cancer, Prostate cancer, Renal cell carcinoma, Renal pelvis and ureter, transitional cell cancer*, Seminoma, Serous tumour, Sertoli cell tumour, Teratoma, Testicular cancer, Transitional cell cancer(urothelial carcinoma), Ureter and renal pelvis, Urethral cancer, Uterine sarcoma, Vaginal cancer, Vulvar cancer, Wilms tumor (nephroblastoma), Esophageal cancer, Head and neck cancer, Nasopharyngeal carcinoma, Oral cancer, Oropharyngeal cancer, Paranasal sinus and nasal cavity cancer, Pharyngeal cancer, Salivary gland cancer, Hypopharyngeal cancer, Acute biphenotypic leukemia, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute myeloid leukemia, Acute myeloid dendritic cell leukemia, AIDS-related lymphoma, Anaplastic large cell lymphoma, Angioimmunoblastic T-cell lymphoma, B-cell prolymphocytic leukemia, Burkitt's lymphoma, Chronic lymphocytic leukemia, Chronic myelogenous leukemia, Cutaneous T-cell lymphoma, Diffuse large B-cell lymphoma, Follicular lymphoma, Hairy cell leukemia, Hepatosplenic T-cell lymphoma, Hodgkin's lymphoma, Intravascular large B-cell lymphoma, Large granular lymphocytic leukemia, Lymphoplasmacytic lymphoma, Lymphomatoid granulomatosis, Mantle cell lymphoma, Marginal zone B-cell lymphoma, Mast cell leukemia, Mediastinal large B cell lymphoma, Multiple myeloma / plasma cell neoplasm, Myelodysplastic syndromes, Mucosa-associated lymphoid tissue lymphoma, Mycosis fungoides, Nodal marginal zone B cell lymphoma, Non-Hodgkin lymphoma, Precursor B lymphoblastic leukemia, Primary central nervous system lymphoma, Primary cutaneous follicular lymphoma, Primary cutaneous immunocytoma, Primary effusion lymphoma, Plasmablastic lymphoma, Sezary syndrome, Splenic marginal zone lymphoma, T-cell prolymphocytic leukemia, Basal cell carcinoma, Squamous cell carcinoma, Squamous cell skin cancer, Skin adnexal tumors (e.g. sebaceous carcinoma), Melanoma, Merkel cell carcinoma, Keratoacanthoma, Sarcomas of primary cutaneous origin (e.g. dermatofibrosarcoma protuberans), Lymphomas of primary cutaneous origin (e.g. mycosis fungoides), Soft Tissue Sarcoma, Angiosarcoma, Fibrosarcoma, Liposarcoma, Malignant peripheral nerve sheath tumor, Synovial sarcoma, Thoracic and respiratory, Adenocarcinoma of the lung, Basaloid squamous cell lung carcinoma, Bronchial adenomas / carcinoids, Giant-cell carcinoma of the lung, Large-cell lung carcinoma, Large cell lung carcinoma with rhabdoid phenotype, Laryngeal cancer, Mesothelioma, Non-small cell lung cancer, Non-small cell lung carcinoma, Pleuropulmonary blastoma, Sarcomatoid carcinoma of the lung, Small cell lung cancer, Squamous-cell carcinoma of the lung, Thymoma and thymic carcinoma, AIDS-related cancers, and Kaposi sarcoma; or combinations thereof.

[0094] Therefore, according to another aspect of the invention there is provided a method of treatment or prevention of cancer in a subject, the method comprising the administration of the nanoparticles and / or composition according to the invention to a subject.

[0095] The disease may be an infectious disease, such as septicaemia. The infectious disease may be a viral, bacterial, fungal or protozoal infection.

[0096] According to another aspect of the invention there is provided the nanoparticle or composition according to the invention for use as a medicament.

[0097] According to another aspect of the invention there is provided the nanoparticle or composition according to the invention for use in the treatment or prevention of cancer.

[0098] In one embodiment, the treatment comprises the administration of a nanoparticle encapsulating a metal particle, such as a gold or iron oxide nanoparticle, and the tissue- localised excitement of the metal particle, such as by application of a laser.

[0099] The treatment may be a combination treatment comprising the administration of two or more types of nanoparticles or compositions according to the invention. In an embodiment wherein at least one of the nanoparticles encapsulates a metal particle, such as a gold or iron oxide nanoparticle (such as a gold nanocluster), the treatment may further comprise a tissue-localised excitement of the metal particle, such as by application of a laser. The wavelength may be capable of excitement of the metal particle, such as a gold nanoparticle. The subject may be mammalian. In a preferred embodiment, the subject is human.

[0100] The subject may be afflicted with a disease or condition to be treated. In one embodiment, the subject has, is suspected to have, or is at risk of, cancer. The cancer may be a solid tumour cancer. In one embodiment, the cancer is breast cancer or neuroblastoma cancer. In one embodiment, the cancer is metastatic such as metastatic breast cancer. The subject may be afflicted with neuroblastoma cancer, pancreatic cancer, ovarian cancer, or prostate cancer.

[0101] In another embodiment, the subject may be afflicted with an infectious disease, such as septicaemia. The infectious disease may be a viral, bacterial, fungal or protozoal infection. In such cases, the pharmaceutically active agent to be encapsulated may be an antimicrobial effective for treating such infections.

[0102] The nanoparticles or composition of the invention may be administered to the patient in a pharmaceutically effective dose. The nanoparticles or composition of the invention may be administered to the patient at a dose of from 100 to 300 mg / m2of active drug (refers to active drug dose, not the nanoparticle dose). The nanoparticles or composition of the invention may be administered to the patient at a dose of about 260 mg / m2of active drug. The dosing may be once or repeated. In one embodiment, the administration is repeated until a desired therapeutic effect is achieved. In one embodiment, the administration is repeated weekly, for example for a period of at least 3 weeks.

[0103] Definitions

[0104] It is understood that Human serum albumin (HSA) is the most abundant protein in plasma (0.6 mM), comprising about 60% of total plasma proteins. It is synthesized in the liver as a pre-pro-protein with two additional segments, the prepeptide and the propeptide. The prepeptide (18 amino acids) constitutes the secretion signal and leads HSA out of the hepatocyte, while the propeptide (6 amino acids) is of unknown function. Both segments are located in the N-terminal portion of the protein and are cleaved before protein secretion, resulting in mature HSA. HSA is a monomeric protein consisting of three homologous domains (domains I, II and III). Among its 585 amino acids it possesses 35 cysteines that form 17 disulfide bonds. These disulfide bonds are essential for the stability of the protein forming 17 intramolecular disulfide bridges that are present primarily between the a-helices. Domain I (Dom I) comprises residues 1 to 195 and stands out for having the only free cysteine of the protein, Cys34. Free cysteine residue (Cys-34), is present in domain I and is conserved across species. This residue is responsible for albumin dimerization during purification by forming an intermolecular disulfide bridge. The free cysteine residue is in a ~ 10 A deep crevice and plays a critical role in the redox properties of albumins shown to be crucial in cell culture .

[0105] Reference to the covalent binding of the hydrophobic polymer, such as PLA, “only at residue Cys34” is understood to mean that no other residue in the serum albumin is covalently bound to the hydrophobic polymer, such as PLA, or any other hydrophobic polymer, i.e. The serum albumin may be conjugated to only a single hydrophobic polymer, such as PLA. There may be one hydrophobic polymer per serum albumin.

[0106] Reference to an “equivalent residue” is understood to mean the same residue that would be aligned with the reference residue, i.e. Cys34 of HSA in an amino acid sequence alignment and / or would be considered as the equivalent residue as Cys34. The equivalent residue may be a cysteine, for example in a similar position to the Cys34 of HSA.

[0107] Where reference is made to a variant polypeptide or nucleotide sequence, the skilled person will understand that one or more amino acid residue or nucleotide substitutions, deletions or additions, may be tolerated, optionally two substitutions may be tolerated in the sequence, such that it maintains its function. The skilled person will appreciate that 1, 2, 3, 4, 5 or more amino acid residues or nucleotides may be substituted, added or removed without affecting function References to sequence identity may be determined by BLAST sequence alignment (www.ncbi.nlm.nih.gov / BLAST / ) using standard / de fault parameters. For example, the sequence may have 99% identity and still function according to the invention. In other embodiments, the sequence may have 98% identity and still function according to the invention. In another embodiment, the sequence may have 95% identity and still function according to the invention. In another embodiment, the sequence may have 90%, 85%, or 80% identity and still function according to the invention. In one embodiment, the variation and sequence identity may be according the full length sequence. In other embodiments, the variation may be limited to non-conserved sequences and / or sequences outside of active sites, such as binding domains. Therefore, an active site or binding site of a protein may be 100% identical, whereas the flanking sequences may comprise the stated variations in identity. Such variants may be termed “conserved active site variants”.

[0108] Amino acid substitutions may be conservative substitutions. For example, a modified residue may comprise substantially similar properties as the wild-type substituted residue. For example, a substituted residue may comprise substantially similar or equal charge or hydrophobicity as the wild-type substituted residue. For example, a substituted residue may comprise substantially similar molecular weight or steric bulk as the wild-type substituted residue. With reference to “variant” nucleic acid sequences, the skilled person will appreciate that 1, 2, 3, 4, 5 or more codons may be substituted, added or removed without affecting function. For example, conservative substitutions may be considered.

[0109] The term “nano”, such as used in “nanoparticle”, is intended to refer to size in the 1- 1000 nm range, preferably 50-500 nm range.

[0110] The skilled person will appreciate that preferred features of any one embodiment and / or aspect of the invention may be applied to all other embodiments and / or aspects of the invention.

[0111] Examples embodying an aspect of the invention will now be described with reference to the following figures:

[0112] Fig. 1. Schematic illustration of HSA-PLA polymeric micelles preparation process, and PTX loading.

[0113] Fig 2. Synthetic process of HSA-PLA including reducing, quenching and conjugation steps. TCEP was used to release the sulfhydryl group of Cys-34, then 4-Azidobenzoic acid was added to neutralize the excess TCEP. At last, PLA was conjugated to the reduced HSA via the maleimide reaction.

[0114] Fig 3. MALDI-TOF spectrum. Aldolase (39212 Da) was used as internal calibrator. (A) Mass spectrum of HSA standard. (B) Mass spectrum of HSA- PLA conjugates. The molecule weight of HSA standard and HSA-PLA conjugate was 66479 ± 35 Da and 67863 ± 282 Da, respectively (p < 0.01).

[0115] Fig 4. FT-IR spectra of HSA standard, MAL-PLA standard and HSA-PLA lyophilized powder. MAL-PLA has one characteristic absorption peak at 1756 cm-1 of ester carbonyl group (C = O), which is present in HSA-PLA FT-IR spectrum and absent in HSA standard spectrum.

[0116] Fig 5. Characterizing HSA-PLA nanoparticles in the aspects of size distribution and zeta-potential. Hydrodynamic diameter distribution of A) HSA standard and B) HSA-PLA NPs and C) zeta potential distribution of HSA standard and HSA-PLA NPs were obtained through Malvern Nano-ZS instrument. HSA molecules in water were multi-distributed. While after the conjugation of PLA to HSA, HSA-PLA nanoparticles were mono-distributed. HSA-PLA nanoparticles had a larger size and more negative zeta potential when compared with HSA standard.

[0117] Fig 6. A) TEM image of human serum albumin molecules. B) TEM image of HSA-PLA nanoparticles.

[0118] Fig 7. Schematic illustration for encapsulating paclitaxel in HSA-PLA nanoparticles through the probe sonication. Aggregated MeOH-PTX micelles can be re-dispersed to the separated MeOH-PTX particles. Then nanoscale MeOH-PTX particles can be delivered into the hydrophobic cores of HSA-PLA nanoparticles during the probe sonication.

[0119] Fig 8. Photographs of distilled water, albumin water solution and albumin nanosuspensions. (A) Appearances of Abraxane®, HSA-PLA and HSA-PLA (PTX) nanosuspensions were milky, off-white liquid while water and HSA water solution were both colourless liquids. Photograph (B) showed the Tyndall effect within colloidal suspensions, path of red laser was visible in Abraxane®, HSA- PLA and HSA-PLA (PTX) colloidal suspensions and invisible in water and HSA solution when red laser pass through these aqueous media. Fig 9. DLS results of each nanosuspension. Hydrodynamic diameter distribution of A) Taxol®, B) HSA-PLA blank nanoparticles, C) Abraxane® and HSA-PLA (PTX) in distilled water. Zeta potential of Abraxane nanoparticles, HSA-PLA blank nanoparticles and HSA-PLA (PTX) in distilled water was presented in graph (D).

[0120] Fig 10. Comparison of Abraxane nanoparticles, HSA-PLA nanoparticles and HSA-PLA (PTX) nanoparticles in terms of morphology, DH, PDI and zeta-potential. The agglomeration of Abraxane nanoparticles indicated that the Abraxane nanoparticles were unstable in comparison of HSA-PLA (PTX) nanoparticles. Moreover, the more negative zeta-potential of PTX-loaded HSA- PLA nanoparticles (-34 mV) should confer a higher colloidal stability compared with Abraxane (-13mV).

[0121] Fig 11. Colloidal stability of HSA-PLA (PTX) nanoparticles in aqueous media. The changes of hydrodynamic diameter and polydispersity index of HSA-PLA (PTX) nanosuspensions were monitored for 7 days and recorded as the graph of hydrodynamic diameter / PDI versus time (A). Hydrodynamic diameter of Abraxane and HSA-PLA (PTX) nanoparticles at different concentrations (B). Abraxane nanoparticles were unstable at the concentration of 0.3 mg / mL while HSA-PLA (PTX) nanoparticles were even stable at the concentration of 0.01 mg / mL.

[0122] Fig 12. Cellular uptake of HSA-PLA (Cy Red) nanoparticles in 4T1 cells after 1-h incubation. From top to bottom, cell images were presented at lOx, 20x and 40x magnification, respectively. Hoechst 33342 was used to stain cell nuclei and emit blue light after the excitation of light with 405 nm wavelength. HSA-PLA (Cy Red) nanoparticles were taken by every 4T1 cell and the red emitted light from the intracellular nanoparticles illuminated the whole cell.

[0123] Fig 13. Pharmacokinetics study of Taxol®, Abraxane® and HSA-PLA (PTX) in SD rats. (A) Profiles of PTX concentration in rat plasma versus time since injection following intravenous administration of Taxol®, Abraxane® and HSA- PLA (PTX) at a paclitaxel dose of 10 mg / kg. (B) PTX contents in the heart, liver, spleen, lungs and kidneys of rats after 72 hours of injection with PTX formulations.

[0124] Fig 14. Bar charts of percentage of injected paclitaxel in per gram tissue at different time points post-injection. The biodistribution of Taxol®, Abraxane® and HSA-PLA (PTX) nanoparticles in 4T1 tumour-bearing mice at different time points after the intravenous administration of each PTX formulation at the dose of PTX 20 mg / kg. Due to the EPR effect, the PTX levels in tumour were higher than PTX contents in organs after the 24-h and 72-h injection. Data are expressed as mean ± SD, n = 3.

[0125] Fig 15. Pharmacokinetic profiles of PTX concentration versus time postinjection for plasma and different tissue compartments. 4T1 tumour-bearing mice were intravenously injected with Taxol®, Abraxane® and HSA-PLA (PTX) at 20 mg / kg paclitaxel, respectively. Data are expressed as mean ± SD, n = 3.

[0126] Fig 16. In vivo anti-tumour study. (A) Tumour growth curves of 4T1 tumourbearing mice after different treatments. (B) Percent weight change in mice after the first injection, where positive changes indicated weight gain and negative changes meant mice weight loss compared to their body weight at the time of the first injection. (C) Tumour size and weight in different treatment groups 10 days after injection. Data are expressed as mean ± SD (n=5). *, p < 0.05. **, p < 0.01. *** ^ p < 0.001. **** , p < 0.0001.

[0127] Fig 17. Schematic illustration of Ac-HSA-PLA polymeric micelles preparation process. After the acetylation of human serum albumin (Ac-HSA), the decorated albumin (Ac-HSA) can then bind with the receptors, such as CD44 and folate receptor. By conjugating PLA to Ac-HSA, the novel tumour targeted nanocarriers, Ac-HSA-PLA nanoparticles, were created.

[0128] Fig 18. Chemical reactions in the synthetic process of acetylated albuminpolylactic acid conjugates (Ac-HSA-PLA). Sulfo-NHS-acetate was used to acetylate human serum albumin to create acetylated human serum albumin (Ac- HSA). Next, the occupied Cys 34 side chain was reduced to the free sulfhydryl group by using reductant TCEP. Then 4-azidobenzoic acid was added to neutralize the excess TCEP molecules and release acidic by-products. At last, maleimide-polylactic acid was conjugated to the reduced Ac-HSA to create Ac- HSA-PLA conjugates.

[0129] Fig 19. Mass spectra of HSA standard, HSA-PLA conjugate and Ac-HSA- PLA conjugate obtained from MALDI-TOF mass spectrometer. After the acetylation, the molecular weight of Ac-HSA was significantly higher than MW of HSA, 66479 ± 35 Dalton vs. 68374 ± 94 Dalton, p < 0.0001. After the conjugation of PLA to Ac-HSA, the MW of Ac-HSA-PLA was 69638 ± 116 Dalton, which was statistically higher than the MW of Ac-HSA, / ? < 0.0001. Data are presented as means ± SD, n = 3, **** , p < 0.0001.

[0130] Fig 20. Characterizing Ac-HSA-PLA nanoparticles in the terms of size distribution and zeta-potential. Hydrodynamic diameter distribution of a) Ac- HSA conjugates and b) Ac-HSA-PLA NPs and c) zeta potential distribution of HSA standard, Ac-HSA conjugates and Ac-HSA-PLA NPs were obtained through Malvern Nano-ZS instrument. Ac-HSA conjugates were polydisperse in water. While after the conjugation of PLA to Ac-HSA polymer, Ac-HSA-PLA nanoparticles were monodispersed. The more negative zeta potential of Ac- HSA-PLA nanoparticles than Ac-HSA conjugates is due to more positively charged amino acids were shielded during the formation of nanoparticles.

[0131] Fig 21. TEM images of HSA-PLA nanoparticles and Ac-HSA-PLA nanoparticles. The well separated Ac-HSA-PLA nanoparticles indicates that the acetylation of HSA does increase the electrostatic repulsion between nanoparticles as well as the colloidal stability compared with HSA-PLA nanoparticles.

[0132] Fig 22. Fluorescence microscopy images of MDA-MB-231 cells respectively incubated with Coumarin-6 labelled HSA-PLA nanoparticles and Coumarin-6 labelled Ac-HSA-PLA nanoparticles. Blue spots = cell nuclei and green dots = dye labelled nanoparticles. Coumarin-6 labelled Ac-HSA-PLA nanoparticles are taken up more efficiently by the cells. Fig 23. Confocal microscopy images of MCF-7 cells respectively incubated with FITC labelled HSA-PLA nanoparticles and FITC labelled Ac-HSA- PLA nanoparticles. Blue spots = cell nuclei and green dots = dye labelled nanoparticles. FITC labelled Ac-HSA-PLA nanoparticles are taken up more efficiently by the cells and tend to accumulate in cell nuclei.

[0133] Fig 24. Confocal microscopy images of BT549 cells respectively incubated with FITC labelled HSA-PLA nanoparticles and FITC labelled Ac-HSA- PLA nanoparticles. Blue spots = cell nuclei and green dots = dye labelled nanoparticles. FITC labelled Ac-HSA-PLA nanoparticles are taken up more efficiently by the cells.

[0134] Fig 25. Cellular uptake studies of HSA standard, HSA-PLA nanoparticles and Ac-HSA-PLA nanoparticles in MDA-MB-231 cells through flow cytometry. (A) The mean FITC fluorescent intensity per cell for different FITC labelled samples after different incubation time with cells. (B) The flow cytometry histogram of each group after 3 hours incubation with cells (Untreated, FITC-HSA, FITC-HSA-PLA nanoparticles, FITC-Ac-HSA-PLA nanoparticles) show higher mean FITC intensity values for FITC-Ac-HSA-PLA particles indicating higher levels of FITC-Ac-HSA-PLA nanoparticles are more associated with the cells. (C) Statistics analysis of FITC mean fluorescence intensity of each group shows that FITC-Ac-HSA-PLA nanoparticles are more associated with the cells, compared with HSA standard and HSA-PLA nanoparticles. Data are expressed as mean ± SD (n = 3). * * * *, p < 0.0001 ; ns r = not statistically significant.#

[0135] Fig 26. Competition inhibition assays for screening the potential receptors of Ac-HSA-PLA nanoparticles. After the preliminary screening, we found that hyaluronic acid and folic acid can significantly influence the cellular uptake of Ac-HSA-PLA nanoparticles and do not affect the uptake of HSA-PLA nanoparticles in MDA-MB-231 cells. And with more addition of folic acid and hyaluronic acid, less Ac-HSA-PLA nanoparticles were taken by MDA-MB-231 cells. Data are presented as means ± SD, n = 3. *, p < 0.05. * * * * , p < 0.0001; ns r = not statistically significant. Fig 27. Ex vivo imaging study on the Tumour-tropism of acetylated albumin nanoparticles compared with albumin nanoparticles. After 2 hours injection of NIR dye labelled nanoparticles, Ac-HSA-PLA (CyRed) and HSA-PLA (CyRed) nanoparticles, the organs, tissues and tumours were extracted and imaged.

[0136] Fig 28. Statistics analysis of the ex-vivo studies. The total radiant efficiency readings of each tissue and tumour were recorded and summarized in the column chart (A). The higher uptake ratio of liver to tumour (B) suggests that less of injected nanoparticles were taken by tumour. Data are presented as means ± SD, n = 3. **, p < 0.01.

[0137] Fig 29. XRD patterns of various samples. From top to bottom, XRD patterns of PTX standard, PTX + Ac-HSA-PLA nanoparticle mixture (1 :9 w / w), PTX loaded Ac-HSA-PLA nanoparticles, Ac-HSA-PLA blank nanoparticles and Abraxane® nanoparticles. Red arrows indicated the characteristic peaks of crystalline paclitaxel.

[0138] Fig 30. TEM images of A) Abraxane®, B) HSA-PLA (PTX) nanoparticles, C) and D) Ac-HSA-PLA nanoparticles. From a wide view of nanoparticles in TEM images, the appearance of Abraxane nanoparticles are apparently different with the morphology of HSA-PLA (PTX) nanoparticles and Ac-HSA-PLA (PTX) nanoparticles, although all of them are spherical nanoparticles.

[0139] Fig 31. Cytotoxicity of the PTX formulations in breast cancer cell lines: MCF7 (A), 4T1 (B), and MDA-MB-231 (C) cells. Wst-1 cell viability assays were performed after 48 hours drug treatment. Data are presented as mean ± SD (n = 3).

[0140] Fig 32. Anticancer activity of Ac-HSA-PLA (PTX) formulation in MDA-MB- 231 tumour model. The 1 x 107MDA-MB-231 cells in 100 pL pH 7.4 PBS were implanted in the right flank of each NOD-SCID mouse on Day 0, the Matrigel was not used in this study. (A) Tumour growth curves of the MDA-MB-231 tumour bearing mice after different treatments. (B) Mice body weight records. (C) Cumulative survival of the tumour bearing mice (Kaplan Meier). Data are presented as mean ± SD (n = 5).

[0141] Fig 33. In vivo anticancer study of HSA-PLA (SN-38) and Ac-HSA-PLA (SN- 38). MDA-MB-231 tumour models were created by subcutaneous injection of MDA-MB-231 cells at right flanks of NOD-SCID female mice. Each group contains 5 mice, n = 5.

[0142] Fig 34. In vivo anticancer study of Ac-HSA-PLGA-AuNC and Ac-HSA-PLA (PTX). 4T1 tumour models were created by subcutaneous injection of 4T1 cells at right flanks of BALB / c female mice. Each group contains 5 mice (n = 5). In the saline group, animals were intratumorally (IT) injected with 30 pL saline and heated 3 h later with an 808 nm laser for 10 min. In treatment groups, on day 0, animals were intratumorally injected with 30 pL of Ac-HSA-PLGA- AuNC nanosuspension, and heated 3 h later with an 808 nm laser for 10 min. Of them, 5 mice were intravenously (i.v.) injected with Ac-HSA-PLA (PTX) nanosuspension at a dose of PTX 160 mg / kg on day 1.

[0143] Summary of example embodiments

[0144] Embodiment 1 example - HSA-PLA project:

[0145] Passively targeting nanocarrier for paclitaxel - HSA-PLA (PTX)

[0146] HSA-PLA (PTX) vs. Abraxane: Demonstrates higher colloidal stability and payload.

[0147] Methods

[0148] BALB / c female mice (6-8 weeks) were subcutaneously injected with 4T1 cells (2X106 / 100 pL DPBS). When the tumour volume was approximately 100 mm3, mice were randomized into 4 groups (5 mice per group, n = 5). HSA / saline (400 mg / kg) as control group, Taxol® (eq. PTX 20 mg / kg), Abraxane® (eq. PTX 40 mg / kg) and HSA- PLA (PTX) (eq. PTX 40 mg / kg) were intravenously injected to each group’s mice, respectively. First injection at Day 0 and second injection at Day 3, in total of two injections. The tumour volume was measured every day by vernier calliper and calculated through the formula, tumour volume = 0.5 x length x width2. Once the tumour volume was around 1200 to 1500 mm3or loss of body weight more than 15% or tumour ulceration happened more than 3 days, mice were immediately euthanized. Results

[0149] According to the tumour growth curves (Figure 16A), tumour growth was significantly suppressed in mice treated with HSA-PLA (PTX) 40 mg / kg compared to mice treated with Abraxane® 40 mg / kg. The final tumour weight of mice treated with HSA-PLA (PTX) was 239.8 ± 43.0 mg, statistically smaller than group treated with Abraxane® 340.6 ± 62.8 mg (p < 0.5, Figure 16C). The reason for the better tumoricidal activity of HSA-PLA (PTX) was explained in the pharmacokinetic studies, as HSA-PLA (PTX) nanoparticles delivered more paclitaxel to tumour compared with Abraxane®. Although the tumour growth of Taxol® treated group was inhibited compared with control group (HSA / saline), the mouse body weight of Taxol® group was continuously decreasing because of unacceptable systemic toxicity caused by Taxol® (Figure 16B).

[0150] Embodiment 2 example - Ac-HSA-PLA project:

[0151] Tumour-tropism nanocarrier for paclitaxel - Ac-HSA-PLA (PTX)

[0152] Methods

[0153] Briefly, 5 tumour bearing mice per group and each treatment group (n = 5) was injected with Abraxane® (eq. PTX 20 mg / kg), HSA-PLA (PTX) and Ac-HSA-PLA (PTX) nanoparticles (eq. PTX 20 mg / kg) every three days with 5 injections in total, respectively. And the tumour volumes and mice body weights were recorded every three days.

[0154] Results

[0155] As shown in Fig 32, the MDA-MB-231 tumours were disappeared after 5 injections of Ac-HSA-PLA (PTX) nanosuspension. In this formal study, the significant difference in tumour size between mice treated with Abraxane® and Ac-HSA-PLA (PTX) nanoparticles can be observed.

[0156] Example 3 - Synthesis of HSA-PLA and Ac-HSA-PLA

[0157] Methods

[0158] 1. Synthesis of the HSA-PLA conjugates

[0159] Step 1: Reduction of human serum albumin A quantity of 800 mg of HSA powder was dissolved in 120 mL 0.1 M sodium phosphate buffer solution (pH 6.8, 1 mM EDTA) and 7 mL of a TCEP solution (10 mg / mL) in water was added to the HSA solution. The above mixture was magnetically stirred for 4 hours under ice bath and under N2 protection.

[0160] Step 2: Quenching TCEP reduction

[0161] A volume of 0.5 mL DMSO containing 50 mg 4-azidobenzoic acid was added dropwise to the above mixture. After 10 min quenching, 40 mL of 0.2 M sodium phosphate buffer (pH 9.3) was added to neutralize acidic by-products.

[0162] Step 3: Conjugating MAL-PLA to the reduced HSA

[0163] Maleimide-PLA (540 mg, Sigma- Aldrich, UK) was dissolved in 15 mL DMSO and then added to the above reduced HSA solution. The resulting mixture was magnetically stirred for 4 hours in at 37 °C in an oil bath with N2 protection.

[0164] Step 4: Purification and lyophilization

[0165] After the conjugation, the mixture was then centrifuged for 5 minutes with 6000rpm (5 cycles) via a Hermle Z232K Centrifuge (Hermle Labortechnik LmbH, Germany). The collected supernatant was transferred to a dialysis cassette (20K MWCO). After 2 days of dialysis against distilled water, the nanosuspension was centrifuged again for 5 times (6000 rpm, 5 min). The purified nanoparticle suspension was snap frozen in -20°C 25% (w / v) NaCl cool medium and then followed by 48-h lyophilization in a freeze dryer (ALPHA 1-4 LDplus, Martin Christ, Germany). The lyophilized powder was collected and stored in a glass vial at room temperature. Synthesis of the Ac-HSA-PLA

[0166] Step 1: Acetylation of human serum albumin

[0167] A quantity (1000 mg) of HSA was dissolved in 150 mL phosphate buffer solution (0.1 mM, pH 8.0). 1000 mg of sulfo-NHS-acetate was dissolved into 20 mL distilled water and this solution was added dropwise to the HSA solution. The resulting mixture was stirred magnetically at room temperature for 2 hours. After acetylation, the acetylated HSA solution was transferred to a dialysis bag and dialyzed against distilled water for 48 hours. After dialysis, the solution was frozen overnight at -20 °C and followed by lyophilization. The powder of the Ac-HSA was obtained after 48 hours freeze-drying and stored in a glass vial at room temperature.

[0168] Then step 2 Reduction of the Ac-HSA, step 3 Quenching TCEP agent, step 4 Conjugation MAL-PLA to the reduced Ac-HSA and step 5 Purification and lyophilization are same with the method of HSA-PLA synthesis. Results

[0169] The Cys-34 thiol groups of commercial albumins are largely occupied, and thus the Cys-

[0170] 34 thiol groups of albumins are required to be reduced. The chemical TCEP was used to free the occupied Cys 34 of HSA and Ac-HSA. Then the maleimide-polylactide can be conjugated to the HSA and Ac-HSA via the Thiol-maleimide “click” chemistry (Fig 2). As shown in Fig 4, a characteristic peak at 1756 cm-1 of PLA’s carbonyl group was found in the HSA-PLA spectrum in comparison of the HSA standard spectrum. After the successful conjugation of PLA to HSA (Fig 5), the molecular weight of HSA-PLA was increased to 67863 ± 282 Da while the molecular weight of HSA standard is 66479 ± 35 Da, (p < 0.01). After the acetylation of HSA, the molecular weight of Ac-HSA was increased to 68374 ± 94 Da. When PLA was conjugated to the Ac-HSA, the molecular weight of the Ac-HSA-PLA was 69638 ± 1 16 Da (Fig 19).

[0171] Example 4 - Preparation of all nanomedicines and their storage stability

[0172] Methods

[0173] HSA-PLA or Ac-HSA-PLA nanoparticle lyophilized powder (100 mg) was dispersed in

[0174] 35 mL distilled water. To this dispersion was added 1 mL MeOH containing 20 mg paclitaxel dropwise. Probe sonication (Soniprep 150plus, MSE, UK) was exploited to load paclitaxel on HSA-PLA or Ac-HSA-PLA nanoparticles. After 2 cycles (5 min sonication plus 3 min rest as one cycle) probe sonication for HSA-PLA or Ac-HSA- PLA nanoparticles and PTX mixture at 5 amplitudes in an ice bath, the resulting PTX- loaded nanoparticles colloidal liquid was then filtered via a 0.45 pm syringe filter for the purification purpose.

[0175] Then filtrate was frozen at -20°C. The frozen drug loaded nanoparticles must be lyophilized (ALPHA 1-4 LDplus, Martin Christ, Germany) in a cold environment (0 °C). Residual MeOH was removed in the vacuum environment during the lyophilization. The lyophilized powder of PTX-loaded NPs was stored in glass vials at room temperature.

[0176] Results

[0177] As shown in the Fig 7, the methanol stabilized paclitaxel was encapsulated in HSA- PLA and Ac-HSA-PLA nanoparticles. The drug content of the HSA-PLA (PTX) and the Ac-HSA-PLA (PTX) nanomedicine is 15.2 ± 0.3 % w / w and 16.2 ± 0.2 % w / w, respectively. As shown in Table 6, the hydrodynamic diameters of the HSA-PLA (PTX) and the Ac-HSA-PLA (PTX) are 173.54 ± 6.51 nm and 199.53 ± 19.74 nm, respectively. The zeta potentials of the HSA-PLA (PTX) and the Ac-HSA-PLA (PTX) are -34.38 ± 1.01 mV and -34.79 ± 1.38 mV, respectively. According to the Table 2 and Table 7, the lyophilized powders of the HSA-PLA (PTX) and the Ac-HSA-PLA (PTX) are stable at least one month at room temperature.

[0178] Example 5 - Cell uptake of nanomedicines and cytotoxicity of nanomedicines.

[0179] Methods

[0180] Briefly, HSA standard, HSA-PLA nanoparticles and Ac-HSA-PLA nanoparticles were labelled with the fluorescent dye FITC, the cellular uptake of these FITC labelled samples was quantified via a flow cytometer.

[0181] The WST-1 reagent (Roche, Sigma Aldrich, UK) was used for the quantification of live cells after the treatment of Taxol®, HSA-PLA (PTX) nanoparticles or Ac-HSA-PLA (PTX) nanoparticles.

[0182] Results

[0183] As shown in Fig 25.C, The mean FITC intensity of the cells treated with the FITC-HSA, FITC-HSA-PLA nanoparticles and the FITC-Ac-HSA-PLA nanoparticles was 2.48 ± 0.10, 2.22 ± 0.20 and 24.93 ± 1.06, respectively. The cellular association of the Ac- HSA-PLA nanoparticles with the MDA-MB-231 is more than 10 times higher than that of the HSA standard and the HSA-PLA nanoparticles.

[0184] The cytotoxicity (IC50) of the Taxol®, the HSA-PLA (PTX) nanoparticles and the Ac- HSA-PLA (PTX) nanoparticles was calculated and listed in Table 8. The IC50 values of the Taxol® in the MCF-7, 4T1 and the MDA-MB-231 cells are equivalent to the PTX concentration of 0.46 ± 0.19 pg / mL, 0.15 ± 0.03 pg / mL and 0.40 ± 0.36 pg / mL, respectively. The low IC50 of the Taxol® in cells is due to the cytotoxic excipient Cremophor EL in the Taxol® formulation. The IC50 values of the Ac-HSA-PLA (PTX) nanoparticles in the MCF7 cells and the MDA-MB-231 cells are 7.83 ± 1.78 pg / mL and 1.60 ± 1.29 pg / mL respectively, which are significantly lower those of the HSA-PLA (PTX) nanoparticles (MCF7, 10.79 ± 0.63 pg / mL, p < 0.05; MDA-MB-231, 19.56 ± 2.00 pg / mL, p < 0.0001). Example 6 - Biodistribution of nanomedicines in 4T1 tumour bearing model.

[0185] Methods

[0186] BALB / c female mice (6-8 weeks, 20 ± 2g) were subcutaneously injected with 4T1 cells (2xl06cells / 100 qL of DPBS) at right flank sites. Ten days after the injection of the 4T1 cells, tumour volume reached approximately 200 mm3. The Taxol® liquid was diluted with the 0.9% NaCl saline to a concentration of PTX 4 mg / mL. The lyophilized powders of the Abraxane® nanoparticles and the HSA-PLA (PTX) nanoparticles were dispersed in the 0.9% NaCl saline according to a concentration of PTX 4 mg / mL, respectively. These mice were then randomly grouped, three mice per group and intravenously injected with 0.1 mL of the prepared PTX injections at an equivalent PTX of 20 mg / kg, respectively. Subsequently mice were anesthetized and sacrificed at time points of 1, 3, 8, 24 and 72 hours after injection of the paclitaxel preparations. The LC-MS / MS (Agilent Technologies, 6460 Triple Quad LC / MS) was used for the quantification of PTX in mice’s blood, organs and tumours.

[0187] Results

[0188] According to the Table 4, the plasma AUC0-72h values for HSA-PLA (PTX) in mice (23.02 ± 1.71 qg.h mL-1) were significantly higher (p < 0.5) than the plasma AUC0- 72h values of Abraxane® in mice (16.12 ± 2.46 qg.h mL-1). As a result, HSA-PLA (PTX) nanoparticles can deliver a significantly higher amount of PTX to tumours when compared to Abraxane®, the tumour AUC0-72h value of HSA-PLA (PTX) (128.64 ± 3.41 qg.h g-1) was significantly higher (p < 0.01) than the tumour AUC0-72h value of Abraxane® (90.05 ± 12.4 qg.h g-1). Moreover, HSA-PLA (PTX) had a similar clearance feature in organs when compared with Abraxane®. As shown in Fig 15, the clearance rates of HSA-PLA (PTX) nanoparticles in mice’s hearts, livers, spleens, lungs and kidneys were similar to the injected Abraxane®, which indicated that HSA-PLA (PTX) nanoparticles may also have the advantage of the low systemic toxicity.

[0189] Example 7 - Biodistribution of Ac-HSA-PLA nanoparticles in mice.

[0190] Methods

[0191] Briefly, the HSA-PLA nanoparticles and Ac-HSA-PLA nanoparticles are labelled with a NIR dye. NOD-SCID female mice (6-8 weeks) were subcutaneously injected with MDA-MB-231 cells (2 x 106 cells per 100 qL DPBS) at right flank. When the tumour volume was approximately 500 mm3, tumour bearing mice (n = 3 per group) were intravenously injected with the HSA-PLA (Cy) and Ac-HSA-PLA (Cy) nanosuspension at a dose of NIR dye 20 pg per mouse, respectively. Mice were euthanatized after 2h injection of the dye-loaded nanoparticles. After 50 mL PBS heart perfusion, tissues including plasma, brains, hearts, livers, spleens, lungs, kidneys, bones and tumours were then extracted and imaged with an excitation wavelength of 700 nm and an emission wavelength of 790 nm in the IVIS® imaging system (IVIS Lumina XRMS, PerkinElmer, Inc., US).

[0192] Results

[0193] From the ex vivo image (Fig 27), the intravenously injected HSA-PLA (Cy) and Ac- HSA-PLA (Cy) nanoparticles were mainly found in the livers of the MDA-MB-231 tumour bearing mice. While the Ac-HSA-PLA (Cy) nanoparticles have shown a higher tumour deposition than that of the HSA-PLA (Cy) nanoparticles.

[0194] Ac-HSA-PLA (PTX) demonstrates higher cancer cell uptake in vitro and in vivo when compared with HSA-PLA nanoparticle and Abraxane nanoparticle.

[0195] The present invention includes the method of the synthesis of the new nanocarriers for the parenteral administration of paclitaxel (PTX), as well as detailed characterization, in vitro and in vivo evaluations of our PTX nano-formulations. We have designed and synthesized two novel self-assembling polymers, human serum albumin-poly(lactic acid), HSA-PLA, and acetylated human serum albumin-poly(lactic acid), Ac-HSA-PLA. These amphiphilic polymers, HSA-PLA and Ac-HSA-PLA can then self-assembly into polymeric nanoparticles of 100 nm in diameter. Next many hydrophobic small molecules, such as paclitaxel, can be incorporated into hydrophobic part of HSA-PLA and Ac-HSA-PLA nanocarriers. Although the commercial PTX injection Abraxane® has shown the lower systematic toxicity than Taxol®, the overall survival of breast cancer patients is not significantly improved. Because the tumour deposition of PTX from Abraxane® in patient is not improved due to the immunosuppression, poor colloidal stability and passive targeting of Abraxane® nanoparticles after IV administration. Here, in our work, we have enhanced the colloidal stability of both nanocarriers (HSA-PLA nanoparticle and Ac-HSA-PLA nanoparticle) for carrying paclitaxel and targeted tumour to deliver PTX by Ac-HSA-PLA nanoparticles, resulting in improved pharmacokinetics and better tumoricidal effect in a 4T1 and MDA-MB-231 tumour-bearing models without additional toxicity when compared with Abraxane®. Thus, our formulations have potential to prolong the life of metastatic breast cancer patients.

[0196] At the same dose, the paclitaxel formulation, HSA-PLA (PTX), exhibited better tumoricidal effect than commercial alternatives (Abraxane® and Taxol®) in tumour bearing mouse. We have proven that Abraxane® (HSA-PTX nanoparticles) has a poor colloidal stability according to the thermodynamics and DLVO theory. Especially at low concentration, when the concentration of Abraxane® nanoparticles is lower than 0.3 mg / mL, Abraxane® nanoclusters (130 nm in diameter) will start to dissociate into separated tiny particles (40 nm in diameter) and then the extremely unstable tiny particles will dissemble into several human serum albumin molecules and release paclitaxel. Unlike Abraxane®, our formulation HSA-PLA (PTX) is still stable at the concentration of 0.01 mg / mL. This higher colloidal stability of HSA-PLA (PTX) promises the entire nanosystem intactly when entering into the large volume of bloodstream to avoid the unnecessary loss when compared with Abraxane®. That’s the reason why HSA-PLA (PTX) has shown more paclitaxel in blood circulation of rats and mice and better tumoricidal effect.

[0197] Based on the colloidally stable nanoplatform HSA-PLA polymeric micelle, we further acetylated human serum albumin and then produced the active targeting nanoparticle with the higher colloidal stability, acetylated HSA-PLA (Ac-HSA-PLA). According to our in vitro studies, the Ac-HSA-PLA nanoparticles are able to bind with folate receptor and CD44 receptor of MDA-MB-231 human triple negative breast cancer cells. And the uptake efficiency of Ac-HSA-PLA nanoparticles was increased around 10-fold when compared with human serum albumin standard and HSA-PLA nanoparticles. Meanwhile, the excellent active targeting of Ac-HSA-PLA nanoparticle to tumour has been revealed in our recent in vivo pilot studies as well. In our pilot anticancer study, the tumour bearing mice injected with Ac-HSA-PLA (PTX) have been cured and no recurrent tumour. We also did the in vivo imaging to see the biodistribution of Ac-HSA- PLA and HSA-PLA nanoparticles, we used the cyanine dye to label the Ac-HSA-PLA and HSA-PLA nanoparticles respectively, and the biodistribution of dye labelled nanoparticles were visualized by IVIS imaging instrument. The accumulation of Ac- HSA-PLA nanoparticles in tumour was much higher than HSA-PLA nanoparticles. It's worth noting that HSA-PLA nanoparticles are unable to actively target the tumour as the Ac-HSA-PLA nanoparticle did, but HSA-PLA nanoparticles also have their own unique advantages such as easy surface functionalization. During the synthesis of HSA- PLA polymer, we utilized the residue of cysteine to react with maleimide-PLA. So, HSA-PLA polymer still has 59 free lysine amine groups for modification for diverse purposes, such as conjugation of stealth polymer to evade immune system, addition of various ligands to target different sites. Acetylated HSA-PLA nanoparticle is one of the successful derivatives of HSA-PLA nanoparticles. Based on HSA-PLA nanoplatform, we can further develop different version advanced nanoparticles for different applications.

[0198] Nanoparticle agglomeration

[0199] As we can see the TEM image of Abraxane (Fig 30. A), Abraxane nanoparticles heavily agglomerate into several irregularly shaped clusters with a diameter around 130 nm, and particle clusters are well separated from each other. While, from the TEM image of Ac- HSA-PLA (PTX) nanoparticles (Fig 30. C), nanoparticles are not aggregated and well- separated from each other. Nanoparticles in TEM image of HSA-PLA (PTX) nanoparticles (Fig 30. B) are also less agglomerated than Abraxane nanoparticles due to the lower surface-to-volume ratio and lower surface energy of each individual nanoparticle. From the aspect of thermodynamics, lowest free energy of the system is the favourable state to keep the colloidal system stable, and thus, agglomeration of unstable nanoparticles is the spontaneous process to reduce their surface free energy to decrease the free energy of whole colloidal system according to the second law of thermodynamics .

[0200] Therefore, the colloidal stability of Ac-HSA-PLA (PTX) nanoparticles should be higher than Abraxane nanoparticles.

[0201] Tumouricidal activity of Ac-HSA-PLA (PTX) nanoparticles Vs Abraxane®

[0202] We did a formal study to evaluate and compare the in vivo tumouricidal activity of Ac- HSA-PLA (PTX) nanoparticles and Abraxane® in MDA-MB-231 tumour bearing NOD- SCID mice. Briefly, 5 tumour bearing mice per group and each treatment group (n = 5) was injected with Abraxane® (eq. PTX 20 mg / kg), HSA-PLA (PTX) and Ac-HSA-PLA (PTX) nanoparticles (eq. PTX 20 mg / kg) every three days with 5 injections in total, respectively. And the tumour volumes and mice body weights were recorded every three days. As shown in Fig 32, the MDA-MB-231 tumours were disappeared after 5 injections of Ac-HSA-PLA (PTX) nanosuspension. In this formal study, the significant difference in tumour size between mice treated with Abraxane® and Ac-HSA-PLA

[0203] (PTX) nanoparticles can be observed.

[0204] Tables Table 2. Storage stability indicators for HSA-PLA (PTX) nanoparticles during a two- month storage period at room temperature. Data are presented as mean ± SD, n = 3.

[0205]

[0206] Table 10 shows successful incorporation of paclitaxel, docetaxel, and cabazitaxel in HSA-PLA nanocarrier.

[0207] Table 10

[0208]

[0209] Table 1 1 shows a summary of IC50 values of various formulations (columns) in different cell lines (rows). Formulations of controls:

[0210] CrEL-PTX: 6 mg PTX in 1 mL of a mixture of Cremophor oil and absolute ethanol (1 : 1);

[0211] PS80-DTX: 20 mg DTX in 1 mL of a mixture of polysorbate 80 and absolute ethanol (1 : 1); PS80-CTX: 20 mg CTX in 1 mL of a mixture of polysorbate 80 and absolute ethanol (1 : 1).

[0212] CPT-1 1 : prepared according to the formulation of CAMPTOSAR®. Briefly, CPT-1 1 (#B2293, ApexBio, US, 20 mg) was dissolved in 1 mL of distilled water containing D- sorbitol (#BP439, Thermo Fisher Scientific, UK, 45 mg) and 0.9 mg of lactic acid (#252476, Sigma Aldrich, UK) in a water bath at 80 °C. After cooling down to room temperature, pH value of CPT-1 1 solution was adjusted to 3.0.

[0213] Table 11

[0214] Table 12 shows some typical physical parameters of the (Ac-)HSA-PLA nanocarriers.

[0215] Table 12

[0216] Table 13 shows a summary of IC50 values of various formulations (columns) in different cell lines (rows). Formulations: CPT-11 : prepared according to the formulation of CAMPTOSAR®. Briefly, CPT-11 (#B2293, ApexBio, US, 20 mg) was dissolved in 1 mL of distilled water containing D- sorbitol (#BP439, Thermo Fisher Scientific, UK, 45 mg) and 0.9 mg of lactic acid (#252476, Sigma Aldrich, UK) in a water bath at 80 °C. After cooling down to room temperature, pH value of CPT-11 solution was adjusted to 3.0.

[0217] HSA-PLA (SN38): SN-38 solution prepared as 20 mg SN-38 in 1 mL MeOH (0.22% w / v NaOH). Ac-HSA-PLA nanoparticles solution was 10 mg / mL in dH2O. SN-38 solution added to Ac-HSA-PLA nanoparticles solution; sonicated, pH adjusted to 7.0.

[0218] Table 13

[0219] Abbreviations

[0220] Ac: Acetyl group

[0221] HSA: Human Serum Albumin

[0222] PLA: Polylactic Acid

[0223] TEM: Transmission Electron Microscopy

[0224] SEQUENCES

[0225] HSA sequence (SEQ ID NO: 1):

[0226] BSA sequence (SEQ ID NO: 2):

Claims

CLAIMS1. A self-assembling conjugate molecule, the conjugate molecule comprising a hydrophobic polymer covalently bound to human serum albumin (HSA) only at residue Cys34; or a hydrophobic polymer covalently bound to a non-human serum albumin protein only at an equivalent residue to Cys34 of HSA.

2. The self-assembling conjugate molecule according to claim 1, wherein the conjugate molecule is acetylated, optionally wherein the human or non-human serum albumin comprises acetylated lysine residues.

3. The self-assembling conjugate molecule according to claim 1 or claim 2, wherein the hydrophobic polymer comprises or consists of a hydrophobic polymer selected from the group consisting of polylactic acid; a poly(l-lactic acid)-containing co-polymer; a polycaprolactone; a poly(orthoester); a poly(phosphoester); a polystyrene; and maleimide PCL; poly(methyl methacrylate) (PMMA); poly (lactic-co-glycolic acid) (PLGA); PEG-polycaprolactone (PEG-PCL); PEG-poly(lactic acid) (PEG-PLA); and PEG-Poly (lactic-co-glycolic acid) (PEG-PLGA); or combinations thereof.

4. The self-assembling conjugate molecule according to any preceding claim, wherein the hydrophobic polymer comprises PLA of the formula [-C(CH3)HC(=O)O-]„, wherein the integer n is any number from 20 to 30.

5. The self-assembling conjugate molecule according to any preceding claim, wherein the hydrophobic polymer is covalently bound to the Cys34 side chain via a maleimide group.

6. The self-assembling conjugate molecule according to any preceding claim, wherein the self-assembling conjugate molecule is assembled into a nanoparticle.

7. A nanoparticle or a composition of nanoparticles, the nanoparticle(s) comprising selfassembling conjugate molecules according to any of claims 1-6.

8. The nanoparticle or composition of nanoparticles according to claim 7, wherein the nanoparticle(s) are from 50nm to 500nm in size; and / or the nanoparticle size distributions have a polydispersity index of less than 1.

9. The nanoparticle or composition of nanoparticles according to claim 7 or claim 8, wherein the nanoparticle(s) may have a zeta potential of from about -5 to about -100 mV.

10. The nanoparticle or composition of nanoparticles according to any of claims 7-9, wherein the nanoparticle(s) are acetylated in their core and on their surface.1 1. The nanoparticle or composition of nanoparticles according to any of claims 7-10, wherein the nanoparticle(s) are further modified with one or more functional groups.

12. The nanoparticle or composition of nanoparticles according to any of claims 7-1 1, wherein the nanoparticle(s) encapsulate one or more small molecules of 2500Da or less.

13. The nanoparticle or composition of nanoparticles according to any of claims 7-12, wherein the nanoparticle(s) encapsulate one or more agents.

14. The nanoparticle or composition of nanoparticles according claim 13, wherein the agent comprises pharmaceutically active agents, diagnostic agents or contrast agents.

15. The nanoparticle or composition of nanoparticles according claim 14, wherein the agent comprises a metal particle.

16. A composition comprising conjugate molecules according to any of claims 1-6.

17. A method of producing a conjugate molecule, the method comprising covalently binding a hydrophobic polymer to human serum albumin (HSA) or non-human serum albumin protein only at residue Cys34; or covalently binding a hydrophobic polymer to a non-human serum albumin protein only at an equivalent residue to Cys34 of HSA.

18. The method according to claim 17, wherein the hydrophobic polymer is covalently bound to the Cys34 side chain of serum albumin via a maleimide group.

19. The method according to claim 17 or 18, wherein the method further comprises acetylating the human or non-human serum albumin.

20. The method according to any of claims 17 to 19, wherein the acetylation of the human or non-human serum albumin is before the hydrophobic polymer is covalently bound to the Cys34.

21. A method of producing a nanoparticle, the method comprising assembling a plurality of the conjugate molecules according to any of claims 1-6 into a nanoparticle.

22. The method according to claim 21, wherein the method comprises the addition of one or more agents for encapsulation into the nanoparticles prior to assembly.

23. The method according to claim 21 or 22, wherein the method comprises the addition of one or more small molecules, pharmaceutically active agents, or metal particles for encapsulation into the nanoparticles prior to assembly.

24. The nanoparticles and / or composition according to any of claims 7-16, for use as a medicament.

25. The nanoparticles and / or composition according to any of claims 7-16, for use in the treatment or prevention of a disease or condition of a subject.

26. The nanoparticles and / or composition for the use of claim 25, wherein the disease is cancer or an infectious disease.

27. A method of treatment or prevention of cancer or infectious disease in a subject, the method comprising the administration of the nanoparticles and / or composition according to any of claims 7-16 to a subject.

28. The method according to claim 27, wherein the nanoparticles encapsulate a metal nanoparticle, and the method further comprises excitement of the metal nanoparticle within a tissue of the subject to be treated.

Images