Field of the Invention

[0001] The present invention relates to a polymer delivery system for RNAi therapies that includes diseases where siRNA may be effective. In particular the invention relates to polymeric structures that self assemble into nano particles for siRNA delivery.

Background of the Invention

[0002] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

[0003] Since its discovery, RNAi has proved to be a powerful tool for gene function studies, and also shows great potential for the treatment of viral diseases, genetic disorders, and cancers. To date, several siRNA treatments have been tested in clinical trials. It is estimated that the market value of RNAi therapy for 2012 is 560 million with 97 potential therapies in various phases of clinical trials. In spite of these exciting prospects, RNAi therapy faces many challenges with respect to in vivo delivery, which is a major hurdle in translating this therapy to the clinic. Firstly, the poor membrane permeability of siRNA by itself limits cellular uptake. Secondly, siRNA is unstable and is rapidly degraded by nucleases, and thirdly, most of the reagents for delivering siRNA such as Lipo/Oligo-fectamines™ are toxic to the cells.

[0004] It has been quoted that "the three biggest problems with RNAi therapeutics remain "delivery, delivery, and delivery". There is therefore a growing interest in the delivery of disease-modifying siRNA agents directly to the tissue or organ of interest. New targets for RNA interference (RNAi)-based therapy are constantly emerging from the increasing knowledge of key molecular pathways paramount in disease.

[0005] Targets for RNA interference (RNAi)-based therapy include the treatment of cancer. In particular, sarcomas i.e. cancers that arise from transformed cells of mesenchymal origin are targets for RNAi therapy due to the possibility of localised treatment. Malignant tumours made of cancerous bone, cartilage, fat, muscle, vascular or hematopoietic tissues are, by definition, considered sarcomas. [0006] Compared with some other neoplasias, such as breast cancer, lung cancer or even cervical cancer, sarcomas have been largely overlooked for the targeted application of RNAi therapy. Recently, however, some interesting and new targets have been identified by RNAi library screening, and were shown to have a profound effect on cancer cell growth with cell apoptosis. One of these promising new targets is Polo-like kinase 1 or PLK1. PLK1-siRNA were tested on several osteosarcoma cell lines in the Applicant's laboratory which confirm this finding. Osteosarcoma is a bone tumour that usually develops during the period of rapid bone growth in adolescence or teenagers maturing into adults. Osteosarcoma is the most common bone tumour in youth and accounts for 60% of primary malignant bone tumours diagnosed in the first two decades of life, with an average age at diagnosis of 15.

[0007] PLK1 is a serine/threonine kinase involved in various mitotic processes, such as centrosome maturation and chromosome segregation. It is also suggested that PLK1 is required for cell cycle progression not only in mitosis but also for DNA synthesis, maintenance of DNA integrity, and prevention of cell death. PLK1 is over-expressed in several tumour types, including human oesophageal cancer, human oral squamous cell carcinoma, hepatocellular carcinoma, and gastric carcinoma. Several studies have demonstrated the potential of this kinase as a therapeutic target for these cancers. For example, siRNA duplexes against PLK1 introduced into four oesophageal cancer cell lines resulted in a significant inhibition of PLK1 expression in the cells, which further caused a dramatic mitotic catastrophe (mitotic cell cycle arrest as well as defects in several mitotic events such as incomplete separation of sister chromatids and failure of cytokinesis) followed by massive apoptotic cell death, eventually resulting in a significant decrease in growth and viability of all four oesophageal cancer cell lines studied.

[0008] Cationic polymers have shown great promise in binding, protection, and delivery of siRNA to cancer cells. Polymers made from AB diblock or ABC triblock copolymers have interesting design capabilities as the polymer can be tailor-made with a cationic A block and a PEG B block (for serum stability and longer systemic half life). The polycation may consist of the well-known polyethylene diamine (PEI), poly(l-lysine), chitosan, poly(dimethylaminoethylmethacrylate) (PDMAEMA) or many other polymer types. These cationic polymers bind to siRNA through electrostatic binding to form polyplex nanoparticles, and as a result of the greater N/P ratio (i.e. the nitrogen (N) atoms on the polymer to the phosphates (P) from the siRNA backbone) as the overall surface charge is positive (Figure 1). These positively charged polyplex nanoparticles bind electrostatically to anionic cell surface proteins and endocytosis occurs, resulting in its rapid uptake into cells. Release from the endosomes (with a low pH environment) is believed to occur because of the high buffering capacity of the amines on the polymer, acting as a proton sponge. This process induces osmotic swelling of the endosome that will rupture to release the siRNA to the cytoplasm. To maintain such a buffering capacity within the endosome, the polymer requires many basic (i.e. cationic) groups, which has the disadvantage of significantly increasing cytotoxicity. For siRNA delivery to be effective for protein regulation where cell death is not required, the polyplex nanoparticles must be non-toxic but still have the ability to escape the endosome. However, although PEGylation through the formation of AB diblock copolymer significantly reduces the cytotoxicity of the cationic polymer, the rate of cellular uptake is very slow and hinders knockdown.

[0009] Once into the cytoplasm, the siRNA must be released from the cationic polymer. The fact that polycations have a high positive charge on the polymer backbone to encapsulate the negatively-charged siRNA actually makes the release difficult. To overcome this difficulty, many polymers have been designed to release via a trigger, including temperature, pH, redox potential, light, electric pulse, enzymatic degradation and salt levels. For example, the incorporation of disulfide linkages in the side chains of different polymers provides, through the intracellular reduction of the disulfide by glutathione and thioredoxin, a trigger release pathway of siRNA into the cytoplasm. Some polycations modified with disulfide linkages such as PEI , poly(2- dimethylaminoethyl methacrylate), and poly(amidoamine) have shown high knockdown efficiency but with high toxicity. Shim at al released siRNA using pH (< 5) labile ketal linkages. However, these cellular triggers are highly variable between cell lines and cellular environments. The focus has now shifted to developing degradable cationic polymers that not only release DNA or siRNA but degrade into benign compounds thereby reducing toxicity from accumulation of the polymer in cells especially after repeat doses. This is increasingly more important in the development of these polymers.

[0010] Applicants have demonstrated the synthesis of a low cytotoxic polycation, poly(2-dimethylaminoethyl acrylate) or PDMAEA that maintains its cationic strength but can degrade to a benign polymer with nontoxic by-products without the need for an internal or external degradation trigger (Biomacromolecules, 2011 , 12, 1876-1882 and Biomacromolecules, 201 1 , 12, 3540-3548). However, this molecule failed to show gene knockdown.

[001 1] In the past 10 years since the Nobel prize-winning discovery of RNA interference (RNAi), billions of dollars have been invested in the therapeutic application of gene silencing in humans. Today, there are promising data from ongoing clinical trials for the treatment of age-related macular degeneration and respiratory syncytial virus. Despite these early successes, however, the widespread use of RNAi therapeutics for disease prevention and treatment requires the development of clinically suitable, safe and effective drug delivery vehicles.

[0012] Therefore, the use of siRNA as therapeutic agents, in particular for cancer, requires the development of more efficient delivery systems.

[0013] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

[0014] It is an object of the invention in a preferred embodiment to synthesise precisely engineered polymeric dendrimers with well-defined chemically reactive functionalities within and on the peripheral layer that self-assemble into nanoparticles with controlled size and surface functionality for siRNA delivery.

[0015] Applicants have recently developed a polymer delivery system that shows great potential for delivering siRNA. This system, a novel polymeric architecture was more effective than the commercial transfection reagent Oligofectamine (Invitrogen). In vitro testing further demonstrated that this system, also known as a nanocarrier system, can effectively deliver siRNA thereby to potentially treat a range of diseases where siRNA may be effective like cancer.

Summary of the Invention

[0016] In a first aspect the invention relates to a block copolymer comprising a cationic polymer capable of binding to siRNA, a pH mediator capable of disrupting endosomes and a polymer capable of binding with cell membrane thereby to release the block copolymer into the cytoplasm and wherein the cationic polymer is capable of degradation thereby to deliver siRNA. [0017] Preferably the block copolymer is a diblock copolymer wherein the cationic polymer capable of binding to siRNA is one block and pH mediator and the polymer capable of binding with cell membrane form a copolymer that is the second block.

[0018] Preferably the cationic polymer is a cationic acrylate polymer. Preferably the acrylate polymer includes amino groups.

[0019] Preferably the pH mediator is an azole, in more preferred embodiments the azole is imidazole.

[0020] Preferably the polymer capable of binding with cell membrane is an acrylate, in preferred embodiments the acrylate is a butyl acrylate.

[0021] In a second aspect the invention provides for a dendritic molecule comprising a core and one or more generational layers wherein each generational layer includes a block copolymer comprising a cationic polymer capable of binding to siRNA, a pH mediator capable of disrupting endosomes and a polymer capable of binding with cell membrane thereby to release the block copolymer into the cytoplasm and wherein the cationic polymer is capable of degradation thereby to release the siRNA.

[0022] In a third aspect the invention provides for a method of treatment or prophylaxis of a disease or for ameliorating the symptoms of a disease comprising administering to a patient in need thereof a therapeutically effective amount of a composition including a block copolymer or dendritic molecule of the first or second aspect.

[0023] The disease may be selected from cancer, or diseases such as fibrotic diseases, neurodegenerative diseases including parkinson's disease and alzheimer's disease, skin diseases, infectious diseases, lung diseases, heart and vascular diseases, cardiovascular diseases, metabolic diseases, neurological diseases, endocrinological diseases, gastroenterological diseases, intestinal disorder, gastrointestinal disorder, hematological diseases, respiratory diseases, muscle skeleton diseases, urological diseases, respiratory disorder, haematological disorder, ocular disorder.

[0024] If the disease is cancer, the cancer may be selected from the following: Anal and colorectal (bowel) cancer (such as squamous cell cancers); Bile duct cancer (such as intraheptatic cancers, extraheptatic bile duct cancers, perihilar tumours); Bladder cancer (such as urothelial cancer, squamous cell carcinomas, adenocarcinomas); Bone cancer (such as sarcomas including osteosarcoma, chondrosarcoma, ewing sarcoma, soft tissue sarcomas); Brain & spinal cord tumours (such as meningiomas, neuromas, cranio-pharyngiomas, pituitary tumours, cystic astrocytomas, astrocytomas, oligodendrogliomas, ependymomas, glioblastomas and mixed gliomas); Breast cancer (such as ductal carcinoma in-situ (DCIS), invasive ductal cancer (IDC) and invasive lobular cancer (ILC)); Carcinoid tumours (such as neuroendocrine tumours), Cervical cancer (such as squamous cell carcinomas, adenocarcinomas); Endocrine cancer (such as thyroid cancer, adrenal gland tumours, multiple endocrine neuroplasias (MEN1 or MEN2), parathyroid gland tumours, pituitary gland tumours); Eye cancer (such as rhabdomyosarcoma, intraocular cancers, retinoblastoma and medulloepithelioma); Gall bladder cancer; Head & neck cancers (such as mouth, nose & throat, salivary gland, larynx, pharynx, sinus and nasopharyngeal, oropharyngeal and hypopharyngeal cancers); Kaposi's sarcoma; Kidney cancer (such as renal cell carcinoma including clear cell carcinoma, papillary, chromophobic oncocytic and sacromatoid, urothelial carcinoma, renal sarcoma, lymphoma, Wilms tumour); Leukaemia (such as myeloid (AML or CML), lymphocytic); Liver cancer (such as hepatocellular carcinoma (HCC), cholangiocarcinoma, angiosarcoma); Lung cancer (such as non-small cell lung cancer (NSCLC) including squamous cell carcinoma, large cell carcinoma, adenocarcinoma, or small cell lung cancer); Lymphoma (such as non-Hodgkin including follicular lymphoma or diffuse large cell lymphoma & Hodgkin lymphoma); Melanoma (such as superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma (LMM); acral lentiginous melanoma); Mesothelioma (such as epithelioid, sarcomatoid, Mesothelioma mixed, pleural or peritoneal); Multiple myeloma; Ovarian cancer (such as epithelial ovarian cancers, germ cell ovarian cancers, sex-cord stromal cancers); Pancreatic cancer (such as exocrine tumours, pancreatic neuroendocrine tumours (PNETs) including gastrinomas insulinomas glucagonomas somatostatinomas VIPomas); Penis cancer; Peritoneal cancer; Prostate cancer (such as benign prostatic hyperplasia); Skin cancers (such as non-melanoma including basal cell carcinoma, squamous cell carcinoma); Soft tissue cancers (such as malignant fibrous histiocytoma (MFH), liposarcoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, primitive neuroectodermal tumour (PNET), gastrointestinal stromal sarcoma (GIST), stromal sarcoma, synovial sarcoma); Stomach & oesophageal cancer (such as adenocarcinoma, lymphoma, gastric stromal tumours, carcinoid tumours); Testicular cancer (such as seminoma, non-seminoma including teratoma, yolk sac tumour, horiocarcinoma and embryonal carcinoma); Thymus cancer; Uterus cancer (such as endometrial cancer, adenocarcinoma of the endometrium, adenosquamous carcinoma, papillary serous carcinoma clear cell carcinoma or uterine sarcoma); Vaginal cancer (such as squamous cell carcinoma, adenocarcinoma); Vulval cancer (such as squamous cell carcinoma, vulvar melanoma, adenocarcinoma, verrucous carcinoma, sarcomas).

[0025] Preferably the cancer is a localised cancer like osteosarcoma.

[0026] In a fourth aspect the invention provides for use of a block copolymer or dendritic molecule of the first or second aspect in the manufacture of a medicament for the treatment or prophylaxis of a disease or for ameliorating the symptoms of a disease.

[0027] The disease may be selected from cancer, or diseases such as fibrotic diseases, neurodegenerative diseases including parkinson's disease and alzheimer's disease, skin diseases, infectious diseases, lung diseases, heart and vascular diseases, cardiovascular diseases, metabolic diseases, neurological diseases, endocrinological diseases, gastroenterological diseases, intestinal disorder, gastrointestinal disorder, hematological diseases, respiratory diseases, muscle skeleton diseases, urological diseases, respiratory disorder, haematological disorder, ocular disorder.

[0028] If the disease is cancer, the cancer may be selected from the following: Anal and colorectal (bowel) cancer (such as squamous cell cancers); Bile duct cancer (such as intraheptatic cancers, extraheptatic bile duct cancers, perihilar tumours); Bladder cancer (such as urothelial cancer, squamous cell carcinomas, adenocarcinomas); Bone cancer (such as sarcomas including osteosarcoma, chondrosarcoma, ewing sarcoma, soft tissue sarcomas); Brain & spinal cord tumours (such as meningiomas, neuromas, cranio-pharyngiomas, pituitary tumours, cystic astrocytomas, astrocytomas, oligodendrogliomas, ependymomas, glioblastomas and mixed gliomas); Breast cancer (such as ductal carcinoma in-situ (DCIS), invasive ductal cancer (IDC) and invasive lobular cancer (ILC)); Carcinoid tumours (such as neuroendocrine tumours), Cervical cancer (such as squamous cell carcinomas, adenocarcinomas); Endocrine cancer (such as thyroid cancer, adrenal gland tumours, multiple endocrine neuroplasias (MEN1 or MEN2), parathyroid gland tumours, pituitary gland tumours); Eye cancer (such as rhabdomyosarcoma, intraocular cancers, retinoblastoma and medulloepithelioma); Gall bladder cancer; Head & neck cancers (such as mouth, nose & throat, salivary gland, larynx, pharynx, sinus and nasopharyngeal, oropharyngeal and hypopharyngeal cancers); Kaposi's sarcoma; Kidney cancer (such as renal cell carcinoma including clear cell carcinoma, papillary, chromophobic oncocytic and sacromatoid, urothelial carcinoma, renal sarcoma, lymphoma, Wilms tumour); Leukaemia (such as myeloid (AML or CML), lymphocytic); Liver cancer (such as hepatocellular carcinoma (HCC), cholangiocarcinoma, angiosarcoma); Lung cancer (such as non-small cell lung cancer (NSCLC) including squamous cell carcinoma, large cell carcinoma, adenocarcinoma, or small cell lung cancer); Lymphoma (such as non-Hodgkin including follicular lymphoma or diffuse large cell lymphoma & Hodgkin lymphoma); Melanoma (such as superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma (LMM); acral lentiginous melanoma); Mesothelioma (such as epithelioid, sarcomatoid, Mesothelioma mixed, pleural or peritoneal); Multiple myeloma; Ovarian cancer (such as epithelial ovarian cancers, germ cell ovarian cancers, sex-cord stromal cancers); Pancreatic cancer (such as exocrine tumours, pancreatic neuroendocrine tumours (PNETs) including gastrinomas insulinomas glucagonomas somatostatinomas VIPomas); Penis cancer; Peritoneal cancer; Prostate cancer (such as benign prostatic hyperplasia); Skin cancers (such as non-melanoma including basal cell carcinoma, squamous cell carcinoma); Soft tissue cancers (such as malignant fibrous histiocytoma (MFH), liposarcoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, primitive neuroectodermal tumour (PNET), gastrointestinal stromal sarcoma (GIST), stromal sarcoma, synovial sarcoma); Stomach & oesophageal cancer (such as adenocarcinoma, lymphoma, gastric stromal tumours, carcinoid tumours); Testicular cancer (such as seminoma, non-seminoma including teratoma, yolk sac tumour, horiocarcinoma and embryonal carcinoma); Thymus cancer; Uterus cancer (such as endometrial cancer, adenocarcinoma of the endometrium, adenosquamous carcinoma, papillary serous carcinoma clear cell carcinoma or uterine sarcoma); Vaginal cancer (such as squamous cell carcinoma, adenocarcinoma); Vulval cancer (such as squamous cell carcinoma, vulvar melanoma, adenocarcinoma, verrucous carcinoma, sarcomas).

[0029] Preferably the cancer is a localised cancer like osteosarcoma.

[0030] In a fifth aspect the invention provides for a block copolymer or dendritic molecule for use in the treatment or prophylaxis of a disease or for ameliorating the symptoms of a disease. [0031] The disease may be selected from cancer, or diseases such as fibrotic diseases, neurodegenerative diseases including parkinson's disease and alzheimer's disease, skin diseases, infectious diseases, lung diseases, heart and vascular diseases, cardiovascular diseases, metabolic diseases, neurological diseases, endocrinological diseases, gastroenterological diseases, intestinal disorder, gastrointestinal disorder, hematological diseases, respiratory diseases, muscle skeleton diseases, urological diseases, respiratory disorder, haematological disorder, ocular disorder.

[0032] If the disease is cancer, the cancer may be selected from the following: Anal and colorectal (bowel) cancer (such as squamous cell cancers); Bile duct cancer (such as intraheptatic cancers, extraheptatic bile duct cancers, perihilar tumours); Bladder cancer (such as urothelial cancer, squamous cell carcinomas, adenocarcinomas); Bone cancer (such as sarcomas including osteosarcoma, chondrosarcoma, ewing sarcoma, soft tissue sarcomas); Brain & spinal cord tumours (such as meningiomas, neuromas, cranio-pharyngiomas, pituitary tumours, cystic astrocytomas, astrocytomas, oligodendrogliomas, ependymomas, glioblastomas and mixed gliomas); Breast cancer (such as ductal carcinoma in-situ (DCIS), invasive ductal cancer (IDC) and invasive lobular cancer (ILC)); Carcinoid tumours (such as neuroendocrine tumours), Cervical cancer (such as squamous cell carcinomas, adenocarcinomas); Endocrine cancer (such as thyroid cancer, adrenal gland tumours, multiple endocrine neuroplasias (MEN1 or MEN2), parathyroid gland tumours, pituitary gland tumours); Eye cancer (such as rhabdomyosarcoma, intraocular cancers, retinoblastoma and medulloepithelioma); Gall bladder cancer; Head & neck cancers (such as mouth, nose & throat, salivary gland, larynx, pharynx, sinus and nasopharyngeal, oropharyngeal and hypopharyngeal cancers); Kaposi's sarcoma; Kidney cancer (such as renal cell carcinoma including clear cell carcinoma, papillary, chromophobic oncocytic and sacromatoid, urothelial carcinoma, renal sarcoma, lymphoma, Wilms tumour); Leukaemia (such as myeloid (AML or CML), lymphocytic); Liver cancer (such as hepatocellular carcinoma (HCC), cholangiocarcinoma, angiosarcoma); Lung cancer (such as non-small cell lung cancer (NSCLC) including squamous cell carcinoma, large cell carcinoma, adenocarcinoma, or small cell lung cancer); Lymphoma (such as non-Hodgkin including follicular lymphoma or diffuse large cell lymphoma & Hodgkin lymphoma); Melanoma (such as superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma (LMM); acral lentiginous melanoma); Mesothelioma (such as epithelioid, sarcomatoid, Mesothelioma mixed, pleural or peritoneal); Multiple myeloma; Ovarian cancer (such as epithelial ovarian cancers, germ cell ovarian cancers, sex-cord stromal cancers); Pancreatic cancer (such as exocrine tumours, pancreatic neuroendocrine tumours (PNETs) including gastrinomas insulinomas glucagonomas somatostatinomas VIPomas); Penis cancer; Peritoneal cancer; Prostate cancer (such as benign prostatic hyperplasia); Skin cancers (such as non-melanoma including basal cell carcinoma, squamous cell carcinoma); Soft tissue cancers (such as malignant fibrous histiocytoma (MFH), liposarcoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, primitive neuroectodermal tumour (PNET), gastrointestinal stromal sarcoma (GIST), stromal sarcoma, synovial sarcoma); Stomach & oesophageal cancer (such as adenocarcinoma, lymphoma, gastric stromal tumours, carcinoid tumours); Testicular cancer (such as seminoma, non-seminoma including teratoma, yolk sac tumour, horiocarcinoma and embryonal carcinoma); Thymus cancer; Uterus cancer (such as endometrial cancer, adenocarcinoma of the endometrium, adenosquamous carcinoma, papillary serous carcinoma clear cell carcinoma or uterine sarcoma); Vaginal cancer (such as squamous cell carcinoma, adenocarcinoma); Vulval cancer (such as squamous cell carcinoma, vulvar melanoma, adenocarcinoma, verrucous carcinoma, sarcomas).

[0033] Preferably the cancer is a localised cancer like osteosarcoma.

[0034] In a sixth aspect the invention relates to use of a block copolymer or dendritic molecule of the first or second aspect for siRNA delivery.

[0035] Preferably the siRNA delivery is to osteosarcoma cells.

[0036] Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

[0037] The invention relates to the unique polymer architectures which comprise the following three components: (1) diblock copolymer producing a degradation polymer and polymer sponge; (2) knockdown; and (3) polymer that can bind to cell membrane. The polymer architectures may be varied by changing aspects such as N/P ratio (differing proton charge) to optimize siRNA delivery. The invention further relates to the use of these polymer architectures for siRNA delivery in the treatment of cancer, in particular to osteosarcoma cells.

Brief Description of the Figures

[0038] Fig. 1 : A scheme showing the binding and release of siRNA in a nanoparticle polyplex.

[0039] Fig. 2: Knockdown efficiency of (a) P(DMAE) ₆₅ (A), (b) P(DMAE) ₆₅ -b-lmPAA ₁₇ (A-B1), (c) P(DMAEA ₆₅ -b-lmPAA ₃ 3 (A-B2) and (d) P(DMAEA ₆₅ -b-lmPAA ₄ 3 (A-B3) complexed with PLK and S10 at N/P ratio 1 , 5 and 10. After 30 min complexation in water, the polymer/siRNA solutions were transfected into the cells for 4 h and then incubated for 48 h. Controls used are untreated (Control), PLK/Oligofectamine complex (Oligofect-PLK), S10/Oligofectamine (Oligofect-S10) complexes, PLK only (PLK only) and S10 only (S10 only). Concentration of PLK and S10 used is 50nM. The data are reported as the mean ± standard error of the mean of three replicates. Values in parenthesis are the N/P ratio.

[0040] Fig. 3: Knockdown efficiency of (a) P(DMAE) ₆₅ A, (b) P(DMAE ₆₅ -b-(lmPAA ₂ o- co-BA ₁₂ )) (A-C1), (c) P(DMAE ₆ 5-b-(lmPAA3rCO-BA ₂ o)) (A-C2) and (d) P(DMAE ₆₅ -b- (lmPAA ₄ 5-co-BA ₂₉ )) (A-C3) complexed with iPLK and S10 at N/P ratio 1 , 5 and 10. After 30 min complexation in water, polymer/siRNA solutions were transfected into the cells for 4 h and then incubated for 48 h. Controls used are untreated (Control), PLK/Oligofectamine complex (Oligofect-PLK), S10/Oligofectamine (Oligofect-S10) complexes, PLK only (PLK only) and S10 only (S10 only). Concentration of PLK and S10 used is 50nM. The data are reported as the mean ± standard error of the mean of three replicates. Values in parenthesis are the N/P ratio.

[0041] Fig. 4: Knockdown efficiency of (a) P(DMAEA)65 (A), (b) P(DMAEAe5-b- (DMA16-CO-BA11)) (A-D1 ), (c) P(DMAEA65-b-(DMA34-co-BA23)) (A-D2) and (d) P(DMAEA65-b-(DMA45-co-BA35)) (A-D3) complexes with PLK and S10 at N/P ratio 1 , 5 and 10. After 30 min complexation of polymer with siRNA in water, the polymer/siRNA solutions were transfected into the cells for 4 h and then incubated for 48 h. Controls used are untreated (Control), PLK/Oligofectamine complex (Oligofect-PLK), S10/Oligofectamine (Oligofect-S10) complexes, PLK only (PLK only) and S10 only (S10 only). Concentration of PLK and S10 used is 50 nM. The data are reported as the mean ± standard error of the mean of three replicates. Values in parenthesis are the N/P ratio.

[0042] Fig. 5: A rapid, reproducible method to produce pure polymeric dendrimers in less than 30 min at room temperature. The methodology also shows the chemical functionality of the ends of the dendrimers to couple targeting agents.

[0043] Fig. 6: Inhibition of OS tumour development by siRNA against PLK1 delivered by H2 polymer.

[0044] Fig. 7: A scheme showing a method of polymer synthesis.

[0045] Fig. 8: SEC traces of (a) P(DMAEAes) (A), (b) P(DMAEA65-b-lmPAAi7) (A- B1 ), (c) P(DMAEA65-b-lmPAA33) (A-B2) and (d) P(DMAEA65-b-lmPAA43) (A-B3). SEC data measured by DMAc with 0.03 wt% of LiCI as eluent, refractive index detector and PSTY standards for calibration. The intensity for different distribution curves was normalized.

[0046] Fig. 9: SEC traces of (a) P(DMAEAes) (A), (b) P(DMAEA65-b-(lmPAA2o-co- BA12)) (A-C1 ), (c) P(DMAEA65-b-(lmPAA3i-co-BA2o)) (A-C2) and (d) P(DMAEAe5-b- (lmPAA45-co-BA29)) (A-C3). SEC data measured by DMAc with 0.03 wt% of LiCI as eluent, refractive index detector and PSTY standards for calibration. The intensity for different distribution curves was normalized.

[0047] Fig. 10: SEC traces of (a) P(DMAEAes) (A), (b) P(DMAEA65-b-(DMAie-co- BAii)) (A-D1 ), (c) P(DMAEA65-b-(DMA34-co-BA23)) (A-D2) and (d) P(DMAEAe5-b- (DMA45-CO-BA35)) (A-D3). SEC data measured using DMAc by 0.03 wt% of LiCI as eluent, refractive index detector and PSTY standards for calibration. The intensity for different distribution curves was normalized.

[0048] Fig. 11 : Shows a mechanism for polymer assembly, binding with SiRNA and release of SiRNA through a self-catalysed degradation of PDMAE.

[0049] Fig. 12: Shows the chemical structures of some block copolymers of the present invention. [0050] Fig. 13: Shows the degradation profile of polymer A-C3 at pH7.6 and 5.5 when complexed with oligo DNA as measured by DLS, and the time for release of the oligo DNA from the polymer carrier.

[0051] Fig. 14: ¹ H NMR of P(DMAEA ₆₅ ) -CI (A) in CDCI ₃ .

[0052] Fig. 15: ¹ H NMR of P(DMAEA ₆₅ -b-lmAA ₄ 3) (A-B3) in Methanol-d ₄ .

[0053] Fig. 16: ¹ H NMR of P(DMAEA ₆ 5-b-(lmPAA ₄ 5-co-BA ₂ 9)) (A-C3) in Methanol-d ₄ .

[0054] Fig. 17: ¹ H NMR of P(DMAEA ₆ 5-b-(DMA ₄ 5-co-BA ₃ 5)) (A-D3) in CDCI ₃ .

[0055] Fig. 18: (A) Time-dependent particles size and (B) agarose gel assay for binding and release of P(DMAEA ₆₅ -b-lmPAA ₄₃ ) (A-B3) complexes with Oligo DNA in Milli-Q water at N/P ratio 10, pH 7.6, room temperature. Oligo DNA concentration is 10 μg/mL. D _h data are reported as an average of five number-average measurements.

[0056] Fig. 19: (A) Time-dependent particles size and (B) agarose gel assay for binding and release of P(DMAEA ₆ 5-b-(lmPAA ₄ 5-co-BA ₂ 9)) (A-C3) complexes with Oligo DNA in Milli-Q water at N/P ratio 10, pH 7.6, room temperature. Oligo DNA concentration is 10 μg/mL. D _h data are reported as an average of five number-average measurements.

[0057] Fig. 20: (A) Time-dependent particles size and (B) agarose gel assay for binding and release of P(DMAEA ₆ 5-b-(DMA ₄ 5-co-BA ₃ 5)) (A-D3) complexes with Oligo DNA in Milli-Q water at N/P ratio 10, pH 7.6, room temperature. Oligo DNA concentration is 10 μg/mL. D _h data are reported as an average of five number-average measurements.

[0058] Fig. 21 : (A) Time-dependent particles size and (B) agarose gel assay for binding and release of P(DMAEA ₆₅ -b-lmPAA ₄₃ ) (A-B3) complexes with Oligo DNA in Milli-Q water at pH value 7.6 and N/P ratio 10 in room temperature. pH value was then changed to 5.5 by adding 10 μΙ_ of buffer (sodium acetate and acid acetic). Oligo DNA concentration is 10 μg/mL. D _h data are reported as an average of five number-average measurements. [0059] Fig. 22: (A) Time-dependent particles size and (B) agarose gel assay for binding and release of P(DMAEA ₆ 5-b-(lmPAA ₄₅ -co-BA ₂ 9)) (A-C3) complexes with Oligo DNA in Milli-Q water at pH value 7.6 and N/P ratio 10 in room temperature. pH value was then changed to 5.5 by adding 10 μΙ_ of buffer (sodium acetate and acid acetic). Oligo DNA concentration is 10 μg/mL. D _h data are reported as an average of five number-average measurements.

[0060] Fig. 23: (A) Time-dependent particles size and (B) agarose gel assay for binding and release of P(DMAEA ₆ 5-b-(DMA ₄₅ -co-BA ₃ 5)) (A-D3) complexes with Oligo DNA in Milli-Q water at pH value 7.6 and N/P ratio 10 in room temperature. pH value was then changed to 5.5 by adding 10 μΙ_ of buffer (sodium acetate and acid acetic). Oligo DNA concentration is 10 μg/mL. D _h data are reported as an average of five number-average measurements.

[0061] Fig. 24: U-20S cell in vitro knockdown efficiency of (a) P(DMAEA ₆₅ -b- lmPAA ₄₃ ) (A-B3), (b) P(DMAEA ₆ 5-b-(lmPAA ₄ 5-co-BA ₂ 9)) (A-C3), and (c) P(DMAEA ₆₅ -b- (DMA ₄₅ -co-BA ₃₅ )) (A-D3) complexes with siRNA targeting PLK and negative siRNA control S10 at N/P ratios of 1 , 5 and 10 after 30 min complexation in water. The polymers were added to the cells and left to transfect for 4 h, and further incubated for 48 h. Controls used were untreated (Control), PLK/Oligofectamine complex (Oligofect- PLK), S10/Oligofectamine (Oligofect-S10) complexes, PLK siRNA only (PLK only) and S10 siRNA only (S10 only). Concentration of PLK and S10 used was 50 nM. The data are reported as the meanis.e.m. of three replicates, (d) U-20S cell viability of P(DMAEA) ₆ 5-P(lmPAA ₄ 5-co-BA ₂ 9) (A-C3) complexes with universal siRNA (Uni) and scrambled siRNA (Scr) at N/P ratio 1 , 5 and 10 after 30 min complexation in water, 4 h transfection and 48 h incubation. Controls used are untreated (Control). Concentration of universal siRNA and scrambled siRNA used is 50 nM. The data are reported as the meanistandard error of the mean of two replicates. Values in parenthesis are N/P ratios.

[0062] Fig. 25: U-20S cell viability in the presence of P(DMAEA) ₆₅ -(lmPAA ₄₅ -co- BA ₂ g)) (A-C3): (A) polymer A-C3 before degradation added to cell after storage for 30 min in water at room temperature, and (B) polymer A-C3 stored for 2 weeks in water at room temperature (i.e. to allow full degradation of PDMAEA) and then added to cells. The polymer solutions were incubated with the cells for 4 h at 37 °C at different concentrations before the cells were washed with PBS, and then 150 μί of DMEM was added to each well. Control represents no polymer addition (i.e. PBS buffer only). The data are reported as the mean ± standard error of the mean of two replicates. Values in parenthesis are the N/P ratio.

Detailed Description of the Invention

[0063] Unless otherwise specified, DMAEA is dimethyl amino ethyl acrylate, IMz is imidazole and BA is butyl acrylate.

[0064] In the most preferred embodiment of the invention, a linear tri-block copolymer P(DMAEA-b-(IMz-b-BA)) denoted here as H2 has been engineered to form about150 nm sized nanoparticles when bound to siRNA or oligo DNA. The degradation of PDMAEA in any aqueous media eliminates cellular triggers usually required to degrade the polymer and release the siRNA, and thus has versatility over a wide range of cell lines and applications as demonstrated in this application.

[0065] The choice of the cationic polymer is based on its ability to bind to siRNA and its ability to degrade to a benign anionic form thereby to deliver the siRNA.

[0066] In vitro studies using P(DMAEA-b-(IMz-co-BA)) when bound to an oligo DNA (an analogue of siRNA) gave very high cellular uptake rates into HeLa cells with low cytotoxicity even at N/P ratios as high as 50. The knockdown efficiency of this polymer bound with siGFP (a siRNA to knockdown GFP production) was 60%, which was slightly better than the oligofectamine control. The di-block was designed such that the positively charged PDMAEA block ionic bound to the negatively siRNA, the PIMz block was used as a pH mediator to disrupt endosomes through osmotic build-up, and the PBA block to bind with the endosome membrane to enhance release of the polyplex nanoparticles into the cytoplasm where the polymer degrades and releases the siGFP.

[0067] The same polymer, P(DMAEA-b-(IMz-co-BA) (H2), used above was also tested to deliver siRNA to silence the PLK1 gene in U-20S cells (osteosarcoma cell line). A previously reported siRNA, S10, that was effective at silencing human papillomavirus E6/E7, was used as a negative control. Polymer delivery of siRNA was tested with different N/P ratios ranging from 5 to 50. The results show that at N/P ratio of 10 and 50 the polymer can effectively deliver PLK1 -siRNA, silence the gene, and reduce cell viability. The results further show that at an N/P ratio of 10, a significant reduction of cell viability (4.15 fold decrease in comparison with the control S10) is observed, which is more effective than the commercial transfection reagent Oligofectamine™ (1.52 fold decrease in comparison with the control S10). Cell death due to the polymer was observed when N/P ratio is 50. Therefore, the best knockdown with low cytotoxicity was observed at the N/P ratio of 10. The above data suggests that the polymer is quite promising as a delivery reagent for RNAi therapy. This simple but elegant polymer chemistry allows the optimum combination of PDMAEA, PIMz and PBA to be used with high knockdown efficiency and low toxicity.

[0068] The novel polymer based nanoparticles described above, can efficiently transfect both cancer cell lines and primary cells (difficult to transfect cells) with less cell cytotoxicity.

[0069] P(DMAEA-b-(IMz-co-BA) (H2) for siRNA delivery therefore showed good knockdown of the targeted protein. In order to achieve (a) further and greater control of the self-assembly of linear polymer architectures with siRNA, (b) to make libraries of this polymer with different ratios of PDMAEA, PIMz and PBA and (c) to post functionalise the polymer with cell targeting agents (e.g. antibody fragments, folic acid etc.) in specific regions, polymeric dendrimers that can be produced in libraries with different chemical compositions and chemical functionality on the peripheral layer (see Figure 5) are also envisaged in accordance with the invention. The optimal size of the polymeric dendrimers self-assemble into near monodisperse nanoparticles is approximately 20 -30 nm. The polymers can be characterised using a suite of known techniques, including size exclusion chromatography (SEC), NMR (one and two dimensional), MALDI-ToF, FTIR, elemental analysis and other mass spectroscopy techniques. The size of the nanocarriers can be determined using dynamic light scattering (DLS), asymmetric field flow fractionation (AFFF) and sizing chromatography (HDC).

[0070] In an effort to develop a polycation able to degrade in aqueous environments without an intracellular trigger, Applicants synthesised poly(2-dimethylaminoethyl acrylate) (PDMAEA) that auto-degrades from its cationic state to an anionic polymer over a few days (Figure 1) using an established method.

[0071] This polymer degrades to poly(acrylic acid) at an identical rate independent of pH or aqueous media, and through ionic repulsion releases the siRNA from the polyplex. The polymer has key attributes to be included as one of the important components in an ideal gene delivery carrier: (a) it maintains its ionic strength for many hours, which is enough time for high cell uptake, (b) it is non-toxic to a wide range of cell lines, (c) degrades to non-toxic by-products and (d) can bind to DNA or siRNA through ionic interactions. The use of such a degradation and release mechanism does not rely on any specific cellular enzymes or properties, and thus offers a more consistent and predictable release profile. Importantly, this polymer can degrade to its benign form if delivered to non-targeted cells. It should be noted that the well used poly(2-dimethylaminoethyl methacrylate) the methacrylate version of PDMAEA, does not degrade in water, and thus does not have the attributes of PDMAEA. The H2 AB diblock copolymer consisting of PDMAEA, PIMz (as the proton sponge), and PBA (poly butyl acrylate) to bind to the cellular membrane and further disrupt the endosome could effectively delivery various siRNAs to give excellent silencing of specific proteins.

[0072] The present invention provides for polymeric dendrimers wherein each generational layer contains polymer chains with very different chemical composition. Typically an example generational layer will consist of homopolymers of poly(dimethylaminoethylmethacrylate (PDMAEA), PIMz as the proton sponge and poly (butyl acrylate) (PBA) coupled using 'click' chemistry. The new synthetic methodology in accordance with this invention will enable a library of dendritic structures each with a different composition thus providing for optimised compositions for a suitable delivery device. Such a combinatorial approach is rare and produces complex polymer architectures in accordance with the present invention.

[0073] Polymeric dendrimers have better controllable binding and self-assembly properties to their linear counterparts. One successful example of the use of polymeric dendrimers by the present applicants was in vaccine delivery. A dendritic structure covalently coupled to small epitopes self-assembled to form nanoparticles in water of 20 nm, inducing a native conformation of antigen.

[0074] The polymers are made rapidly, in pure form and with reproducible structure from batch to batch. Applicants methodology to make polymeric dendrimers (consisting of quite complex architectures, see Figure 3) with all of the above attributes (reference) allows a combinatorial approach to building a wide range of polymeric dendrimers with a wide range of chemical compositions and architectures for optimizing targeting to specific cells, transfection and knockdown of specific proteins. For example, antibody fragments or other biological molecules such as folate or sugars will be used to target cancer osteosarcoma cells. For in vitro experiments the dendrimers are labelled with fluorescent dyes (e.g. cherry red, GFP).

[0075] Cellular studies included investigating intracellular trafficking of nanoparticles containing Cy3-labeled siRNA in OS cells ((U-20S) using confocal fluorescence microscopy. A time course study is first performed, starting at 1 h, to look for the punctate fluorescence within the apical regions of cells, defining cellular borders, and then again at 4 h, 8h, and 24 h, to look for a fluorescent signal within all areas of the cytoplasm. Briefly, at respective time points ceils cultured on the coversiips will be fixed in the 4% paraformaldehyde and the cell nuclei will be stained with DAPi and cytoplasm with FITC conjugated actin antibodies. Confocal microscopy is used to observe the siRNA diffusion (red against blue and green). Controls are cells transfected with polymer or Cy3 siRNA only. A more diffused fluorescence appearance compared to images taken at 1 h, is evidence of a possible particle dissociation and siRNA release.

[0076] For assessment of OS phenotype after PLK inhibition using polymers, U-20S cells (OS cell line) are cultured in monolayers in 6 well plates. When the cells reach 70% confluence they are transfected with PLK-siRNA using the polymers as a delivery system. After 72 h of transfection, MTT ceil proliferation and ceil viability assay (Cell- titer Gio assay) is performed to further identify an optimal N/P ratio in U-20S cells that gives maximum reduction of cell viability but minimum cytotoxicity of the polymer itself. S10 siRNA is used as a negative control. The N/P ratio is optimized ranging from 10/1 to 40/1. In addition to cell viability assay, RT-qPCR and immuno-biotting is used to measure mRNA and protein levels of PLK after 48h transfection and to quantitatively compare gene silencing efficacy delivered by Oiigofectamine.

[0077] The PLK gene knockdown induced cell death or apoptosis is assessed by microscopic examination of their morphology and by colony-forming assay for cell growth viability. FACS-based Annexin V-FITC (or PE) assay can also be used to detect early apoptosis. Caspase 3/7 assay and PARP protein Western blot are also used to analyze apoptosis after treatment.

[0078] Novel polymer based nanoparticles are also effective in delivering the siRNA in vivo in osteosarcoma animal models. By blocking the important genes related to osteosarcoma pathogenesis, it is possible to reduce disease progression. This part of study uses OS animal models. The animal model is created using xeno-transplant mouse tumour model.

[0079] In RNAi therapy for osteosarcoma a xeno-transplant mouse tumour model of OS is used to test the RNAi therapy. OS U-20S cells are injected subcutaneously on the back or flank of immunodeficient (Rag-/-) mice. The mice are locally (intra-tumourly) injected with the polymer-siRNA against PLK-1 at different doses. A control group is injected with polymer or siRNA alone. Three injections are administered over three weeks (once a week). Tumour growth can be monitored regularly by both measuring the size using an in vivo Imaging System (KODAK) as established before, and by measuring the final tumour weight. Alternatively, tumour size will be measured using calipers and expressed in volume (mm ³ ). For round tumours, the diameters will be measured and for flat tumours the width, length, and thickness will be measured. All measurements will be repeated three times and the volume of each tumour will be calculated as the mean of three measurements. When the experiment is terminated, tumour tissues will be collected for histology examinations of cell death, apoptosis, and proliferation (Ki-67).

[0080] This present invention therefore provides for novel polymer nano-particles for efficient local siRNA delivery. Local delivery to site specific tissues eliminates many of the systemic delivery problems, including targeted delivery to specific tissues and then to specific cells. This delivery system is a major step forward for the current siRNA delivery technology and could potentially be used for the treatment for localised diseases such as osteosarcoma. As nanopolymers can be modified to carry other molecule such as antibodies, this technique platform will contribute greatly to the future development of cell population targeted therapies.

Examples

[0081] Materials included dioxane (99%), carbondisulfide (99%), 1 -butanethiol (99%), methyl bromopropionate (98%), 3-(1 H-imidazol-1-yl)propan-1-amine (98%) and dimethyl sulfoxide (DMSO, >99.9%) were used as received from Sigma-Aldrich. n- Hexane (>98%) and tetrahydrofuran (THF, >99.9%) were used as received from Merck. 2-(dimethylamino) ethyl acrylate (98%, Sigma-Aldrich) and butyl acrylate (99%) were passed through a column of basic alumina (activity I) to remove inhibitor. Azobisisobutyronitrile (AIBN) was recrystallized twice from methanol prior to use. MilliQ Water (18.2 MQcm-1) was generated using a Millipore MilliQAcademic Water Purification System. 9-27 Oligo DNA, siBgal, siGFP was synthesized by Invitrogen, 9- 27F+R-MW=14998 (23bp), Sense: 5'-GTCAGAAATAGAAACTGGTCATC-3' Antisense: 5'-GATGACCAGTTTCTATTTCTG-AC3' . siBgal sequence: AATTATGCCGATC GCGTCACA. siGFP (GFP-targeted siRNA) sequence: 5'-GCACGACUUCUUC AAGUCCUU-3'. Oligofectamine, a standard transfection reagent, was purchased from Invitrogen (Carlsbad, California). All other chemicals and solvents used were of at least analytical grade and used as received.

Synthesis

[0082] Figure 7 indicates polymer synthesis for siRNA delivery and relates to polymers synthesis for siRNA delivery by SET-LRP at room temperature using (i) Cu powders (size <0.475 μηι) and CuCI ₂ /Me ₆ TREN in isopropanol and (ii) CuCI and Me ₆ TREN in methanol

[0083] Synthesis of N-(3-(1 H-imidazol-1 -yl)propyl)acrylamide (ImPAA). 3-(1 H- imidazol-1-yl)propan-1-amine (15.8g, 0.13 mol) and sodium bicarbonate (13.8g 0.16 mol) were dissolved in Mili-Q water (60 mL). The solution was cooled to 0.5°C in an ice batch. Acryloyl chloride (11.4g, 0.13 mol) was added dropwise for about 1 h. The solution was stirred at room temperature for over night (about 15h). Salts were filtered and the product was extracted by chloroform (5 x 100 mL). The organic phase were combined and dried over anhydrous MgS0 ₄ for 1 h. The salt was filtered and the solvent was evaporated. The product was purified by column chromatography (1 :3 methanol/dichloro methane on silica, second band, Rf = 0.67). ¹ H NMR (MeOD) δ (ppm) 1.9 (q, J = 4.5 Hz, 2H, -CH ₂ -CH ₂ -CH ₂ -), 3.14 (t, J = 4.5 Hz, 2H, -CH ₂ -CH ₂ -N-), 3.96 (t, J = 4.5 Hz, 2H, -CH ₂ -CH ₂ -NH-), 5.56 (d, J = 4.5 Hz, 1 H, double bond), 6.12 (d, J = 4.5 Hz, 1 H, double bond), 6.86 (s, J = 4.5 Hz, 1 H, -CH=CH-N-), 7.05 (s, J=4.5 Hz, 1 H, -N-CH=CH-), 7.57 (s, J = 4.5 Hz, 1 H, -N-CH-N-).

[0084] Synthesis of Poly(2-Dimethylaminoethyl Acrylate) (PDMAEA-CI) Macro- CTA by SET-LRP. CuCI ₂ /Me ₆ TREN (32 mg, 8.8 x 10 ^"5 mol), Me ₆ TREN (61 mg, 2.6 x 10 ^"4 ), ethyl 2-chloropropanoate (120 mg, 8.8 x 10 ^"4 mol) and 2-Dimethylaminoethyl Acrylate (18.9g, 0.132 mol) were dissolved in isopropanol (10 mL) and the solution was purged Argon for 30 min at room temperature. Cu powder (size < 0.475 μηι) was added under Argon flow. After the polymerisation over 7h at room temperature, the solution was diluted by acetone (100 mL) and passed through Al ₂ 0 ₃ to remove copper. Acetone was removed by evaporator and the product was obtained by precipitating in a large excess of cold n-hexane (500 mL), and then isolated by centrifugation. The operation of redissolving by acetone (10 mL) and precipitation in n-hexane (500 mL) was repeated three times. The polymer was dried under high vacuum for 48 h at room temperature to give a yellow oily product (yield 70%). The conversion of polymerization was 40% as determined from ¹ H NMR spectroscopy. Mn = 4400, PDI = 1.29 (SEC-RI calibrated using PSTY Standards and used DMAc + 0.03 wt% LiCI as eluent).

[0085] Synthesis of PDMAEA-b-PlmPAA by SET-LRP (A-B _x ). A typical polymerisation is as follows: PDMAEA-CI Macro-CTA (0.5g, 5.5 x 10 ^"5 mol), ImPAA (0.537g, 3.0 x 10 ^"3 mol), and Me ₆ TREN (0.012g, 5.0 x 10 ^"5 mol) were dissolved in methanol (2 mL) and the solution was purged Argon for 30min. CuCI (0.005 g, 5.0 x 10 ^"5 mol) was added under Argon flow. The reaction was stirred at room temperature for 15 h, diluted by acetone (10 mL) and passed through Al ₂ 0 ₃ (basic) to remove copper. The solvent was removed by evaporator and the product was recovered by precipitating in acetone: n-hexane mixture (10 mL, 4:6) and isolated by centrifuging. The operation of redissolving by acetone (2 mL), precipitation in acetone: n-hexane mixture (10 mL, 4:6) and centrifuging was repeated three times. The product was dried under high vacuum for 48 h to give a pair yellow sticky solid (yield 30%). The conversion of polymerization as determined from ¹ H NMR spectroscopy was 40% . Mn = 24000, PDI = 1.21 (SEC-RI calibrated using PSTY Standards and used DMAc + 0.03 wt% LiCI as eluent).

[0086] Three different molecular weights of second block copolymers (ImPAA-co-BA) were synthesised by changing the ratio of monomers to Macro-CTA as details data shown below in Table 1. Table 1

Code PDMAEA-CI (g) ImPAA (g) CuCI (mg) Me ₆ TREN (mg) MeOH (mL)

A-B1 0.5 0.269 5 12 1.5

A-B2 0.5 0.537 5 12 2

A-B3 0.5 0.806 5 12 2

[0087] Synthesis of PDMAEA-b-P(lmPAA-co-BA) by SET-LRP (A-C _x ). A typical polymerisation is as follows: PDMAEA-CI Macro-CTA (0.840g, 8.4 x 10 ^"5 mol), ImPAA (1.500g, 8.4 x 10 ^"3 mol), BA (0.540g, 4.2 x 10 ^"3 mol) and Me ₆ TREN (0.039g, 1.7 x 10 ^"4 mol) were dissolved in methanol (2 mL) and the solution was purged Argon for 30min. CuCI (0.017g, 1.7 x 10 ^"4 mol) was added under Argon flow. The reaction was stirred at room temperature for 15 h, diluted by acetone (10 mL) and passed through Al ₂ 0 ₃ (basic) to remove copper. The products were dialysed against methanol (3 x 250ml) to remove monomer. The solvent was removed by rotavap and the product was recovered by precipitating in acetone n-hexane mixture (10 mL, 4:6) and isolated by centrifuging. The operation of redissolving by acetone (2 mL), precipitation in acetone: n-hexane mixture (10 mL, 4:6) and centrifuging was repeated three times. The product was dried under high vacuum for 48 h to give a pair yellow sticky solid (yield 34%). The conversion of polymerization as determined from ¹ H NMR spectroscopy was 31 % and 40% for ImPAA and BA, respectively. Mn = 24000, PDI = 1.25 (SEC-RI calibrated using PSTY Standards and used DMAc + 0.03 wt% LiCI as eluent).

[0088] Three different molecular weights of second block copolymers P(lmPAA-co- BA) were synthesised by changing the ratio of monomers to Macro-CTA as details data shown below in Table 2. Table 2

Code PDMAEA-CI (g) ImPAA (g) BA (g) CuCI (mg) Me ₆ TREN MeOH

(mg) (mL)

A-C1 0.931 1.0 0.358 18 43 3

A-C2 0.840 1.50 0.540 17 39 2

A-C3 0.559 1.50 0.536 1 1 26 2

[0089] Synthesis of PDMAEA-b-P(lmPAA-co-DMA) by SET-LRP (A-D _x ). A typical polymerisation is as follows: PDMAEA-CI Macro-CTA (1.458g, 1.6 x 10 ^"4 mol), DMA (0.722g, 7.3 x 10 ^"3 mol), BA (0.933g, 7.3 x 10 ^"3 mol) and Me ₆ TREN (0.075g, 3.3 x 10 ^"4 mol) were dissolved in methanol (2.5 mL) and the solution was purged Argon for 30min. CuCI (0.032g, 3.3 x 10 ^"4 mol) was added under Argon flow. The reaction was stirred at room temperature for 15 h, diluted by acetone (10 mL) and passed through Al ₂ 0 ₃ (basic) to remove copper. The products were dialysed against methanol (3 x 250ml) to remove monomer. The solvent was removed by rotavap and the product was recovered by precipitating in acetone n-hexane mixture (10 mL, 4:6) and isolated by centrifuging. The operation of redissolving by acetone (2 mL), precipitation in acetone: n-hexane mixture (10 mL, 4:6) and centrifuging was repeated three times. The product was dried under high vacuum for 48 h to give a pair yellow sticky solid (yield 28%). The conversion of polymerization as determined from ¹ H NMR spectroscopy was 78% and 51 % for DMA and BA, respectively. Mn = 15700, PDI = 1.23 (SEC-RI calibrated using PSTY Standards and used DMAc + 0.03 wt% LiCI as eluent).

[0090] Three different molecular weights of second block copolymers P(lmPAA-co- DMA) were synthesised by changing the ratio of monomers to Macro-CTA as details data shown below in Table 3. Table 3

Code PDMAEA-CI (g) DMA (g) BA (g) CuCI (mg) Me ₆ TREN MeOH (mL)

(mg)

A-D1 1 .458 0.481 0.622 32 75 2.5

A-D2 1 .458 0.722 0.933 32 75 2.5

A-D3 1 .458 0.962 1 .244 32 75 2.5

Further synthesis and physicochemical properties of polymers

[0091] Single electron transfer-living radical polymerization (SET-LRP) of DMAEA produced PDMAEA (Table 4 below) with a number-average molecular weight (Mn) of 4,200 and a polydispersity index (PDI) of 1.29 determined by size exclusion chromatography based on polystyrene standards and neglecting differences in polymer hydrodynamic volume (see Figs 18-10). A more accurate value of 9,430 determined by ¹ H NMR showed that the polymer consisted of 65 DMAEA units. This polymer was previously tested as a delivery vehicle for siRNA, showing >90% cell uptake after 4 h and low cytotoxicity, and thus was used as the first block in this work.

[0092] Table 4 below shows molecular weights, polydispersities (PDI), and Ή NMR data of SET-LRP polymerization of P(DMAEA) (A), P(DMAEA-b-lmPAA) (A- B1 ,A-B2,A-B3), P(DMAEA-b-(lmPAA-co-BA)) (A-C1 .A-C2, A-C3) and P(DMAEA-b- (DMA-co-BA)) (A-D1 , A-D2, A-D3).

a SEC data measured using DMAc with 0.03 wt% of LiCI as eluent, refractive index detector and PSTY standards for calibration.

b P(DMAEA)-CI repeating units was calculated from the integral area of two peaks at 1.2 ppm and 2.5 ppm using the following equation:

NDMAEA=(|2.5X3)/(II .2X2).

c Repeating units of ImPAA were calculated based on 65 repeating unit of P(DMAEA)-CI by using the integral area of a peak at 2.5 ppm (I2.5) and 7.0 (I7.0) and the following equation: NimPAA= (b ox 65 x 2)/(l2.s).

d Repeating units of BA were calculated based on 65 repeating unit of P(DMAEA)-CI by using the integral area of a peak at 0.9 ppm (I0.9) and 2.5 (I2.5) and the following equation: NBA= (ΙΟ.Θ Χ 65 x 2)/(l2.sx 3).

e Repeating units of DMA were calculated based on 65 repeating unit of P(DMAEA)-CI by using the integral area of a peak at 2.5 ppm (I2.5) and 2.9 (I2.9) and the following equation: NDMA= (I2.9x 65 x 2)/(l2.s x 6).

f Mn determined by iH NMR were calculated by using the following equation: Mn = 136 + (NDMAEA X 143) + (NimPAA X 179) + (NBA X 128) + (NDMA X 99).

[0093] PDMAEA ₆ 5 was extended with blocks containing units of ImPAA, BA and/or dimethylacrylamide (DMA) comonomers as shown in Fig. 12 using the SET-LRP technique. Conversion of monomer to polymer was kept low (<40%) to avoid high levels of radical-radical termination and further maintain a high -C1 chain-end functionality to produce block copolymers (A-B1 to A-D3) with PDIs below 1.3. The number of repeating units for all comonomers is given in Fig. 12 and the Table 4 based on ¹ H NMR analysis (see Figs 14-17).

[0094] Complexation of an oligo DNA (23 bp) with the A-B polymer series in water showed an increase in size (i.e. diameter, D _h ) from ~5 to 200nm with an increase in the N/P ratio (i.e. nitrogen to phosphorus ratio) from 0 to 10 (see Table 5 below).

*N/P ratio = 0 is only polymer dissolved in water at the concentration 1 mg/mL.

Particle size of PDMAEA (A), P(DMAEA-b-lmPAA) (A-B1 , A-B2, A-B3), P(DMAEA-b- (ImPAA-co-BA)) (A-C1 , A-C2, A-C3) and P(DMAEA-b-(DMA-co-BA)) (A-D1 , A-D2, A- D3) complexes with Oligo DNA in Milli-Q water at different N/P ratios (1 , 5 and 10). Oligo DNA concentration is 10 μg/mL. D _h data are reported as an average of five number-average measurements. [0095] A similar trend was found for the A-C and A-D series, but here the initial D _h (at N/P = 0) showed that the incorporation of the hydrophobic monomers, BA and ImPAA, in the second block gave small polymeric micelles of -15 to 20nm (A-C1 to A- C3). The A-C3 polymer candidate showed that at an N/P ratio of 10 at pH 7.6, these small 20nm nanoparticles aggregated with the oligo DNA to result into a narrow particle size distribution close to 200nm in size as shown in Fig. 13. Leaving the complex of A- C3/ oligo DNA in water at pH 7.6 resulted in its degradation after 17 h, whereby the size decreased from 200 to 20nm and the oligo DNA was fully released (see the Agarose gel in Fig. 13). A similar profile was found when the complex was kept at pH 5.5, with full release of the oligo DNA after 25 h and a size decrease to 5 nm (Fig. 13) corresponding to the size of an individual polymer coil (i.e. unimer) in solution. This result suggests that the siRNA (with a similar size to the oligo DNA) can still be complexed with the polymer in the low pH environment of the endosome, and if escape from the endosome is faster than the release time of -17 h, the siRNA has a great chance of being released within the cytosol. The similar degradation profiles of A-C3 in pH 5.5 and 7.6 further supported a non-triggered and timed-release mechanism of the siRNA from the polymer carrier. In addition, the degradation profiles for A-B3 and A-D3 at pH 7.6 and 5.5 were all similar to A-C3, suggesting that only the degradation of PDMAEA to PAA plays the dominant role for release of oligo DNA (see Figs 18-23).

BIOLOGY ASSAY

[0096] GFP knockdown assay. HeLa-GFP cells (2.5 x 10 ⁴ ) were seeded into 24 well plates for 24 h before transfection. siRNAs (0.533 μg, 4x10 ^"11 mol, Sigma, Sydney) were complexed with polymers at different N/P ratios (1 to 50, as indicated) in a total volume of 150 μΙ of water and the mixtures were allowed to complex without stirring for 30 min at room temperature. OptiMem (Invitrogen, Sydney, Australia) was added to a final volume of 1 mL before polymers/siRNA complexes were added to the cells (250 μΙ_ per well). Cell without siRNA (Untreated), and Oligofectamine/siRNA was used as the control. The plates were incubated at 37 ^° C for 4 h before complexes were removed; cells were washed with PBS and 200 μΙ_ complete DMEM (Gibco) was added to each well. The cells were incubated for 48 h, harvested by adding 300 μΙ_ 0.25% trypsin- EDTA (Invitrogen), spun at 400g for 5 min to pellet the cells, and resuspended in 100 μΙ_ 2% paraformaldehyde. GFP knockdown in the cells was examined on cells by analysing samples on FACSCanto (BD) and data were analysed using ACSDiVa software (BD). [0097] Cytotoxicity assay. HeLa cells (0.5 x 10 ⁴ ) were seeded into 96 well plates for 24 h before transfection. Control wells containing media without cells were used to obtain a value for background luminescence. Oligo DNA (0.533 μg, 4x10 ^"11 mol) was mixed with polymers at different N/P ratios (1 to 50, as indicated) in a total volume of 100 μΙ of water and allowed to complex for 30 min at room temperature before OptiMem (Invitrogen, Sydney, Australia) was added to a final volume of 1 ml_. Cell without Oligo DNA (Untreated), cell with Oligo DNA (naked), and Oligofectamine/Oligo DNA was used as the control. Polymers/Oligo DNA complexes were added to the cells (100 μΙ_ per well) and plates were incubated at 37°C for 4 h before complexes were removed, cells were washed with PBS and 150 μΙ_ complete DMEM (Gibco) was added to each well. The cells were incubated for 48 h before Cell Titre Glo (Promega, Sydney, NSW, Australia) cell viability assay was subsequently performed according to the manufacturer's instructions.

[0098] This target was then used as a model system to screen the polymer nanocarrier delivery of siRNA (see Preliminary Data below for the knockdown results). This in vitro work represents a promising method of delivery since at the initial stages of osteosarcoma, the disease is mostly localised to within the disease site.

[0099] Preliminary in vivo work is indicated in Fig. 6. The figure indicates significant inhibition of OS tumour development by siRNA against PLK1 delivered by H2 polymer. In xeno-transplant mouse model, OS cells were injected to mouse subcutaneously without treatment (KHOS/NP) or treated with polymer only (polymer), polymer+ siRNA against PLK1 (polymer +siPLK1), and polymer + negative siRNA S10 (polymer+siS10). The tumour sizes were measured at day 26 after injection. Each dot represents a mouse.

[00100] In vitro RNAi-mediated knockdown of PLK1 in osteosarcoma.

Osteosarcoma is a bone cancer prevalent in young people, with poor survival rates. Polo-like kinase 1 (PLK1) has an important role in maintaining tumourgenic phenotype of osteosarcoma cells. Its knockdown using siRNA delivery is expected to induce selective growth arrest and cell death. The present inventors used the U-20S cell line as an in vitro model system to test our polymers (from Fig. 12) for knockdown using a previously screened siRNA36. The knockdown of U-20S cells was evaluated using the polymer-loaded siRNA complexes of the present invention via a cell viability assay. Four siRNAs were used: (i) siRNA targeting PLK1 , (ii) universal negative control (Mission siRNA, Sigma-Aldrich), (iii) scrambled siRNA for PLK, and (iv) a negative control S10 siRNA that targets another pathway. The polymer-loaded siRNA complexes were also compared with oligofectamine/siRNA complexes, in which oligofectamine is regarded as the gold standard for siRNA delivery. Solutions of the polymer and siRNA in PBS buffer at various N/P ratios (that is, 0 to 10) were mixed and incubated for 30 min at room temperature. The complex was then added to the cells in complete DMEM media at 37°C, and after 4 h, the cells were washed to remove any complex not taken up by the cells and incubated for another 48 h. The concentration of siRNA used in this assay was 50 nM.

[00101] It can be seen that polymer A loaded with siRNA by itself (that is, PDMAEA65) showed little toxicity and little or no knockdown compared with oligofectamine-loaded siRNA targeting PLK1 (see Fig 2a). Incorporating an ImPAA second block (A-B series) to act as either a proton sponge or fusogenic polymer also showed little toxicity and little or no cell death irrespective of the molecular weight of the second block when loaded with PLK1 siRNA (Fig. 24b and Figs. 2b-d). The results suggest that even with the inclusion of ImPAA (similar functional group to histidine), the polymer carrier could not escape the endosome. Polymer series A-C incorporating not only ImPAA but hydropobic BA was synthesized to further assist in endosome escape. The cell viability data (Fig. 24b and Fig. 3) using A-C2 and A-C3 loaded with PLK1 siRNA showed little or no cell death at N/P ratios below 10. At an N/P ratio of 10, both polymers loaded with PLK1 siRNA complexes showed excellent knockdown (>80%) and little toxicity. This amount of cell viability loss specific to targeting the PLK1 pathway was substantially better than oligofectamine/siRNA. To test whether cell death was due to the combination of ImPAA and BA in the second block or as a result of only BA, we synthesized the A-D polymer series consisting of BA and DMA in the second block. The results for A-D3 (Fig. 24c) and A-D1 and A-D2 (Fig. 3) showed that without ImPAA there was no observed cell death. The combination of ImPAA and BA in the second block demonstrates that both comonomers act synergistically to allow escape of the polymer/siRNA complex from the endosome into the cytosol, most probably due to the combined ionic and hydrophobic interactions with the endosome membrane. To confirm that cell death was primarily due to siRNA targeting PLK, we tested two different control siRNAs (universal negative control (denoted as Uni) and a scrambled siRNA (denoted as Scr)) delivered with polymer A-C3, and the data given in Fig. 24d shows that there is no knockdown with either of these siRNAs. Further, the cell viability of polymer A-C3 alone before (Fig. 25A) and after (Fig. 25B) degradation (that is, self- catalyze to poly(acrylic acid)) of the PDMAEA block in A-C3 showed little or no cell toxicity even at an NP ratio of 20. These results collectively demonstrate the excellent knockdown potential of A-C3 with little or no cytotoxicity.

[00102] Escape from the endosome is thus a key feature of our polymer carrier that allows effective delivery of the siRNA to the cytosol. Two mechanisms have been proposed for pH-responsive polymers (for example, with imidazole groups) to facilitate escape: (i) an osmotic gradient caused by a flux of counter ions to maintain the ionic strength in the endosome and thus induce endosome lysis, or (ii) interaction of the imidazole groups with the endosome membrane. The results obtained by the present inventors showed that the siRNA targeting PLK gave -80% knockdown with little or no toxicity from the polymer A-C3. The requirement for the combination of ImPAA and BA in the second block (A-C series) demonstrates the capability of these monomer units to fuse with the endosome membrane and facilitate escape. The ImPAA (that is, imidazole) side groups become highly protonated when the pH drops below its pKa of ~6, which should enable these positive charges to interact with the negatively charged endosome membrane and induce a bilayer phase separation. The BA monomer units should further interact with the membrane through hydrophobic interactions and facilitate fusion with the membrane leading to escape. As described earlier, the fusogenic second block in A-C2 and A-C3 is located in the core of the micelle with a size of ~20nm (See Table 5 above), and must therefore be exposed to the surface of the micelles when the pH drops in the endosome to interact with the endosome membrane, mimicking structural reorganization found for the influenza virus. When the polymer without siRNA was self-assembled in water at pH 7.6, small micelles formed with a size of ~20nm as seen in the Table 6 below.

Table 6

hydrodynamic diameter, D _h (nm) (PDI in parentheses)

PH

A-B3 A-C3 A-D3

7.6 7.638 22.70 12.04

(0.292) (0.373) (0.286)

5.5 4.012 4.571 1 1 .13

(0.177) (0.604) (0.197)

7.6 6.732 21 .50 14.74

(0.335) (0.415) (0.300) [00103] Particle size of P(DMAEA ₆₅ -b-lmPAA ₄ 3) (A-B3), P(DMAEA ₆₅ -b-(lmPAA ₄₅ -co- BA ₂₉ )) (A-C3) and P(DMAEA ₆ 5-b-(DMA ₄ 5-co-BA ₃ 5)) (A-D3) in Milli-Q water pH 7.6 and pH 5.5. D _h data was reported as a number-average hydrodynamic diameter from five measurements at room temperature. Polymer concentration was 1 mg/mL. The measurements were done after stored 30 mins at pH 7.6 and room temperature. Solution pH changed by HCI (0.1 M) and NaOH (0.1 M).

[00104] When the pH was decreased to 5.5, the polymer was fully water soluble and the polymer chains dissociated from the micelle forming unimers of ~5 nm. Increasing the pH back to 7.6 resulted in reconstitution (self-assembly) of the unimers back to small ~20nm particles. The polymer (A-C3)/oligo DNA complex showed no change in particle size when the pH was decreased from 7.6 to 5.5 (Fig. 13). These results suggested that the 20nm polymer particles when complexed with the siRNA formed a large 200nm aggregate, and when in the endosome, the size of the aggregate did not change due to the strong ionic binding between the siRNA and polymer chains even though the ImPAA groups were now cationic and hydrophilic. Our observations are consistent with the fully water soluble ImPAA and BA second block reorganizing in the endosome to be exposed to the exterior of the aggregate, allowing fusion and escape from the endosome membrane. Once in the cytosol, release of the siRNA occurs after 17 h, which acts to interfere with the specific mRNA.