WO2016000070A1 - Hydrophobically derivatized hyperbranched polyglycerol for intravascular drug delivery - Google Patents

Abstract

Herein are provided derivatized hyperbranched polyglycerols (HPGs) for use as agents for the delivery of a drug or other biologically active moiety to a target tissue to treat indications such as cancer, which may be useful in the treatment of or the manufacture of a medicament, in the preparation of a pharmaceutical composition for the treatment of indications such cancer. Also provided are derivatized HPGs for use as a pre-treatment or co- treatment to improve drug uptake in a tissue. Furthermore, there are provided methods of making derivatized HPGs. In particular embodiments, the HPGs are derivatized with C₁₀ alkyl chains. Such derivatized HPGs may be suitable for intravascular administration.

Description

HYDROPHOBICALLY DERIVATIZED HYPERBRANCHED POLYGLYCEROL FOR

INTRAVASCULAR DRUG DELIVERY

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 62/019,082 filed on June 30, 2014, which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

This invention relates to therapeutics, their uses and methods for drug delivery. In particular, the invention relates to polymers based on derivatized hyperbranched polyglycerols and methods for intravascular delivery of biologically active moieties to biological tissues.

BACKGROUND

Chemotherapy is often employed in treating cancer patients; however, the efficacy of anti-cancer drugs is often limited by associated adverse effects or poor pharmacokinetics. For example, docetaxel ("DTX"), a microtubule inhibitor, is a widely prescribed drug for the treatment of a broad range of cancers as a single agent as well as for combination therapy. However, owing to its hydrophobic nature, DTX has poor water solubility and requires solubilisation with surfactants such as polysorbate 80 (Tween 80) that results in side effects such as acute hypersensitivity infusion reactions and hemolysis. In addition, DTX has also been reported to cause serious side effects, which include peripheral neuropathy, neutropenia and musculoskeletal toxicity.

Various nanocarriers have been developed in response to these obstacles. Nanoparticles ("NPs") can improve the solubility of poorly water-soluble drugs, extend the circulation half- life of the drug in plasma, and provide controlled drug release that can result in enhanced therapeutic benefits in cancer treatment. Accumulation of NPs in solid tumors is achieved through a phenomenon called the enhanced permeability and retention ("EPR") effect, which results from the typically leaky vasculature (increased permeation) and the impaired lymphatic drainage (increased retention) in the tumors. Consequently, NPs tend to remain intravascular in other tissues, yet they leak out of the vasculature in tumors and accumulate in the extra- vascular space. The EPR effect is a molecular size-dependent phenomenon, where NPs with sizes above about 40 kDa usually display prolonged circulation due to slow renal clearance and possess the potential to gradually permeate tumors in a selective fashion. However, when administered intravascularly, the nanocarriers may encounter serum protein adsorption, leading to sequestration by the mononuclear phagocyte system ("MPS") and undesirable accumulation in the liver.

Hyperbranched polyglycerol ("HPG") is a versatile nano-sized macromolecular platform that can be synthesized in a single step, resulting in polymers of controlled molecular weights with low polydispersity (Kainthan, R.K., et ah Macromolecules 2006; 39: 7708-7717; Imran ul-haq, M., et ah J. Polym. Set Part A: Polym. Chem. 2013; 51 : 2614-2621; Wilms, D., et ah Macromolecules 2009; 42: 3230-3236). Due to its structural similarities to polyethylene glycol ("PEG"), HPG possesses intrinsic ability to avoid MPS uptake, yet it is thermally and oxidatively more stable than PEG (Kainthan, R.K., et al. Biomaterials 2007; 28: 4581-4590). Furthermore, the hydroxyl groups of HPG can be derivatized with a variety of functional groups and hydrophobic molecules may be encapsulated in the hydrophobic core of an HPG NP (WO2006/130978).

SUMMARY

This disclosure is based in part on the use of derivatized hyperbranched polyglycerol ("HPG") nanoparticles ("NPs") as agents for the intravascular delivery of a biologically active moiety, such as a drug, to a biological tissue, such as a tumor, as well as other indications wherein delivery of a drug or other biologically active moiety to a tissue or cell is desired.

HPG NPs disclosed herein may be used as an intravascular carrier for a

chemotherapeutic agent or other biologically active moiety and for the preparation of a therapeutic medicament for the intravascular delivery of such drugs or moieties to a tumor or other tissues and/or cells. In particular, the HPG NPs disclosed herein may be used as an amphiphilic carrier for a chemotherapeutic agent for the treatment of cancer.

In various aspects, the claimed invention relates to a HPG nanoparticle for use in intravascular delivery of a biologically active moiety to a biological tissue, the HPG nanoparticle comprising a core comprising HPG derivatized with C₈-C₁₈ alkyl chains and a shell comprising at least one hydrophilic substituent bound to hydroxyl groups of the core, wherein the at least one hydrophilic substituent comprises polyethylene glycol ("PEG"), methoxy polyethylene glycol ("MPEG"), or hyperbranched polyglycerol (HPG). Where the hydrophilic substituent bound to hydroxyl groups of the core is HPG, such HPG bound to hydroxyl groups of the core is not derivatized with alkyl chains.

In various aspects, the claimed invention relates to a method for intravascular delivery of a biologically active moiety to a biological tissue, the method comprising administering a HPG nanoparticle loaded with the biologically active moiety to a subject, wherein the HPG nanoparticle comprises a core comprising HPG derivatized with C₈-C₁₈ alkyl chains and a shell comprising at least one hydrophilic substituent bound to hydroxyl groups of the core, wherein the at least one hydrophilic substituent comprises PEG or MPEG, or HPG. Where the hydrophilic substituent bound to hydroxyl groups of the core is HPG, such HPG bound to hydroxyl groups of the core is not derivatized with alkyl chains.

In various aspects, the claimed invention relates to use of a HPG nanoparticle for intravascular delivery of a biologically active moiety to a biological tissue, the HPG

nanoparticle comprising a core comprising HPG derivatized with C -C₁₈ alkyl chains and a shell comprising at least one hydrophilic substituent bound to hydroxyl groups of the core, wherein the at least one hydrophilic substituent comprises PEG, MePEG, HPG. Where the hydrophilic substituent bound to hydroxyl groups of the core is HPG, such HPG bound to hydroxyl groups of the core is not derivatized with alkyl chains. Also claimed is use of such HPG NPs for manufacture of a medicament for such intravascular delivery of a biologically active moiety to a biological tissue.

In various aspects, the claimed invention relates to a pharmaceutical composition for use in intravascular delivery of a biologically active moiety to a biological tissue, the pharmaceutical composition comprising a HPG nanoparticle and a biologically active moiety, the HPG nanoparticle comprising a core comprising HPG derivatized with C₈-C₁₈ alkyl chains and a shell comprising at least one hydrophilic substituent bound to hydroxyl groups of the core, wherein the at least one hydrophilic substituent comprises PEG, MPEG, or HPG. Where the hydrophilic substituent bound to hydroxyl groups of the core is HPG, such HPG bound to hydroxyl groups of the core is not derivatized with alkyl chains.

In various embodiments, the at least one hydrophilic substituent comprises PEG.

In various embodiments, the C₈-C₁₈ alkyl chains are C₈ and C₁₀ alkyl chains or C₁₀ alkyl chains.

In various embodiments, the biologically active moiety is a hydrophobic drug. In one embodiment, the hydrophobic drug is a chemotherapeutic agent. In another embodiment, the chemotherapeutic agent is a taxane or an analog thereof. In a further embodiment, the chemotherapeutic agent is docetaxel ("DTX") or an analog thereof.

In various embodiments, the HPG nanoparticles may comprise about 0.05 to about 1 moles of the at least one hydrophilic substituent per mole of the HPG component of the core (i.e. the monomer unit glycidol in the polymer, and not including alkyl chains). In some embodiments, the HPG nanoparticles may comprise about 0.5 to about 0.6 moles of the at least one hydrophilic substituent per mole of the HPG component of the core. In some

embodiments, the HPG nanoparticles may comprise 0.57 moles of the at least one hydrophilic substituent per mole of the HPG component of the core.

In various embodiments, the HPG nanoparticles may comprise about 0.02 to about 1 moles of the C -C₁₈ alkyl chains per mole of the HPG component of the core. In some embodiments, the HPG nanoparticles may comprise about 0.4 to about 0.5 moles of the C₈-C₁₈ alkyl chains per mole of the HPG component of the core. In some embodiments, the HPG nanoparticles may comprise 0.44 moles of the C₈-C₁₈ alkyl chains per mole of the HPG component of the core.

The number average molecular weight of the hyperbranched polyglycerol nanoparticle may be about 10 kDA to about 1 M Da. The number average molecular weight of the hyperbranched polyglycerol nanoparticle may be about 50 kDA to about 150 kDa. The number average molecular weight of the hyperbranched polyglycerol nanoparticle may be about 70 kDA to about 128 kDa.

The polydispersity index of the hyperbranched polyglycerol nanoparticle may be about 1 to about 10. The polydispersity index of the hyperbranched polyglycerol nanoparticle may be about 1 to about 1.5. In one embodiment, the composition of the HPG nanoparticles by weight is 19.8wt% HPG, 18.9 wt% Cio alkyl chains, and 61.3 wt% MPEG.

In various embodiments, the biological tissue is a tumor.

The HPG NPs disclosed herein may be described through a common nomenclature which identifies the basic hyperbranched structure, the core attributes, and the surface attributes as follows:

HPG-core(x)-shelli(yl)-shell₂(y2)... -shell_n(yn) (I) which designates a polymer consisting of HPG, comprising a core derivatized with a substituent selected from hydrophobic groups such as C₈, C₁₀, C₁₂, C₁₄, C₁₆ or C₁₈ alkyl groups that are either linear or branched or contain aryl substituents, wherein the amount of the core substituent is x, expressed in number of moles or as a percentage. The polymer also has n substituents on the shell, such as HPG, PEG or MPEG, as disclosed herein. Each shell substituent may be designated as being present in a certain amount yl, y2 oryn and can be expressed in number of moles or as a percentage. In some notations, general classes can be designated in the same manner, but without explicitly identifying the amounts of each. In addition, when the shell is PEG or MPEG, it may be further defined by the chain length of this polymeric component, for example, MPEG350, PEG200, etc. For the general class however, the molecular weight may be omitted.

The polymers disclosed herein are meant to include all racemic mixtures and all individual structural isomers or variants, in particular as defined by the branch patterns within the HPG structure, or in terms of the physical attachment of the surface substituents to the HPG.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows schematic diagrams of HPG-C₁₀-HPG, HPG-C₁₀-PEG and HPG-104;

Figure 2 shows in vitro toxicity assays with human dermal neonatal fibroblasts

("HDFn"), human dermal microvascular endothelial cells ("HDMEC") and human umbilical vein endothelial cells ("HUVEC") incubated with various concentrations of HPG-C₁₀-HPG for 72 h; Figure 3 shows in vitro toxicity assays with HDFn, HDMEC and HUVEC incubated with various concentrations of HPG-Qo-PEG for 72 h;

Figure 4 shows histopathological analyses of mice tissues 34 days following intravenous administration of PBS (pH 7.4) or HPG-C₁₀-PEG;

Figure 5 shows body weights of mice injected with PBS (pH 7.4) or HPG-Cio-PEG

(lOO mg/kg);

Figure 6 shows docetaxel ("DTX") release from HPG-C₁₀-PEG in PBS (pH 7.4) at

37 °C;

Figure 7 shows DTX release from HPG-d₀-HPG in PBS (pH 7.4) at 37 °C;

Figure 8 shows in vitro toxicity assays with HDFn, pancreatic cancer cells ("PANC 1"), breast cancer cells ("MDA-MB-231") and non-small cell lung cancer cells ("HCC 827") incubated with various concentrations of DTX and DTX/HPG-C₁₀-HPG for 72 h;

Figure 9 shows ratios of IC₅₀ between cancer cells to HDFn exposed to DTX or DTX/HPG-C₁₀-HPG;

Figure 10 shows relative cell viability of colorectal cancer cells ("HT-29"), HCC 827 and pancreatic cancer cells ("BxPC-3") exposed to DTX or DTX/HPG-C₁₀-PEG at different concentrations for 72 h;

Figure 11 shows relative cell viability of HUVEC and HDFn exposed to DTX or DTX/HPG-C₁₀-PEG at different concentrations for 72 h;

Figure 12 shows proliferation (XTT) assays of HDFn and HT-29 exposed to DTX or

DTX/HPG-Cio-PEG for 3 h at 4 °C or 37 °C;

Figure 13 shows scintillation count of ³H-DTX taken up by HDFn and HT-29 cells; Figure 14 shows biodistribution of HPG-C₁₀-HPG in three xenograft models with scintillation counts expressed as percent injected dose ("ID") per gram of tissue together with results of statistical analyses;

Figure 15 shows biodistribution of HPG-C₁₀-PEG in mice with subcutaneous models of HT-29 and HCC 827;

Figure 16 shows tumor-to-blood ratios at 24 h and 72 h for HT-29 and HCC 827;

Figure 17 shows tumor-to-liver ratios at 24 h and 72 h for HT-29 and HCC 827;

Figure 18 shows tumor-to-spleen ratios at 24 h and 72 h for HT-29 and HCC 827; Figure 19 shows tumor-to-muscle ratios at 24 h and 72 h for HT-29 and HCC 827;

Figure 20 shows biodistribution of HPG-C₁₀-HPG, HPG-C₁₀-PEG and HPG-104 after 24 h or 72 h in NOD-SCID mice implanted with BxPC-3 (results of statistical analyses are provided below each graph);

Figure 21 shows tumor-to-normal tissue ratios for HPG-C₁₀-HPG, HPG-C₁₀-PEG and

HPG-104 (the line indicates the mean value and results of statistical analyses are provided below each graft);

Figure 22 shows relative tumor volume as a function of number of days after implant for mice with BxPC-3 tumors treated with either a low dose or high dose of DTX in a clinical formulation or DTX/HPG-C₁₀-PEG;

Figure 23 shows a 1H NMR spectrum (D₂0, 300 MHz) of HPG-C₁₀-PEG; and

Figure 24 shows a GPC-MALLS profile of HPG-C₁₀-PEG in 0.1 N NaN0₃.

DETAILED DESCRIPTION

Any terms not directly defined herein shall be understood to have the meanings commonly associated with them as understood by a person of ordinary skill in the art.

Polymer nanoparticles ("NPs") disclosed herein include those shown in Figure 1, which all appear to be related to hyperbranched polyglycerol ("HPG") NPs. The terms "nanoparticle" or "nanoparticles" are used as they are normally understood to a person of ordinary skill in the art and often refer to materials, structures or objects having at least one dimension that is between about 0.1 nm and about 100 nm. Synthesis of HPG NPs has been previously described, including the production of amphiphilic copolymers and amphophilic block copolymers, including derivatization with various functional groups and/or the production of copolymers and block copolymers (such as the addition of alkyl groups through ester linkages and the addition of polyalkylene glycol groups).

In various aspects, the HPG NPs disclosed herein may include HPG NPs derivatized with substituents having functional groups such that the HPG NPs may be used for

intravascular delivery of a biologically active moiety to a biological tissue of a subject. The phrase "intravascular delivery" is used as it is normally understood to a person of ordinary skill in the art and often refers to administration of a biologically active moiety to a subject by entry into one or more blood vessels.

Generally, the HPG NPs disclosed herein have a "core", which includes an initiator (for example, trimethyloyl propane (TMP)) and HPG. The HPG core may be derivatized with C₈- C₁₈ alkyl chains. When the HPG core is derivatized with alkyl chains, the "HPG component" of the core is intended to refer only to the monomer unit glycidol in the polymer, and is not intended to include the alkyl chains with which the HPG is derivatized. The "core" may be prepared by adding a glycerol epoxide (the hyperbranching component monomer) to an alkyl epoxide (which imparts the hydrophobic nature to the "core"). In some embodiments, the "core" may be enclosed in a "shell", wherein the shell comprises at least one hydrophilic substituent bound to hydroxyl groups of the core. The HPG nanoparticles may comprise about 0.05 to about 1 moles of the at least one hydrophilic substituent per mole of the HPG component of the core. In some embodiments, the HPG nanoparticles may comprise about 0.5 to about 0.6 moles of the at least one hydrophilic substituent per mole of the HPG component of the core. In some embodiments, the HPG nanoparticles may comprise 0.57 moles of the at least one hydrophilic substituent per mole of the HPG component of the core. During shell formation, it may be possible for a portion of the "shell" substituents to react with hydroxyl groups located towards the centre of the NP. Even if such reactions occur, the core maintains its hydrophobic character. The HPG NPs disclosed herein may include C₈-C₁₈ alkyl chains, or other similar alkyl chains. The term "alkyl" is used as it is normally understood to a person of ordinary skill in the art and often refers to monovalent saturated aliphatic hydrocarbon groups having from one to 20 carbon atoms, unless otherwise defined. The hydrocarbon may be either straight-chained or branched and may contain cycloaliphatic or aryl substituents. Alkyl chains may be selected from one or more of C₈-C₁₈ alkyl chains. Alternatively, the alkyl chains may be selected from C₈, Cg, C₁₀, C_11} C₁₂, C₁₄, C₁₆ or C₁₈ alkyl chains. Alternatively, the alkyl chains may be selected from C₈ and/or C₁₀ alkyl chains. The alkyl chain or chains may comprise about 0.02 to about 1 moles of the C₈-C₁₈ alkyl chains per mole of the HPG component of the core. In some embodiments, the HPG nanoparticles may comprise about 0.4 to about 0.5 moles of the C₈-C₁₈ alkyl chains per mole of the HPG component of the core.. In some embodiments, the HPG nanoparticles may comprise 0.44 moles of the C₈-C₁₈ alkyl chains per mole of the HPG component of the core. In some embodiments, the composition of the HPG nanoparticles by weight is 19.8wt% HPG, 18.9 wt% C₁₀ alkyl chains, and 61.3 wt% MPEG.

The composition of the HPG nanoparticles may be determined by NMR spectroscopy.

The alkyl chain or chains selected for the core and their amount may depend on the intended use for the HPG NP.

The term "initiator" is used as it is normally understood to a person of ordinary skill in the art and often refers to a small molecule comprising an alkyl component and more than one, and preferably more than two, hydroxyl groups. However, the initiator may have three or four or more hydroxyl groups. An example of an initiator is trimethyloyl propane (TMP).

The terms "amphiphilic", "amphiphilic polymer" or "amphiphilic NP" are used as they are normally understood to a person of ordinary skill in the art and often refer to the presence of both a hydrophobic and hydrophilic moiety in a single molecule. Hydrophobic refers to any substance or portion thereof which is more soluble in a non-polar solvent than in a polar solvent. Hydrophobicity can be conferred by the inclusion of non-polar groups in a molecule, including, but not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic or heterocyclic group(s). Hydrophilic refers to any substance or portion thereof which is more soluble in a polar solvent than in a non-polar solvent. Hydrophilic characteristics derive from the presence of polar or charged groups such as carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxyl and other similar groups. The hydrophilic portion may comprise MPEG, PEG or HPG.

The term "polyethylene glycol" or "PEG" is used as it is normally understood to a person of skill in the art and often refers to such compounds having a molecular weight between about 200 to about 20,000, depending on the number of ethylene oxide units in the polymer chain. Preferred molecular weights are from about 200 to about 400, about 200 to about 1000 and about 200 to about 2000, although molecular weights of about 2000 to about 8000 may also be used.

The term "methoxypoly(ethylene oxide)" or "MPEG" is used as it is normally understood to a person of skill in the art and often refers to such compounds having a molecular weight between about 350 to about 10,000, depending on the number of ethylene oxide units in the polymer chain. Preferred molecular weights are from about 350 to about 550, about 350 to about 750 and about 350 to about 2000, although molecular weights of about 2000 to about 5000 may also be used.

Methods of preparing or synthesizing the HPG NPs disclosed herein will be understood by a person of ordinary skill in the art having reference to known chemical synthesis principles. For example, HPG NPs can be synthesized by the anionic ring opening multibranching polymerization (ROMBP) of glycidol alone (to form HPG- 104 which does not contain any hydrophobic core or hydrophilic shell) or glycidol with octadecyl glycidyl ether or glycidol with octadecyl glycidyl ether and MPEG. Thus, (HPG-C₁₀-HPG (which has a hydrophobic core and an HPG shell) may be synthesized in two-steps in a single reaction pot by the anionic ring opening polymerization of a glycidol with octadecyl glycidyl ether ( to form the hydrophobic core) followed by the polymerization of glyciol alone (to form the HPG shell). HPG-Qo-PEG may be synthesized by the anionic ring opening polymerization of glycidol with an epoxide-containing co-monomer by a two-step procedure. In the first step, a mixture of glycidol and octadecyl glycidyl ether (ODGE) is polymerized using trimethylol propane (TMP) as the initiator at 95 °C. In the second step, polymerization is continued in the same reaction pot by the addition of ct-epoxy-co-methoxy polyethylene glycol (MPEG₄₀o-epoxide) to afford HPG-C₁₀-PEG NPs with a hydrophobic core and a hydrophilic PEG shell. Characteristics of HPG-104, HPG-Cio-PEG and HPG-C₁₀-HPG are listed in Table 1. These HPG NPs may have similar molecular weights (about 100 kDa) with very low polydispersity and similar size (hydrodynamic radius of approximately 5 nm). HPG-C₁₀-PEG is comprised of 22 mol% of the C₁₀ alkyl chains (hydrophobic moiety) and 28.5 mol% of PEG chains, as determined by 1H NMR spectroscopic analysis.

Table 1. Physical Characteristics of HPG NPs

The embodiments of the HPG NPs disclosed herein include all possible stereochemical alternatives, including those illustrated or described herein. In some embodiments, the HPG NPs disclosed herein include isomers such as geometrical isomers having different branch patterns. The HPG NPs synthesized by methods disclosed herein are random branching HPG NPs and will contain glycerol monomers that are fully reacted, e.g., linked in three directions, or partially reacted, being linked to another monomer in one or two directions. The presence of each branching architecture may be confirmed by analytical techniques (e.g., 2D NMR HSQC experiments).

In some embodiments, the HPG NPs disclosed herein may be in the solvent addition form. The HPG NPs may be associated with a non-stoichiometric amount of a solvent, water and/or buffers, typically expressed as a weight or volume percent. The solvent may be, for example, and without limitation, a pharmaceutically acceptable solvent or other biocompatible solvent including ethanol, DMSO, propylene glycol, glycerol, PEG200, PEG300, Transcutol or Solutol.

The HPG NPs disclosed herein may be administered to a subject. The phrase "subject" is used as it is normally understood to a person of ordinary skill in the art and may include a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, rabbit, etc. The subject may be suspected of having or at risk for having a cancer or other disease that can be treated by intravascular delivery of a biologically active moiety to a biological tissue. A cancer (for example, lung, pancreatic, breast or colon cancers) or an inflammatory disease as well as other indications may be desired targets of the HPG NPs disclosed herein for the parenteral delivery of a drug or other biologically active moiety. Compositions and HPG NPs according to embodiments disclosed herein may be administered in any of a variety of known parenteral routes. For example, the HPG NPs may be administered parenterally, i.e., by intra-articular, intravenous, subcutaneous, intramuscular, intraperitoneal, intracerebral, or

intracerebroventricular administration.

The biologically active moiety can be of any class of hydrophobic molecule which can be solubilized and held within the hydrophobic core of the HPG NP. Compound solubility can be measured and defined as per the United States Pharmacopoeia / The National Formulary standards and guidelines. In one embodiment, small molecules, proteins, antibodies, peptides or pharmaceutically acceptable salts thereof may be held within the hydrophobic core. In one embodiment, the hydrophobic molecules may be molecularly targeted inhibitors, e.g. PI3K, CDK, PLK inhibitors. In another embodiment, the HPG NPs disclosed herein may be used for intravascular delivery of a chemotherapeutic agent to a tumor. The chemotherapeutic agent may be a taxane. The chemotherapeutic agent may be docetaxel ("DTX") or an analog thereof. The chemotherapeutic agent may be paclitaxel or an analog thereof. The biologically active moiety may be mitomycin or an analog thereof. Mitomycin may include all mitomycin analogs. Mitomycin and analogs thereof may include, for example, mitomycin A, mitomycin B, mitomycin C, mitomycin D, mitomycin F, mitomycin G, mitomycin H, mitomycin K and analogs thereof. The biologically active moiety may be mitomycin C. The biologically active moiety may be mitomycin F. The biologically active moiety may be valrubicin. The biologically active moiety may be vinblastine. The biologically active moiety may be cisplatin. The biologically active moiety may be methotrexate. The biologically active moiety may be doxorubicin or an analog thereof. The biologically active moiety may be epirubicin. The biologically active moiety may be gemcitabine. The biologically active moiety may be everolimus. The biologically active moiety may be suramin. The biologically active moiety may be a combination of moieties. The combination of moieties may be methotrexate, vinblastine, and doxorubicin (M-VAC). The combination of moieties may be M-VAC and cisplatin. The combination of moieties may be two or more of valrubicin, cisplatin, paclitaxel and DTX.

The chemotherapeutic agent may hydrophobically bind with the core of the HPG NPs disclosed herein, allowing for the delivery of the chemotherapeutic agent to a tumor. The chemotherapeutic agent may then be released. Alternatively, the drug/HPG NP complex may be taken up by cancer cells and/or stromal cells in the tumor microenvironment and therapeutic agent may be then released. In various aspects, the hydrophilic shell may act to shield the hydrophobic core and solubilize the HPG NPs in the blood of the subject.

In another aspect, the HPG NPs disclosed herein may be used as an intravascular formulation for the treatment of a cancer. The HPG NPs described herein may be used to treat lung cancer, pancreatic cancer, colon cancer prostate cancer, ovarian cancer, skin cancer, breast cancer, head and neck cancer, liver cancer, stomach cancer, sarcomas, or brain cancer. In general, a solid tumor may be treated with a HPG NP disclosed herein.

When injected intravascularly, the HPG NPs will inevitably encounter the normal cells of the body, particularly endothelial cells that line the blood vessels. Therefore, NPs for intravascular drug delivery systems should show low toxicity to non-cancerous cells, including primary human cells. The toxicity of the HPG NPs disclosed herein was tested to two different types of endothelial cells, human umbilical vein endothelial cells ("HUVEC") and human dermal microvascular endothelial cells ("HDMEC"). The toxicity of the HPG NPs disclosed herein was also tested to fibroblasts (e.g., human dermal neonatal fibroblasts

("HDFn")) as they are one of the most abundant cells in the body. Different concentrations of HPG-Cio-HPG and HPG-C₁₀-PEG were incubated with the cell lines for 72 h and XTT assays were carried out to assess cell viability. The results of the in vitro assay of HPG-C₁₀-HPG to assess the toxicity of this HPG NP to HUVEC, HDMEC and HDFn are shown in Figure 2. The IC₅₀ values are shown in Table 2. No ill effect on cell proliferation was observed at 1 mg/mL of HPG-Cio-HPG to all three cell types. Toxicity of HPG-C₁₀-HPG to HUVEC is evident at 5 mg/mL; however, the effect on proliferation of HDFn and HDMEC was not as significant at this concentration. Notably, the lower plateau was not reached with the concentrations of HPG-C₁₀-HPG tested for HDFn, therefore, the IC50 value for this cell line could not be calculated. For HPG-C₁₀-PEG, the results are shown in Figure 3. Cytotoxicity was observed with HDFn and HUVEC at 0.5 mg/mL, while it was absent in HDMEC. At 1 mg/mL, cytotoxicity was apparent in all the primary cell lines tested. The IC₅₀ values of HPG-C₁₀-PEG were found to be 0.96, 1.2 and 0.56 mg/mL for HDFn, HDMEC and HUVEC, respectively. However, HPG-C₁₀-PEG exhibited much lower cytotoxicity compared to other polymeric NPs such as polyamidoamine (PAMAM) dendrimers; for example, the cytotoxicity observed for generation 4 PAMAM dendrimers ranged from 50-300 μg/mL (Zhang, X., et al. J. Control Release 2014; 174: 209-216).

Table 2. IC50 values for in vitro toxicity assays with human primary cell lines incubated with HPG-do-HPG for 72 h

The toxicity of the HPG NPs disclosed herein was also tested on mice after intravascular administration. Normal C57/BL6 mice were intravenously injected with 100 mg/kg of HPG-Qo-PEG, and no detectable toxicity was evident from histopathological examination of organs (Figure 4) and body weights (Figure 5). Hematological analysis of the whole blood samples collected from the mice also did not show any significant difference in the major hematological parameters between the control and test groups (Table 3). The hematological evaluation is critical for safety since intravascular delivery of hydrophobic biologically active moieties is generally designed to be long-circulating and hence have long blood residence times. These results indicate that the HPG NPs disclosed herein can be safely injected intravenously into mice without any adverse effects and may be blood compatible. Thus, the HPG NPs disclosed herein may be intravascularly injected into a subject without any or minimal adverse effects.

Table 3. Hematology data of mice injected with PBS or HPG-C₁₀-PEG

The HPG NPs disclosed herein may be loaded with a biologically active moiety. The biologically active moiety may interact with the hydrophobic core of the HPG NP and be encapsulated within the hydrophilic shell of the HPG NP. The content and type of alkyl chains at the core may affect the solubility of the HPG NPs and the ability of HPG NPs to carry a hydrophobic drug. The higher the content of alkyl chains, the more likely it is that a hydrophobic drug will be carried in the core of the HPG NP. However, if the content of alkyl chains is too high, the HPG NP will no longer be soluble in the blood stream. In various embodiments, the content of alkyl chains in the HPG NP is about 0.02 to about 1 moles of alkyl chains per mole of the HPG component of the core. In some embodiments, the HPG nanoparticles may comprise about 0.4 to about 0.5 moles of alkyl chains per mole of the HPG component of the core.. In some embodiments, the HPG nanoparticles may comprise 0.44 moles of alkyl chains per mole of the HPG component of the core.

The HPG NPs disclosed herein and loaded with a chemotherapeutic agent may show a prolonged drug release profile. For example, drug loading experiments were undertaken to load up to 5 wt % of ³H-DTX into HPG-C₁₀-PEG, with the highest stability of the formulation achieved at 1% loading, which translates to a DTX:HPG-C₁₀-PEG ratio of 1 : 1. An in vitro drug release profile in PBS (pH 7.4) at 37 °C from HPG-C₁₀-PEG is shown in Figure 6. The release profile of ³H-DTX suggests that the drug was released in a sustained and controlled manner over about 17 days, with 50% of the drug released in about 3 days and 90% of the drug released within about 15 days. Similar results for an in vitro drug release profile in PBS (pH 7.4) at 37 °C from HPG-C₁₀-HPG are shown in Figure 7. The maximum drug loading that could be stably achieved was 1 wt %/ of HPG. Incorporated ³H-DTX was released with an initial burst phase and then a subsequent sustained release at 37 °C in PBS. Fifty percent of the drug was released by about 5 days and 90 % of the drug was released by about 13 days. The % drug release data fit well with a 2-phase decay curve (Graphpad®) with R^z = 0.96.

The HPG NPs disclosed herein and loaded with a chemotherapeutic agent such as DTX may show a relative protective effect with non-cancerous cells, potentially widening the therapeutic index of the HPG NPs disclosed herein. In one aspect, the HPG NPs disclosed herein which are loaded with a chemotherapeutic agent may effectively kill cancer cells while showing less effect on normal cells. In another aspect, HPG NPs disclosed herein and loaded with a chemotherapeutic agent may show a preferential protective effect on primary noncancerous cells. Cellular uptake rates of the HPG NPs comprising HPG-C10-PEG appears similar for both primary cells and cancer cells, indicating that the enhanced killing of cancer cells compared to primary cells may be related to differences in how HPG NPs are processed intracellularly. On the other hand, the cellular uptake rates of HPG-C10-HPG by normal and cancer cells differ, which suggests that cellular uptake also depends on the structure and composition of the HPG NPs. The ability of the HPG NPs disclosed herein and loaded with DTX to kill cancer cells was assessed with XTT assays. Human primary cells and a panel of cancer cells were incubated with different concentrations of DTX-loaded HPG-C₁₀-HPG ("DTX/HPG-C₁₀- HPG"). XTT assays were also carried out after incubating cancer cells and human primary cells with DTX-loaded HPG-C₁₀-PEG ("DTX/HPG-C₁₀-PEG") at various concentrations (0- 10000 nM) for 72 h. Exposure of cells to free DTX was performed in parallel for both assays for comparison.

As shown in Figure 8, a relative protective effect with HDFn was observed with DTX/HPG-Qo-HPG which is not as apparent with cancer cells (non-small cell lung cancer cells ("HCC 827"), pancreatic cancer cells ("PANC 1") and breast cancer cells ("MDA-MB- 231")). To simulate the in vivo situation, ratios of IC₅₀ values between cancer cells and HDFn to DTX or DTX/HPG-C₁₀-HPG were assessed. Ideally this ratio for a given drug will be large, indicating that the drug is toxic to cancer cells yet non-toxic to normal cells. Conversely, a ratio of 1 suggests that the drug is equally toxic to both cancer and normal cells. When the ratio is less than 1, the drug is more toxic to normal cells than to cancer cells. The ratios can be plotted graphically and tabulated, as shown in Figure 9 and Table 4. For free DTX, the IC₅₀ value for HDFn was lower than those for HCC 827 and PANC 1, indicating that DTX can be more toxic to normal fibroblasts than to cancer cells. MDA-MB-231 cells were more susceptible to DTX than HDFn. However, the IC₅₀ ratios of DTX for the three cancer cell lines tested were very close to 1. For DTX/HPG-C₁₀-HPG, the IC₅₀ ratios for HCC 827, PANC 1 and MDA-MB-231 cells were 2.1, 3.7 and 3.3, respectively. These values may indicate that cancer cells are more susceptible to killing by DTX/HPG-Cio-HPG than the non-cancerous HDFn cells. Without being bound by any particular theory, this change of susceptibility may be due to a change of cellular interaction with DTX/HPG-Cio-HPG, leading to potential increase in therapeutic index. Table 4. Values of IC₅₀ for primary cells (HDFn) and cancer cells (PANC 1, MDA-MB-231 and HCC 827) exposed to DTX or DTX/HPG-Ci₀-HPG and ratios of IC₅₀ for HDFn to IC₅₀ for cancer cells exposed to DTX or DTX/HPG-C₁₀-HPG

For experiments conducted with DTX/HPG-C₁₀-PEG, DTX, a drug that targets microtubules, displayed similar toxicity to all cells tested (Figures 10 and 1 1), as expected. However, DTX/HPG-C₁₀-PEG killed cancer cells (colorectal cancer cells ("HT-29"), HCC 827 and pancreatic cancer cells ("BxPC-3")) with similar effect as the free DTX (Figure 10) while the primary human cells (HUVEC and HDFn) were not as susceptible to killing by DTX/HPG- Cio-PEG (Figure 1 1). The HPG NPs disclosed herein may show a protective effect on normal cells compared to free DTX. The protective effect observed in normal human cells was consistently absent in the three cancer cell lines tested. These results suggest that the HPG NPs disclosed herein may enhance the therapeutic ratio for the chemotherapeutic drug by killing the cancer cells while protecting the normal cells from cytotoxic effects of DTX.

The differential killing effects of DTX/HPG-Cio-PEG suggest two non-exclusive possibilities. Firstly, the uptake of DTX/HPG-Qo-PEG differs between the cancer cells and the primary human cells thereby leading to a difference in toxicity effects. Secondly, there is a difference between the processing of DTX/HPG-C^-PEG in the cancer cells, when compared to the non-cancerous cells, so that the availability of DTX is different. To understand if the uptake mechanism of DTX/HPG-C₁₀-PEG is energy-dependent, cellular uptake experiments were performed at different temperatures. HDFn and HT-29 cells were exposed to DTX/HPG- Cio-PEG at either 4 °C (ice) or 37 °C for 3 h. Experiments with free DTX were performed in parallel for comparison. Significantly, more cells were killed when the cells were exposed to DTX/HPG-C₁₀-PEG at 37 °C, when compared to exposure at 4 °C, suggesting that there was more uptake of DTX/HPG-C₁₀-PEG at the higher temperature (Figure 12). This was true for both the normal cells and the cancer cells, indicating that DTX/HPG-C₁₀-PEG was taken up in an energy-dependent manner in both cell types. Moreover, similar cell killing by the free DTX was observed for exposure at 4 °C or 37 °C, which may show that cellular uptake of free DTX is not energy-dependent and is presumably through passive diffusion of the drug into the cells, similar to other chemotherapeutic agents.

To determine if the rate of uptake differs between primary and cancer cells, these cells were exposed to HPG-C₁₀-PEG loaded with tritiated DTX ("³H-DTX/HPG-C₁₀-PEG"). After 3 h of exposure, the cells were washed and lysed for scintillation count. The percent uptake of H-DTX/HPG-C₁₀-PEG was the same between the non-cancerous cells and the cancer cells, whether 100,000 or 50,000 cells were seeded for the experiments (Figure 13). There was also no statistical difference between the percent uptake of free ³H-DTX between these cells. The results suggest that both HDFn and HT-29 cells take up ³H-DTX/HPG-C₁₀-PEG at the same rate. Therefore, without wishing to be bound by any particular theory, the preferential killing effect of DTX/HPG-Cjo-PEG on cancer cells may result from different cellular processing mechanisms, instead of differences in cellular uptake. The foregoing results suggested that both types of cells (HDFn and HT-29) take up DTX/HPG-C₁₀-PEG to a similar extent with a similar rate. These results may indicate that a different processing mechanism of DTX/HPG- Cio-PEG inside the cancer cells and normal cells results in differential cytotoxic effect.

Tumor uptake and biodistribution studies are critical predictors of therapeutic efficacy as well as normal organ toxicity. A major limitation of NPs for intravascular drug delivery has been their uptake by the mononuclear phagocyte system ("MPS") as a result of rapid sequestration of NPs from the blood by hepatic midzonal and periportal Kupffer cells

(Moghimi, S. M., et al. Pharmacol. Rev. 2001; 53: 283-318). Typical NPs undergo high uptake into the organs of the MPS such as the liver and spleen. Previous studies with PEGylated NPs of similar size (approximately 10 nm) have shown mean liver and spleen uptake of

approximately 25% ID/g at 24 h in mice (Cao, T., et al. Biomaterials 2013; 34: 7127-7134). Also, BIND-014, a PLGA-PEG NP for DTX delivery has shown considerable accumulation in the spleen (up to 20% ID/g) (Hrkach, J., et al. Sci. Transl. Med. 2012; 4: 128-139). This nonspecific uptake may result in declining blood drug concentration incapable of maintaining therapeutically effective drug concentrations in the tumors. The HPG NPs disclosed herein may have a more favourable biodistribution profile due to reduced MPS clearance and long circulation half-life. In various embodiments, the HPG NPs disclosed herein may persist in the blood circulation of a subject and may also show low accumulation in the liver and spleen of the subject.

The HPG NPs disclosed herein may accumulate in a tumor. However, if the hydrophobic core is not sufficiently shielded by the hydrophilic shell, HPG NPs may accumulate in the liver and spleen. Without being bound by any particular theory, it appears that without significant shielding, the hydrophobic core of the HPG NPs disclosed herein interacts with cells of the MPS.

Figure 14 shows blood and tumor distributions of radio-labelled HPG-C₁₀-HPG in three subcutaneous xenograft models (HCC 827, HT-29 and BxPC-3). Results for a similar study with HPG-Cio-PEG are shown in Figure 15. These xenograft models were chosen to reflect the heterogeneity of the tumor microenvironments. BxPC-3 is a xenograft model with a low degree of vascularization and vascular permeability. On the other hand, HT-29 has high vascularity and increased vascular permeability. HCC 827 is a non-small cell lung carcinoma that is known to recruit fibroblasts.

With marked differences between the xenograft models, similar patterns of

biodistribution were observed with radiolabeled HPG-C₁₀-HPG. Blood-resident HPG-Qo-HPG decreased through the observed time points (6, 24 and 72 h), yet around 3% injected dose ("ID")/g of HPG-C₁₀-HPG was retained in the tumors. With varying degrees of HPG-C₁₀-HPG accumulating at the brain, lungs, heart, kidneys and pancreas, a significant decrease of HPG- Cio-HPG remaining in these tissues over the observed time points was noted.

For HPG-C₁₀-PEG, similar patterns of biodistribution were observed for two xenograft models (HT-29 and HCC 827) (Figure 15). The amount of HPG-C₁₀-PEG in the circulation decreased significantly from 24 h to 72 h post-injection, while the accumulation of HPG-C₁₀- PEG in the tumors was sustained. The mean liver and spleen uptake for HPG-C₁₀-PEG in the xenograft models ranged between 9-16% and 4-7 % ID/g, respectively, while the mean tumor uptake of HPG-C₁₀-PEG was in the range of 3-6 % ID/g at the 24 h and 72 h time points.

Comparing to HPG-C₁₀-HPG, , biodistribution studies showed that accumulation of HPG-C₁₀- HPG in the liver and the spleen reached approximately 30 ID/g and 10 ID/g, respectively. which are significantly higher than the accumulation of HPG-C₁₀-PEG in these organs.

Tumor-to-blood ratios indicated increasing accumulation in the tumors over time (Figure 16). The significant tumor uptake was likely attributable to the ability of the HPG-Cio-PEG to circulate in the blood for a long duration as well as to the small hydrodynamic diameter of approximately 12 nm of HPG-C₁₀-PEG. Tumor-to-liver and tumor-to-spleen ratios of HPG- Cjo-PEG (Figures 17 and 18, respectively) also suggest relatively low elimination by the MPS on the basis that there was minimal difference between the two xenograft models. Long circulation half-life of up to 34 h has previously been observed for a similar class of NPs (Kainthan, R. K., et al. Biomaterials 2008; 29: 1693-1704). Very little HPG-C₁₀-PEG accumulated in skeletal muscles, which is evident with the large tumor-to-muscle ratios in both models (Figure 19).

These studies also demonstrate that the biodistribution of HPG-C₁₀-PEG is similar to that of albumin (Matsumura, Y. and Maeda, H. Cancer Res. 1986; 46: 6387-6392). This finding is significant since albumin has been established as an effective NP drug carrier for delivery of the anti-cancer drug paclitaxel (nab-paclitaxel, Abraxane) (Gradishar, W. J., et al. J. Clin. Oncol. 2009; 27: 3611-3619 and Von Hoff, D. D., et al. J. Clin. Oncol. 2011; 29: 4548- 4554). Therefore, HPG-C₁₀-PEG may act as a drug carrier in a similar way, while being easier to produce and a more versatile carrier for intravascular drug delivery. Moreover, HPG- o- PEG may allow for bulk production at an acceptable cost while also addressing the safety concerns over prion and viral transmission in the blood supply associated with animal-derived albumin.

Without being bound by any particular theory, the biodistribution profile of the HPG NPs disclosed herein may be achieved through the enhanced permeability and retention

("EPR") effect. Figure 20 shows the results of biodistribution at 24 and 72 h post-injection for HPG-Cio-HPG, HPG-C₁₀-PEG and HPG-104. The biodistribution studies were performed with the same tumor xenograft model (BxPC-3 subcutaneously implanted into NOD-SCID mice). Similar to the biodistribution with HT-29 and HCC 827 xenograft models, HPG-Cio-HPG accumulated heavily at the liver and the spleen. With PEGylation, the accumulation of HPG- C₁₀-PEG at the liver and the spleen was significantly reduced and the proportion of blood- resident HPG-C₁₀-PEG at 24 and 72 h was increased significantly. With neither the hydrophobic core nor the hydrophilic shell, HPG- 104 showed the lowest accumulation at the liver and the spleen. The results suggest that the unfavourable accumulation of HPG-C₁₀-HPG at the liver and spleen may be due to the presence of the hydrophobic core, whose effect is partially shielded by PEGylation (HPG-C₁₀-PEG) and its removal (HPG-104) results in a significant decrease of accumulation at these organs.

In various embodiments, the HPG NPs disclosed herein may deliver chemotherapeutic agents to tumors while sparing the normal tissue (such as the liver) to reduce or minimize nonspecific toxicity. HPG-C₁₀-HPG, HPG-C₁₀-PEG and HPG-104 may all accumulate in tumors at similar levels at 24 and 72 h (e.g., 3-6% ID/g). However, the ratio of % ID/g of the HPG NPs between the liver and the tumor for each mouse implanted with an HPG NP may be a useful indicator of specific accumulation. Figure 21 shows the tumor-to-liver, tumor-to-spleen, tumor-to-heart and tumor-to-muscle ratios of HPG-C₁₀-HPG, HPG-C₁₀-PEG and HPG-104 for assessing specific tumor accumulation. As expected, based on the biodistribution data, the tumor-to-liver and tumor-to-spleen ratios of HPG-C₁₀-HPG was the lowest among the three HPG NPs; however, the tumor-to-heart ratio of HPG-C₁₀-HPG was the highest with good tumor-to-muscle ratio. Comparing HPG-C₁₀-PEG and HPG-104, the latter has a better tumor- to-liver ratio, comparable tumor-to-spleen and tumor-to-heart ratios, yet an inferior tumor-to- muscle ratio. Therefore, these data suggest that the hydrophobicity of the HPG NPs may affect the specific accumulation at a tumor (as compared with the liver).

In various embodiments, HPG NPs disclosed herein and loaded with a

chemotherapeutic agent may deliver the chemotherapeutic agent to a tumor and decrease a volume of that tumor. For example, as shown in Figure 22, mice treated with three weekly injections (Q7dx3) a low dose (5 mg/kg) ("LD") of DTX delivered intravenously as DTX in a clinical formulation (sourced from Hospira) or HPG-C₁₀-PEG loaded with comparable amount of DTX (DTX/HPG-Cio-PEG) showed a decreased relative tumor volume compared to those mice treated with PBS (pH 7.4). However, tumor volume for mice treated with DTX/HPG-Cio- PEG was significantly lower than for mice treated with the clinical formulation of DTX at the low dose. For high doses of DTX (15 mg/kg) ("HD"), three out of five mice treated with the clinical formulation of DTX lost greater than 20% of their body weight and needed to be euthanized about 60 days following implantation of the BxPC-3 cells. In contrast, HDs of DTX incorporated into HPG-Cio-PEG could be delivered to a tumor and effectively decreased the relative tumor volume, indicating that DTX-HPG-C₁₀-PEG may be more efficacious than clinical formulations of DTX.

Suitable pharmaceutical compositions comprising the HPG NPs disclosed herein may be formulated by means known in the art and their mode of intravascular administration and dose determined by the skilled practitioner. Many techniques known to one of skill in the art are described m Remington: the Science and Practice of Pharmacy by Alfonso Gennaro, 20 ed., Lippencott Williams & Wilkins (2000).

An "effective amount" of a pharmaceutical composition disclosed herein includes a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as decreased cancer cell proliferation, increased life span or increased life expectancy. A therapeutically effect amount of a HPG NP incorporating a biologically active agent may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the biologically active agent to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the formulation are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as the prevention or the prevention of the progression of an indication. Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease.

It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. The amount of composition may vary according to factors such as the disease state, age, sex and weight of the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage.

In some embodiments, HPG NPs disclosed herein may be used, for example, and without limitation, in combination with other treatment methods. For example, HPG NPs disclosed herein incorporating a chemotherapeutic agent may be used as neoadjuvant (prior), adjunctive (during), and/or adjuvant (after) therapy in combination with other therapies known to one of ordinary skill in the art, including additional chemotherapies, radiotherapy, and/or surgery.

Various alternative embodiments and examples are disclosed herein. These

embodiments and examples are illustrative and should not be construed as limiting the scope of the invention.

EXAMPLES

All reagents and chemicals were purchased from Sigma- Aldrich, Canada (Oakville, Ontario) and used without further purification unless mentioned. Glycidol was purified by distillation under reduced pressure before use and stored over molecular sieves at 4°C.

Octyl/decyl glycidyl ether (ODGE) was purified by distillation under reduced pressure, a- Epoxy-co-methoxy polyethylene glycol was synthesized as per published procedure (Cho, K., et al. Macromol. Rapid Commun. 1999, 20: 598-601). Tritiated methyl iodide was purchased from ARC Radiochemical (St. Louis, MO) as solution in toluene and was used directly after dilution in dimethylsulfoxide.

1H spectra were recorded in D₂0 on Bruker Avance 300 MHz NMR spectrometer. Absolute molecular weights of the polymers were determined by Gel Permeation

Chromatography (GPC) on a Waters 2695 separation module fitted with a DAWN EOS multi- angle laser light scattering (MALLS) detector coupled with Optilab DSP refractive index detector, both from Wyatt Technology. Hydrodynamic radii (R_h) of the polymers were measured using quasi-elastic light scattering (QELS) detector coupled to the MALLS detector. GPC analysis was performed using Waters ultrahydrogel columns (guard, linear and 120) and 0.1 N NaN0₃ (10 mM phosphate buffer) as the mobile phase. All animals were bred in the Animal Resources Centre at the British Columbia Cancer Research Centre and all experimental procedures were done in accordance with ethical guidelines set by the Animal Care Committee and the Canadian Council on Animal Care.

EXAMPLE 1: Synthesis and characterization of HPG NPs (HPG-C₁₀-HPG, HPG-

C₁₀-PEG and HPG-104)

Synthesis ofHPG-C₁₀-HPG

Polymerization was carried out in a three-necked round-bottomed flask fitted with a mechanical stirrer. The initiator, trimethylol propane (TMP, 0.12 g) was added into the flask under argon atmosphere and reacted with potassium methylate (150 xL, 25 wt% solution in methanol) at 65°C for 30 min. Methanol was removed under vacuum for 4 h. The temperature was then increased to 95°C and a mixture of glycidol (2.5 mL) and ODGE (4 mL) was added slowly using a syringe pump over a period of 18 h. Potassium hydride (3 drops in mineral oil) was added and the reaction mixture was stirred for 4 h. Glycidol (3 mL) was added slowly over a period of 15 h. After complete addition of the monomer, the reaction mixture was stirred for an additional 5 h. The polymer was dissolved in methanol and twice precipitated from excess diethyl ether. The polymer was then dissolved in water and dialyzed against water using MWCO 10000 cellulose acetate membrane for two days with periodic changes in water. The dry polymer was recovered by freeze drying.

^!H NMR (D₂0, 300 MHz): δ ppm 0.86 (CH₃ from alkyl chain), 1.2-1.6 (-CH₂ from alkyl chain), 3.4-4.0 (main chain protons from HPG). C₁₀ alkyl content: 11 mol%. GPC- MALLS (0.1 N NaN0₃): M_n 128000; M_w/M_n 1.03; R_h 4.5 nm. Synthesis ofHPG-C₁₀-PEG

Polymerization was carried out in a three-necked round-bottomed flask fitted with a mechanical stirrer. The initiator, trimethylol propane (TMP, 1.2 g) was added into the flask under argon atmosphere and reacted with potassium methylate (1.5 mL, 25 wt% solution in methanol) at 65°C for 15 min. Methanol was removed under reduced pressure for 4 h. The temperature was then increased to 95°C and a mixture of glycidol (26 mL) and ODGE (26 mL) was added slowly using a syringe pump over a period of 24 h. After the addition was complete, the reaction mixture was stirred for an additional 3 h. a-Epoxy-co-methoxy polyethylene glycol (mPEG₄oo-epoxide, 54 mL) was added slowly over a period of 24 h. After complete addition of the monomer, the reaction mixture was stirred for an additional 3 h. The polymer was dissolved in methanol and the solution was passed through Amberlite-IER-120 resin to remove the potassium ions. The unreacted ODGE was removed by extraction with hexane. The polymer was precipitated from excess diethyl ether, dissolved in water and dialyzed against water using MWCO 10000 cellulose acetate membrane for 2 days with periodic changes in water. The dry polymer was recovered by freeze drying.

1H NMR (D₂0, 300 MHz): δ ppm 0.86 (CH₃ from alkyl chain), 1.2-1.6 (-CH₂ from alkyl chain), 3.35 (-OCH₃ from PEG), 3.4-4.0 (main chain protons from HPG and PEG) (Figure 23). PEG content: 28.5 mol%; C₁₀ alkyl content: 22 mol%. GPC-MALLS (0.1 N NaN0₃): M„ 82600; M_w/M_n 1.16; R_h 5.9 nm (Figure 24). Synthesis ofHPG-104-HPG

The polymer was synthesized using a similar procedure as that described above with only glycidol as the monomer. The polymer does not contain C₁₀ alkyl groups or PEG.

GPC-MALLS (0.1 N NaN0₃): M„: 104000, M_w/M_n: 1.15, R_h: 5.1 nm.

EXAMPLE 2: Docetaxel loading of HPG-C₁₀-HPG and HPG-C₁₀-PEG

DTX loading into HPG-C₁₀-HPG and HPG-C₁₀-PEG was carried out as described previously (Mugabe, C, et al. Int. J. Pharm. 2011 ; 404: 238-249). Briefly, an appropriate amount of HPG-C₁₀-HPG or HPG-C₁₀-PEG was weighed in a 4 mL vial and dissolved in 1 mL of methanol or acetonitrile, respectively. To determine optimal drug loading, as part of separate experiments, DTX at up to 5 % w/w (of HPG-C₁₀-HPG or HPG-C₁₀-PEG) was added to the vial. The vial was placed in an oven set at 60 ⁰ C to allow methanol or acetonitrile to evaporate. A stream of nitrogen was passed inside the vial for removal of residual solvent. The resultant DTX-loaded HPG-C₁₀-HPG or HPG-C₁₀-PEG matrix was reconstituted with PBS (pH 7.4) to obtain a clear solution. EXAMPLE 3: In vitro XTT (proliferation) assay

HDFn, HUVEC and HDMEC cells were purchased from Life Technologies (Carlsbad, CA). Only early passages of the cells were used in experiments. All cancer cells (BxPC-3, HT-29, HCC 827, PANC1 and MDA-MB-231) were purchased from American Type Cell Culture (ATCC). All cells were cultured according to the recommendation of the suppliers and tested regularly for mycosplama contamination (InvivoGen, San Diego, CA).

Primary or cancer cells were seeded one day before experimentation in 96-well plates. On the next day, the medium was removed and replaced with medium alone or medium containing various concentrations of HPG-C₁₀-HPG or HPG-C₁₀-PEG for in vitro toxicity assay. Alternatively, medium containing different concentrations of DTX (Polymed

Therapeutics, Houston, TX) or DTX-loaded HPG NPs were added to the cells to evaluate cell kill efficiency. After 72 h of incubation, the media in the wells was removed and replaced with fresh media. XTT mixture (Roche, Mannheim, Germany) was added to each well and the cells were incubated for 4 h at 37 °C before the absorbance readings at 450 nm were taken.

Absorbance readings were normalized with those at 650 nm of each well and at 450 nm with wells containing only medium and XTT reagent. Cell viability was normalized to absorbance readings of cells incubated with medium alone. All conditions were performed in 6 replicates and three independent experiments were done. Values of IC50 were computed with Graphpad Prism®. Error bars shown in Figures 2, 3, 8, 10 and 11 are standard errors of the mean.

Cell kill assays were performed with cells exposed to DTX (Polymed Therapeutics,

Houston, TX) or DTX-loaded HPG-C₁₀-PEG or DTX-loaded HPG-C₁₀-HPG for 72 h. The media was then removed and replaced with fresh media. XTT assays were then carried out as described above. EXAMPLE 4: In vivo tolerability evaluation

Female C57/B6 mice were intravenously injected with 100 mg/kg of HPG-C₁₀-PEG or PBS (pH 7.4) at an injection volume of 100 μ1/20 g through the tail vein. Body weights of mice and signs of ill health were monitored for more than 30 days. There were no behavioural or pathological signs in the animals throughout the study period. The mice were then euthanized and all the major organs such as brain, lungs, heart, stomach, small intestine, ovaries, kidneys, spleen, urinary bladder, liver and skeletal muscle were harvested. The organs were fixed with 10 % formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and evaluated by a board-certified pathologist. Samples of whole blood were obtained through cardiac puncture immediately after euthanasia and collected in K₂EDTA-coated tubes. Blood hematology was analyzed with scil Vet abc (Vet Novations, Barrie, ON, Canada). Five mice were included in each group. Error bars shown in Figure 5 are standard deviations.

EXAMPLE 5: Release of DTX from DTX-loaded HPG-Cio-HPG and DTX-loaded

HPG-Cio-PEG

For determination of drug release, HPG-C₁₀-HPG and HPG-C₁₀-PEG were each loaded with DTX (as described above) spiked with tritium-radiolabeled DTX ( H-DTX, Moravek Biochemicals, Brea, CA). The formulation (1 mL) was placed into biodialyser units (Sigma- Aldrich, Oakville, Ontario) comprising 10,000 Da cut off membranes. The biodialyser units were placed in 500 mL PBS (pH 7.4) at 37 °C under continuous stirring and drug release was measured at pre-determined time points. At each time point, 1 mL of release medium was aliquoted into a scintillation vial containing 5 mL of scintillation cocktail (Fisher Scientific, New Jersey) and counted on a scintillation counter (Beckman Coulter, LS6500). Percent cumulative release of DTX was calculated from three experiments carried out in duplicate. Sink conditions were maintained by periodic replacement of the buffer solution. Error bars in Figures 6 and 7 are standard errors of the mean.

EXAMPLE 6: In vitro DTX/HPG-C₁₀-PEG cell uptake experiments

HDFn and HT-29 cells were seeded in 96-well plates the day before experimentation. Cells were pre-incubated on ice (4 °C) or at 37 °C for 3 h, after which the culture media was removed and replaced with media containing DMSO, dilutions of DTX or DTX-loaded HPG- Cto-PEG. Cells were further incubated on ice or at 37 °C for 3 h before the media was removed and washed once with media. Six replicates were included for each test condition. One hundred microliters of fresh media were dispensed into each well and the cells were incubated for 72 h before XTT assays were carried out. Four independent experiments were carried out, and significant difference was determined with paired (with the same experiment) t-test. Error bars in Figure 12 are standard errors of the mean.

To determine DTX uptake by the cells, HDFn and HT-29 cells were seeded in 12-well plates at 50,000 or 100,000 cells/well. On the next day, the media was removed and the media containing H-DTX or Ή-DTX-loaded HPG-C₁₀-PEG was added to the wells. Triplicates were performed for each test condition and three independent experiments were carried out. After 3 h of incubation at 37 °C, the cells were washed with PBS (pH 7.4) and solubilised with NaOH. The lysate samples were mixed with scintillation cocktail and scintillation counts were taken as readout of ³H-DTX taken up by the cells. Percent uptake was computed by scintillation counts of cell lysate normalized to scintillation counts of amount of ³H-DTX or ³H-DTX-loaded HPG- C₁₀-PEG applied to the wells. Error bars in Figure 13 are standard errors of the mean.

EXAMPLE 7: Biodistribution studies

Radiolabeling of HPG-C₁₀-PEG was performed by partial conversion of the hydroxyl groups to methoxide groups using tritiated methyl iodide (C³H₃I). Two hundred milligrams of HPG-C₁₀-PEG was dissolved in 2 mL of l-methyl-2-pyrrolidinone (NMP) and approximately 5% of the hydroxyl groups were deprotonated using sodium hydride. A calculated amount of tritiated methyl iodide (toluene solution) dissolved in DMSO was added to this solution so as to achieve methylation of approximately 1% of the hydroxyl groups. The reaction mixture was stirred at room temperature for 15 h, 15 mL of water was added and the labelled HPG NP was purified by dialysis against water using MWCO 1000 dialysis membrane until the dialysate contained low amounts of radioactivity, this took approximately 48 h. The HPG NP solution was then filtered through 0.2 μιτι syringe filter and the HPG NP weight was determined from the total volume and the HPG NP concentration from a known volume of the solution after freeze-drying. The HPG NP solution for the animal studies was prepared by the addition of appropriate amounts of NaCl and water to achieve the desired osmolarity and the specific activity was measured by scintillation counting.

Male non-obese diabetic-severe combined inrmunodeficient ("NOD-SCID") mice of 8- 12 weeks of age were implanted subcutaneously with 5 x 10⁶ cancer cells per mouse and monitored. When the tumours reached the size of about 200-400 mm³, tritium-labelled HPG- Cio-HPG, HPG-Cio-PEG or HPG-104 was injected intravenously through the tail vein of each mouse (200 μ1/20 g mouse, 20 mg/kg). At 6, 24 and 72 h post-injection, the mice were euthanized with blood, tumors and major organs removed for processing and scintillation counts. Three subcutaneous xenograft models were employed (HCC 827, BxPC-3 and HT-29).

The tumors and organs were incubated in Solvable® (Perkin Elmer) at 50 °C overnight.

After the complete digestion of the tissues, the samples were allowed to cool before the addition of 50 μΐ, of 200 mM EDTA, 200 uL 30% H₂0_2> and 25 μΐ 10 N HC1 to each sample. The samples were then incubated at room temperature for 1 h prior to the addition of 5 mL of scintillation cocktail per sample. Samples were analyzed by scintillation counting. For reference, 20 μΕ of the tritiated HPG stock in triplicate was analyzed by scintillation counts for specific activity determination. All samples were counted at least 1 week after the processing. Distribution of the tritiated HPG in tissues are reported as percent injected dose per gram of tissue (% ID/g). Unpaired, two-tailed, Student's t-test was used to compare data and significant difference was interpreted as p-value <0.05. Error bars in Figures 14 to 20 are standard deviations. In Figure 21 , the tumor-to-liver ratio of the % ID/g for each mouse was plotted and the lines represent the mean values.

EXAMPLE 8: Efficacy study with DTX/HPG-Cio-PEG

NOD-SCID mice of 8-12 weeks of age were implanted subcutaneously with 5 x 10⁶ BxPC-3 cells per mouse and monitored. After 44 days, a low dose (5 mg/kg) or high dose (15 mg/kg) of the clinical formulation of DTX (again, sourced from Hospira) or DTX/HPG-C₁₀- PEG was injected intravenously through the tail vein of each mouse. Follow-up doses were also administered 51 days and 58 days following implantation of the cancer cells. For comparison, a control group was used which received PBS (pH 7.4). The relative tumor volume was monitored until approximately 75 days following implantation of the cancer cells. The number in parentheses in the legend of Figure 22 indicates the number of mice in each group and the number in the boxes in Figure 22 indicates the number of mice remaining in the study. The treatment groups were as follows: 1. Control: mice were treated with phosphate-buffered saline only (sham treatment);

2. DTX-LD: mice were treated with Taxotere at 0.5 mg/mL, 200 uL/20 g mouse weight;

3. DTX-HD: mice were treated with Taxotere at 1.5 mg/mL, 200 uL/20 g mouse weight;

4. DTX/HPG-LD: mice were treated with HPG-C₁₀-PEG loaded with 0.5 mg/mL of Docetaxel, 200 uL/20 g mouse weight; and

5. DTX/HPG-HD: mice were treated with HPG-C₁₀-PEG loaded with 1.5 mg/mL of Docetaxel, 200 uL/20 g mouse weight.

Three out of five mice treated with a high dose of Taxotere lost >20% body weight and needed to be euthanized.

Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. The word "comprising" is used herein as any open-ended term, substantially equivalent to the phrase "including, but not limited to", and the word "comprises" has a corresponding meaning. As used herein, the singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a thing" includes more than one such thing.

Citation of references herein is not an admission that such references are prior art to the present invention nor does it constitute any admission as to the contents or date of these documents. The invention includes all embodiments and variations substantially as

hereinbefore described and with reference to the examples and drawings.

Claims

WHAT IS CLAIMED IS:

1. A hyperbranched polyglycerol nanoparticle for use in intravascular delivery of a biologically active moiety to a biological tissue, the hyperbranched polyglycerol comprising: a core comprising a first hyperbranched polyglycerol derivatized with C₈-Ci₈ alkyl chains; and

a shell comprising at least one hydrophilic substituent bound to hydroxyl groups of the core, wherein the at least one hydrophilic substituent comprises polyethylene glycol (PEG), methoxy polyethylene glycol (MPEG), a second hyperbranched polyglycerol, or a combination thereof.

2. The hyperbranched polyglycerol nanoparticle of claim 1, wherein the second hyperbranched polyglycerol is not derivatized with alkyl chains.

3. The hyperbranched polyglycerol nanoparticle of claim 1, wherein the at least one hydrophilic substituent comprises PEG.

4. The hyperbranched polyglycerol nanoparticle of claim 1, 2, or 3, wherein the C₈-C₁₈ alkyl chains are C₈ and C₁₀ alkyl chains.

5. The hyperbranched polyglycerol nanoparticle of any one of claims 1 to 4, wherein the C₈-C₁₈ alkyl chains are C₁₀ alkyl chains.

6. The hyperbranched polyglycerol nanoparticle of any one of claims 1 to 5, wherein the biologically active moiety is a hydrophobic drug.

7. The hyperbranched polyglycerol nanoparticle of claim 6, wherein the hydrophobic drug is a chemotherapeutic agent.

8. The hyperbranched polyglycerol nanoparticle of claim 7, wherein the chemotherapeutic agent is a taxane or an analog thereof.

9. The hyperbranched polyglycerol nanoparticle of claim 7, wherein the chemotherapeutic agent is docetaxel or an analog thereof.

10. The hyperbranched polyglycerol nanoparticle of any one of claims 1 to 9, wherein the hyperbranched polyglycerol nanoparticle comprises about 0.05 to about 1 mole of the at least one hydrophilic substituent per mole of the hyperbranched polyglycerol component of the core.

11. The hyperbranched polyglycerol nanoparticle of any one of claims 1 to 9, wherein the hyperbranched polyglycerol nanoparticle comprises about 0.5 to about 0.6 moles of the at least one hydrophilic substituent per mole of the hyperbranched polyglycerol component of the core.

12. The hyperbranched polyglycerol nanoparticle of any one of claims 1 to 9, wherein the hyperbranched polyglycerol particle comprises about 0.57 moles of the at least one hydrophilic substituent per mole of the hyperbranched polyglycerol component of the core.

13. The hyperbranched polyglycerol nanoparticle of any one of claims 1 to 12, wherein the hyperbranched polyglycerol nanoparticle comprises about 0.02 to about 1 mole of the Cg-C₁₈ alkyl chains per mole of the hyperbranched polyglycerol component of the core.

14. The hyperbranched polyglycerol nanoparticle of any one of claims 1 to 12, wherein the hyperbranched polyglycerol nanoparticle comprises about 0.4 to about 0.5 moles of the C₈-C₁₈ alkyl chains per mole of the hyperbranched polyglycerol component of the core.

15. The hyperbranched polyglycerol nanoparticle of any one of claims 1 to 12, wherein the hyperbranched polyglycerol nanoparticle comprises about 0.44 moles of the C₈-C₁₈ alkyl chains per mole of the hyperbranched polyglycerol component of the core.

16. The hyperbranched polyglycerol nanoparticle of any one of claims 1 to 15, wherein number average molecular weight of the hyperbranched polyglycerol nanoparticle is about 10 kDA to about 1 M Da.

17. The hyperbranched polyglycerol nanoparticle of any one of claims 1 to 15, wherein number average molecular weight of the hyperbranched polyglycerol nanoparticle is about 50 kDA to about 150 kDa.

18. The hyperbranched polyglycerol nanoparticle of any one of claims 1 to 15, wherein number average molecular weight of the hyperbranched polyglycerol nanoparticle is about 70 kDA to about 128 kDa.

19. The hyperbranched polyglycerol nanoparticle of any one of claims 1 to 18, wherein the polydispersity index of the hyperbranched polyglycerol nanoparticle is about 1 to about 10.

20. The hyperbranched polyglycerol nanoparticle of any one of claims 1 to 18, wherein the polydispersity index of the hyperbranched polyglycerol nanoparticle is about 1 to about 1.5.

21. The hyperbranched polyglycerol nanoparticle of any one of claims 1 to 20, wherein the biological tissue is a tumor.

22. A method for intravascular delivery of a biologically active moiety to a biological tissue, the method comprising:

administering a hyperbranched polyglycerol nanoparticle loaded with the biologically active moiety to a subject, wherein the hyperbranched polyglycerol nanoparticle comprises:

a core comprising a first hyperbranched polyglycerol derivatized with Cg-Cig alkyl chains; and

a shell comprising at least one hydrophilic substituent bound to hydroxyl groups of the core, wherein the at least one hydrophilic substituent comprises polyethylene glycol (PEG), methoxy polyethylene glycol (MPEG), a second hyperbranched polyglycerol , or a combination thereof.

23. The method of claim 22, wherein the second hyperbranched polyglycerol is not derivatized with alkyl chains.

24. The method of claim 22 or 23, further comprising incorporating the biologically active moiety into the core of the hyperbranched polyglycerol nanoparticle.

25. The method of claim 22, 23, or 24, wherein the biologically active moiety is a hydrophobic drug.

26. The method of claim 25, wherein the hydrophobic drug is a chemotherapeutic agent.

27. The method of claim 26, wherein the chemotherapeutic agent is a taxane or an analog thereof.

28. The method of claim 27, wherein the chemotherapeutic agent is docetaxel or an analog thereof.

29. The method of any one of claims 22 to 28, wherein the biological tissue is a tumor.

30. The method of any one of claims 22 to 29, wherein the at least one hydrophilic substituent comprises PEG.

31. The method of any one of claims 22 to 30, wherein the Cg-Ci alkyl chains are C₈ and C₁₀ alkyl chains.

32. The method of any one of claims 22 to 30, wherein the C₈-C₁₈ alkyl chains are Ci₀ alkyl chains.

33. The method of any one of claims 22 to 32, wherein the hyperbranched polyglycerol nanoparticle comprises about 0.05 to about 1 mole of the at least one hydrophilic substituent per mole of the hyperbranched polyglycerol component of the core.

34. The method of any one of claims 22 to 32, wherein the hyperbranched polyglycerol nanoparticle comprises about 0.5 to about 0.6 moles of the at least one hydrophilic substituent per mole of the hyperbranched polyglycerol component of the core.

35. The method of any one of claims 22 to 32, wherein the hyperbranched polyglycerol particle comprises about 0.57 moles of the at least one hydrophilic substituent per mole of the hyperbranched polyglycerol component of the core.

36. The method of any one of claims 22 to 35, wherein the hyperbranched polyglycerol nanoparticle comprises about 0.02 to about 1 mole of the C₈-C₁₈ alkyl chains per mole of the hyperbranched polyglycerol component of the core.

37. The method of any one of claims 22 to 35, wherein the hyperbranched polyglycerol nanoparticle comprises about 0.4 to about 0.5 moles of the C -C₁₈ alkyl chains per mole of the hyperbranched polyglycerol component of the core.

38. The method of any one of claims 22 to 35, wherein the hyperbranched polyglycerol nanoparticle comprises about 0.44 moles of the C₈-C₁₈ alkyl chains per mole of the hyperbranched polyglycerol component of the core.

39. The method of any one of claims 22 to 38, wherein number average molecular weight of the hyperbranched polyglycerol nanoparticle is about 10 kDA to about 1 M Da.

40. The method of any one of claims 22 to 38, wherein number average molecular weight of the hyperbranched polyglycerol nanoparticle is about 50 kDA to about 150 kDa.

41. The method of any one of claims 22 to 38, wherein number average molecular weight of the hyperbranched polyglycerol nanoparticle is about 70 kDA to about 128 kDa.

42. The method of any one of claims 22 to 41, wherein the polydispersity index of the hyperbranched polyglycerol nanoparticle is about 1 to about 10.

43. The method of any one of claims 22 to 41, wherein the polydispersity index of the hyperbranched polyglycerol nanoparticle is about 1 to about 1.5.

44. Use of a hyperbranched polyglycerol nanoparticle for intravenous delivery of a biologically active moiety to a biological tissue, the hyperbranched polyglycerol nanoparticle comprising:

a core comprising a first hyperbranched polyglycerol derivatized with C₈-C₁₈ alkyl chains; and

a shell comprising at least one hydrophilic substituent bound to hydroxyl groups of the core, wherein the at least one hydrophilic substituent comprises polyethylene glycol (PEG) or methoxy polyethylene glycol (MPEG), a second hyperbranched polyglycerol, or a combination thereof.

45. Use of a hyperbranched polyglycerol nanoparticle in preparation of a medicament for intravenous delivery of a biologically active moiety to a biological tissue, the hyperbranched polyglycerol nanoparticle comprising:

a core comprising a first hyperbranched polyglycerol derivatized with C₈-C₁₈ alkyl chains; and

a shell comprising at least one hydrophilic substituent bound to hydroxyl groups of the core, wherein the at least one hydrophilic substituent comprises polyethylene glycol (PEG), methoxy polyethylene glycol (MPEG), a second hyperbranched, or a combination thereof.

46. The use of claim 44 or 45, wherein the second hyperbranched polyglycerol is not derivatized with alkyl chains.

47. The use according to claim 44, 45, or 46, wherein the biologically active moiety is incorporated into the core of the hyperbranched polyglycerol nanoparticle.

48. The use according to any one of claims 44 to 47, wherein the biologically active moiety is a hydrophobic drug.

49. The use according to claim 48, wherein the hydrophobic drug is a chemotherapeutic agent.

50. The use according to claim 49, wherein the chemotherapeutic agent is a taxane or an analog thereof.

51. The use according to claim 49, wherein the chemotherapeutic agent is docetaxel or an analog thereof.

52. The use according to any one of claims 44 to 51, wherein the biological tissue is a tumor.

53. The use according to any one of claims 44 to 52, wherein the at least one hydrophilic substituent comprises PEG.

54. The use according to any one of claims 44 to 53, wherein the C₈-C₁₈ alkyl chains are C₈ and do alkyl chains.

55. The use according to any one of claims 44 to 53, wherein the C₈-C₁₈ alkyl chains are do alkyl chains.

56. The use according to any one of claims 44 to 55, wherein the hyperbranched polyglycerol nanoparticle comprises about 0.05 to about 1 mole of the at least one hydrophilic substituent per mole of the hyperbranched polyglycerol component of the core.

57. The use according to any one of claims 44 to 55, wherein the hyperbranched polyglycerol nanoparticle comprises about 0.5 to about 0.6 moles of the at least one hydrophilic substituent per mole of the hyperbranched polyglycerol component of the core.

58. The use according to any one of claims 44 to 55, wherein the hyperbranched polyglycerol particle comprises about 0.57 moles of the at least one hydrophilic substituent per mole of the hyperbranched polyglycerol component of the core.

59. The use according to any one of claims 44 to 58, wherein the hyperbranched polyglycerol nanoparticle comprises about 0.02 to about 1 mole of the Cg-C₁₈ alkyl chains per mole of the hyperbranched polyglycerol component of the core.

60. The use according to any one of claims 44 to 58, wherein the hyperbranched

polyglycerol nanoparticle comprises about 0.4 to about 0.5 moles of the C₈-C₁₈ alkyl chains per mole of the hyperbranched polyglycerol component of the core.

61. The use according to any one of claims 44 to 58, wherein the hyperbranched

polyglycerol nanoparticle comprises about 0.44 moles of the C₈-C₁₈ alkyl chains per mole of the hyperbranched polyglycerol component of the core.

62. The use according to any one of claims 44 to 61, wherein number average molecular weight of the hyperbranched polyglycerol nanoparticle is about 10 kDA to about 1 M Da.

63. The use according to any one of claims 44 to 61, wherein number average molecular weight of the hyperbranched polyglycerol nanoparticle is about 50 kDA to about 150 kDa.

64. The use according to any one of claims 44 to 61 , wherein number average molecular weight of the hyperbranched polyglycerol nanoparticle is about 70 kDA to about 128 kDa.

65. The use according to any one of claims 44 to 64, wherein the polydispersity index of the hyperbranched polyglycerol nanoparticle is about 1 to about 10.

66. The use according to any one of claims 44 to 64, wherein the polydispersity index of the hyperbranched polyglycerol nanoparticle is about 1 to about 1.5.

67. A pharmaceutical composition for use in intravascular delivery of a biologically active moiety to a biological tissue, the pharmaceutical composition comprising a hyperbranched polyglycerol nanoparticle and a biologically active moiety, the hyperbranched polyglycerol nanoparticle comprising:

a core comprising a first hyperbranched polyglycerol derivatized with C₈-C₁₈ alkyl chains; and

a shell comprising at least one hydrophilic substituent bound to hydroxyl groups of the core, wherein the at least one hydrophilic substituent comprises polyethylene glycol (PEG), methoxy polyethylene glycol (MPEG), a second hyperbranched polyglycerol, or a combination thereof.

68. The pharmaceutical composition of claim 67, wherein the second hyperbranched polyglycerol is not derivatized with alkyl chains.

69. The pharmaceutical composition of claim 67 or 68, wherein the biologically active moiety is incorporated into the core of the hyperbranched polyglycerol.

70. The pharmaceutical composition of claim 67, 68, or 69, wherein the biologically active moiety is a hydrophobic drug.

71. The pharmaceutical composition of claim 70, wherein the hydrophobic drug is a chemotherapeutic agent.

72. The pharmaceutical composition of claim 71, wherein the chemotherapeutic agent is a taxane or an analog thereof.

73. The pharmaceutical composition of claim 71, wherein the chemotherapeutic agent is docetaxel or an analog thereof.

74. The pharmaceutical composition of claim 71, 72, or 73, wherein the biological tissue is a tumor.

75. The pharmaceutical composition of any one of claims 71 to 74, wherein the at least one hydrophilic substituent comprises PEG.

76. The pharmaceutical composition of any one of claims 67 to 75, wherein the C₈-C₁₈ alkyl chains are C₈ and C₁₀ alkyl chains.

77. The pharmaceutical composition of any one of claims 67 to 75, wherein the C₈-C₁₈ alkyl chains are C₁₀ alkyl chains.

78. The pharmaceutical composition of any one of claims 67 to 77, wherein the

hyperbranched polyglycerol nanoparticle comprises about 0.05 to about 1 mole of the at least one hydrophilic substituent per mole of the hyperbranched polyglycerol component of the core.

79. The pharmaceutical composition of any one of claims 67 to 77, wherein the

hyperbranched polyglycerol nanoparticle comprises about 0.5 to about 0.6 moles of the at least one hydrophilic substituent per mole of the hyperbranched polyglycerol component of the core.

80. The pharmaceutical composition of any one of claims 67 to 77, wherein the

hyperbranched polyglycerol particle comprises about 0.57 moles of the at least one hydrophilic substituent per mole of the hyperbranched polyglycerol component of the core.

81. The pharmaceutical composition of any one of claims 67 to 80, wherein the hyperbranched polyglycerol nanoparticle comprises about 0.02 to about 1 mole of the C₈-C₁₈ alkyl chains per mole of the hyperbranched polyglycerol component of the core.

82. The pharmaceutical composition of any one of claims 67 to 80, wherein the

hyperbranched polyglycerol nanoparticle comprises about 0.4 to about 0.5 moles of the C₈-C₁₈ alkyl chains per mole of the hyperbranched polyglycerol component of the core.

83. The pharmaceutical composition of any one of claims 67 to 80, wherein the

hyperbranched polyglycerol nanoparticle comprises about 0.44 moles of the C₈-C₁₈ alkyl chains per mole of the hyperbranched polyglycerol component of the core.

84. The pharmaceutical composition of any one of claims 67 to 83, wherein number average molecular weight of the hyperbranched polyglycerol nanoparticle is about 10 kDA to about 1 M Da.

85. The pharmaceutical composition of any one of claims 67 to 83, wherein number average molecular weight of the hyperbranched polyglycerol nanoparticle is about 50 kDA to about 150 kDa.

86. The pharmaceutical composition of any one of claims 67 to 83, wherein number average molecular weight of the hyperbranched polyglycerol nanoparticle is about 70 kDA to about 128 kDa.

87. The pharmaceutical composition of any one of claims 67 to 86, wherein the

polydispersity index of the hyperbranched polyglycerol nanoparticle is about 1 to about 10.

88. The pharmaceutical composition of any one of claims 67 to 86, wherein the

polydispersity index of the hyperbranched polyglycerol nanoparticle is about 1 to about 1.5.