Technical Field

The present invention generally relates to a core- shell nanoparticle for drug delivery. The present invention also relates to a process for forming a core-shell nanoparticle for drug delivery.

Background

The use of nanotechnology is strongly expressed in areas of medicine, especially drug carriers. One of the main uses of such carriers is in the treatment of cancer. Conventionally, chemotherapeutic methods distribute their side effects throughout a body, affecting both targeted cancer and normal cells. In general, these carriers are less than lOOnm, and possess the ability to deliver selected drugs to disease sites by preferentially accumulating in tumors .

Various types of drug carriers have been developed, and may be classified as the following: liposomes, nanoparticle albumin-bound technology, dendrimers, metallic nanoparticles and molecular targeted nanoparticles . Liposomes are vesicles with a core/shell type structure containing a single or multiple bilayered membrane structure of lipids. In nanoparticle albumin-bound technologies, albumin is used as a therapeutic carrier for the delivery of hydrophobic chemotherapeutics . Dendrimers are well-defined and regularly-branched macromolecules generally synthesized from elements like sugars, nucleotides and amino acids. Metallic nanoparticles, commonly developed from inert metals like gold or titanium, are usually physically in the form of nanoshells. Molecular targeted nanoparticles are based on the functionalization of the surfaces of these nanoparticles with ligands that may bind to tumor-specific surface markers.

In addition to the earlier-described methods of passive targeting, a new generation of medical nanosystems has been developed to permit the active targeting of tumors .

Several concepts of active targeting have been investigated - at a molecular level, vector surfaces have been functionalized with targeting ligands complementary to specific or over-expressed -receptors on cancer cells; at a macroscopic level, for instance, by means of an external magnetic field (e.g. in magnetic resonance imaging, MRI ) , and based on the association of a drug/magnetic nanoparticle combination. The magnetic nanoparticles commonly used are based on non-toxic iron oxides.

Liposomes, emulsions and micelles have been used in incorporating both a therapeutic drug and an imaging probe. In many of these systems, the drug and imaging probe are contained physically. This may lead to a rapid release of the contained entities when introduced into a host and lead to instability of the nanoparticle-carrier and/or incongruence in the bio-distributions of the drug and imaging probe.

Overall, for a combined drug/imaging probe carrier to be effective, synergy must exist between the desired effects of magnetic-guiding/monitoring and drug therapy at the targeted site. Other considerations include that of the potential extent to which selected drug/imaging agents can be loaded within the carrier, surface properties (e.g., hydrophobicity or hydrophillicity) and particle size of the carriers . There is a need to provide a combined drug/imaging probe carrier that overcomes, or at least ameliorates, one or more of the disadvantages described above.

There is a need to provide a process for forming the combined drug/imaging probe carrier.

Summary

According to a first aspect, there is provided a core- shell nanoparticle for drug delivery comprising a polymeric shell encapsulating a core comprising a drug and an imaging agent .

Advantageously, the drug and imaging agent may be loaded within the core of the polymeric nanoparticle via chemical binding. As a result of the chemical binding and/or the presence of the polymeric shell, neither the drug nor the imaging agent will be released during circulation in the blood vessels when the core-shell nanoparticle is administered into the body. In addition, with both the drug . and . imaging agent loaded within the core, the properties of the polymeric nanoparticle (particularly the stability of the polymeric nanoparticles ) will not be affected by the loading of the core contents. This is in comparison to prior art methods in which the drug and/or imaging agent are present on the surface of the drug carrier such that the properties of the drug carrier are altered, causing the drug carrier to aggregate easily in the blood and become entrapped by the reticuloendothelial system (RES).

Advantageously, due to the core-shell nature of the polymeric nanoparticle in which the drug and imaging agent are encapsulated in the core by the polymeric shell, the presence of the polymeric shell may protect the core contents from early and undesired release in the body.

Advantageously, the monomers making up the polymer may be tailored to protect the contents of the core from first pass metabolism or the RES as well as present functional groups for conjugating or binding with the drug and/or imaging agent. The hydrophilic monomer present in the polymeric shell may protect the core contents from interaction with protein or other components in the blood and entrapment by the RES. Hence, the presence of the polymeric shell may aid in prolonging the lifetime of the core-shell nanoparticles when circulating in the blood so that the core-shell nanoparticles are able to reach the target site such as tumour tissue.

In addition, the monomer and/or drug may contain a pH- sensitive linking group. The pH-sensitive linking group may aid in delivering the core-shell nanoparticle to a targeted site such as a tumour. Since tumour tissues normally have a weak acidic environment, the presence of the pH-sensitive linking group may aid in the faster release of the drug in the weak acidic environment.

Advantageously, the core-shell nanoparticle may be stable at room temperature to moderately high temperature (such as from 20°C to 45°C) for months.

Advantageously, the core-shell nanoparticle may have strong passive targeting ability as a result of their stable and well-protected structure. The core-shell nanoparticle may be capable of coordinated movement in vitro as well as in vivo. Due to the protection provided by the polymeric shell, the core-shell nanoparticle can circulate in the blood for a longer period of time such that the core-shell nanoparticle can- send the drug and imaging agent to a target site (such as tumour tissues) more efficiently as compared to prior art drug carriers.

Advantageously, a high loading of drug and/or imaging agent may be achieved in the core.

Advantageously, due to the presence of the drug and imaging agent such as a magnetic nanoparticle in the core, real-time monitoring of the therapeutic effect and magnetically guided targeting may be enabled. The release of the core contents can be coordinated, giving rise to substantially similar biodistribution of the core contents. Accordingly, synergistic functions of the drug and imaging agent at the therapeutic site may be attainable.

According to a second aspect, there is provided a process for forming a . core-shell nanoparticle for drug delivery, comprising the steps of: (a) conjugating a drug to a polymer comprising a hydrophilic monomer and an aromatic acid monomer; and (b) introducing an imaging agent to said conjugated drug-polymer under conditions to thereby form said core-shell nanoparticle having said drug and imaging agent in the core.

The process may optionally exclude physical methods of entrapping or encapsulating the drug and imaging agent togethe .

Advantageously, the process may allow for chemical binding of the drug and imaging agent to the polymer during formation of the core-shell nanoparticle. Hence, the nanoparticle may be stable and may release the drug and imaging agent in a synergistic manner with substantially similar biodistribution. Def ini tions

The following words and terms used herein shall have the meaning indicated:

The term "conjugate" is to be interpreted broadly to refer to the entity formed as a result of covalent attachment of a molecule, for example, a drug, to a corresponding monomeric molecule that has functional groups that can bind with the drug. The formed entity can be termed as "conjugated drug-polymer" or "conjugated drug- monomer" (as appropriate) or variations thereof.

The term "imaging agent" is to be interpreted broadly to refer to any substance designed to target a physiological function in a subject in vivo or sample in vitro and which can be detected upon administration to the subject or test sample.

The term "nano-sized" is to be interpreted broadly to define a size range which is less than about lOOOnm, particularly less than about 200 nm, or more particularly between about 1 nm to about 100 nm.

The term "nanoparticle" is to be interpreted broadly to refer to a particle which has a dimension in the nano- size range, or less than about lOOOnm, particularly less than about 200 nm, or more particularly between about 1 nm to about 100 nm. Where the nanoparticle is not a spherical particle, the above dimension may refer to the dimension of an equivalent spherical particle. Hence, the dimension may refer to the diameter of the nanoparticle (or equivalent spherical particle thereof) .

The word "substantially" does not exclude "completely" e.g. a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from the definition of the invention .

Unless specified otherwise, the terms "comprising" and "comprise", and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, unrecited elements.

As used herein, the term "about", in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value,, and even more typically +/- 0..5% of the stated value.

Throughout this disclosure., certain embodiments■ may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on- the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range .

Certain embodiments may also be described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Detailed Disclosure of Embodiments

Exemplary, non-limiting embodiments of a core-shell nanoparticle for drug delivery and process of forming the same will now be disclosed.

The core-shell nanoparticle comprises a polymeric shell encapsulating a core comprising a drug and an imaging agent .

The process for forming the core-shell nanoparticle for .drug delivery comprises the steps of (a) conjugating a drug to a polymer comprising a hydrophilic monomer and an aromatic acid monomer; and (b) introducing an imaging agent to said conjugated drug-polymer under conditions to thereby form said core-shell nanoparticle having said drug and imaging agent in the core.

The polymeric shell may comprise a polymer. The polymer may comprise a hydrophilic monomer. The polymer may comprise an aromatic acid monomer. The polymer may comprise an aromatic amide monomer capable of being conjugated to the drug (hereby termed "conjugated drug-monomer") .

The hydrophilic monomer may protect the polymeric nanoparticle from entrapment by the RES. Any monomer that confers a hydrophilic property to the resultant polymer may be used. Exemplary hydrophilic monomer may be a monomer based on an alkenyl ether, an allyl ether, an alkylene glycol and/or a phenyl ether. In particular, the hydrophilic monomer ay be selected from the group consisting of 4-vinylbenzyl methoxy triethylene glycol ether, hydroxypolyethoxy allyl ether, ethylene glycol, ethylene glycol linked to a styrene-containing monomer and allyl phenyl ether.

The aromatic acid monomer may , be any aromatic acid- containing monomer that is capable of conjugating with the drug as well as bind with the imaging agent. Advantageously, the polymeric shell may comprise an aromatic acid monomer selected to chemically bond with the drug in said core. The aromatic acid monomer may be benzoic acid such as 4-vinyl benzoic acid.

The polymer may be a di-block polymer comprising monomers of the hydrophilic monomer and the aromatic acid monomer. In one embodiment, the polymer may be a di-block polymer comprised of the alkenyl ether-based monomer (such as 4-vinylbenzyl methoxy triethylene glycol ether) and the aromatic acid monomer (such as 4-vinyl benzoic acid). The mol% of the hydrophilic monomer in the resultant polymer may be in the range of about 60 to about 70 mol.% while the mol% of the aromatic acid monomer accounts for the remaining 30 to 40 mol% . The diblock polymer may be synthesized via nitroxide-mediated radical polymerization. The hydrophilic monomer may be added to an initiator in an appropriate solvent to initiate the hydrophilic monomer. Any unreacted monomer may be removed. The obtained polymer (made up of poly (hydrophilic monomer)) in a suitable solvent ^' may be used as a macroinitiator to initiate another monomer, such as the aromatic acid monomer, to build the diblock polymer (or the triblock polymer, as will be explained further below) .

The conjugated drug-monomer may be any monomer that has an amide group that can conjugate with a drug. An exemplary conjugated drug-monomer is 4-vinylbenzoic amide conjugated with a drug. Where the drug is doxorubicin, the monomer may be 4-vinylbenzoic doxorubicin amide.

The polymer may be a tri-block polymer comprising the hydrophilic monomer, the aromatic acid monomer and - the conjugated drug-monomer. In one embodiment, the polymer may comprise a tri-block polymer comprised of- the alkenyl ether-based monomer (such as 4-vinylbenzyl methoxy triethylene glycol ether) , the aromatic acid monomer (such as 4-vinyl benzoic acid) and the conjugated drug-monomer (such as 4-vinylbenzoic amide conjugated with a drug) . The moll of the hydrophilic monomer in the resultant polymer may be in the range of about 70 to about 90 mol%, the mol% of the aromatic acid monomer may be in the range of about 5 to 25 mol% and the mol% of the conjugated drug-monomer may be in the range of about 3 to about 5 mol%.

In order to form the tri-block polymer, the di-block polymer may be reacted or polymerized with the conjugated drug-monomer. Alternatively,, poly (hydrophilic ether) as mentioned above may be mixed with the aromatic acid monomer and conjugated drug-monomer to form the tri-block polymer. The tri-block polymer may be one which has the drug and the carboxylic acid groups in different portions of the polymer .

The drug of the core may be selected to be capable of being chemically bonded to monomers of the polymeric shell. The drug may be a chemotherapeutic drug or an anti-cancer drug. The drug may be an alkylating agent, an antimetabolite, an ^■ anthracycline, a plant alkaloid, a topoisomerase inhibitor and other antitumor agents. It is to be noted that the choice of drug is not limited as long as it is able to conjugate or bind to one of the monomers in the polymer. It is also to be noted that the person skilled in the art would know what monomer should be used when considering the type of drug to be encapsulated in the core. The drug may comprise moieties selected from the group consisting of amino groups, amine groups, ether groups and carbonyl groups, that can be conjugated via amidation reaction or hydrazine bonding to the monomer. Other anti-cancer drugs can also be used as long as the drug can be conjugated to a monomer. Advantageously, one or more of the amino groups or amine groups or carbonyl groups chemically bonds with the carboxylic acid moiety of the aromatic acid monomer.

The alkylating agent may be selected from the group consisting of cyclophosphamide, mechlorethamine , uramustine, melphalan, chlorambucil, ifosfamide, carmustine, lomustine, streptozocin, busulfan, thiotepa, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, procarbazine, altretamine, decarbazine, mitozolomide , temozolomide and analogues thereof.

The antimetabolite may be selected from the group consisting of- methotrexate, 5-fluorouracil , 6- mercaptopurine, 6-thioguanine and cytarabine.

The anthracycline may be selected from the group consisting of daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, mitoxantrone .

The ^' plant alkaloid may be selected from vincristine, vinblastine, vinorelbine, vindesine, paclitaxel and docetaxel .

The topoisomerase inhibitor may be selected from the group consisting of irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate and teniposide. It is to be appreciated that a mixture of two or more of the above drugs can be conjugated to the polymer and hence be encapsulated in the core. In order to conjugate a mixture of drugs, the monomer or monomers should be chosen as appropriate in order to present functional groups or binding groups that can bind with the various types of drugs.

The drug may be linked with a pH-sensitive linking group (such as hydrazone or cis-aconityl group) . The pH- sensitive linking group may aid in the release of the drug in an appropriate pH environment. The pH environment may be a weakly acidic environment, which is normally found in tumor tissues. Advantageously, the presence of the pH- sensitive linking group may aid in the release of the drug at a targeted site, such as tumour tissues. The percentage release of the drug from the nanoparticle in the weakly acidic environment may be in the range of about 35% to about 60%.

The drug may be conjugated to the polymer by two approaches. The first approach involves conjugation onto the diblock polymer directly by partial reaction of binding groups with the drug via a suitable reaction to obtain a diblock polymer with the drug randomly distributed on the aromatic acid monomer segment. When the drug used is doxorubicin, doxorubicin can be conjugated with the diblock polymer directly by partial reaction of carboxylic acid groups with doxorubicin via amidation to obtain a conjugated doxorubicin-diblock polymer in which doxorubicin may be randomly distributed on the aromatic acid monomer segment. Suitable catalyst (s) for catalyzing the amidation reaction between the doxorubicin and diblock polymer may be used. The catalysts may be N, W-dicyclohexylcarbodiimide, N-hydroxysuccinimide and triethylamine.

The second approach involves polymerizing the conjugated drug-monomer with either the diblock polymer or a mixture of poly (hydrophilic ether) and aromatic acid monomer to form the tri-block polymer as explained above. The drug is then present in different parts of the triblock polymer.

The imaging agent may be selected from the group consisting of metals (such as transition metals), radioactive isotopes and radioopaque agents (e.g., gallium, technetium, indium, strontium, iodine, barium, bromine and phosphorus-containing compounds), radiolucent agents, contrast agents, dyes- (e.g., fluorescent dyes and chromophores ) and enzymes that catalyze a colorimetric or fluorometric reaction.

The imaging agent of the core may be selected to be capable of being chemically bonded to monomers of the polymeric shell.

The imaging agent may be a MRI imaging agent, such as a magnetic nanoparticle.

The magnetic nanoparticle may comprise a transition metal of the Periodic Table of Elements. The transition metal may be selected from the group consisting of cobalt, iron, platinum, gadolinium, manganese and mixtures thereof. The magnetic nanoparticle may be selected from the group consisting of gadolinium (III) chelate, superparamagnetic iron oxide, ultrasmall superparamagnetic iron oxide, superparamagnetic iron platinum, paramagnetic managanese chelate, manganese iron oxide, iron cobalt, iron platinum and cobalt platinum. The polymer may be loaded with the imaging agent such as magnetic nanoparticles via reaction of the magnetic nanoparticles with the carboxylic acid groups on the polymer. The magnetic nanoparticles may be loaded in the polymeric nanoparticle by a -, dialysis procedure. The dialysis procedure may be a three-step procedure. The first dialysis may be carried out to exchange the fatty acid (such as oleic acid) that may be present on the magnetic nanoparticles with an aromatic hydrocarbon (such as toluene) . The second dialysis may be carried out to remove the aromatic hydrocarbon and fatty acid by exchanging with an organosulphur compound (such as dimethyl sulphoxide). The third dialysis may be carried out to remove the organosulphur compound by exchanging with water.

The loading of the drug within the core, may be in the range selected from the group consisting of about 5 wt% to about 35 wt%, about 5 wt% to about 10 wt%, about 5 wt% to about 15 wt%, about 5 wt% to about 20 wt%, about 5 wt% to about 25 wt%, about 5 wt% to about 30 wt%, about 10. wt% to about 35 wt%, about 15 wt% to about 35 wt%, about 20 wt% to about 35 wt%, about 25 wt% to about 35 wt% and about 30 wt% to about 35 wt%. In one specific polymer, the drug loading in the core is about 30 wt%. In another polymer, the drug loading in the core is about 8 wt% to about 9 wt% while in another polymer, the drug loading in the core is about 12 wt% to about 13 wt%.

The loading of the imaging agent within the core may be in the range selected from the group consisting of about 25 wt% to about 70 wt%, about 25 wt% to about 30 wt%, about 25 wt% to about 35 wt%, about 25 wt% to about 40 wt%, about 25 wt% to about 45 wt%, about 30 wt% to about 50 wt%, 25 wt% to about 55 wt%, 25 wt% to about 60 wt%, 25 wt% to about 65 wt%, about 35 wt% to about 70 wt%, about 40 wt% to about 70wt% and about 45 wt% to about 70wt%, 50 wt% to about 70 wt%, 55 wt% to about 70 wt%, 60 wt% to about 70 wt% and 65 t% to about 70 wt% . For a specific polymer, the magnetic nanoparticle loading is in the range of about 28 wt% to about 30 wt% . ■

The loaded nanoparticle may have a particle size in the range selected from the group consisting of about 90 nm to about 160 nm, 90 nm to about 100 nm, 90 nm to about 110 nm, 90 nm to about 120 nm, 90 nm to about 130 nm, 90 nm to about 150 nm, 100 nm to about 160 nm, 110 nm to about 160 nm, 120 nm to about 160 nm, 130 nm to about 160 nm, 140 nm to about 160 nm and 150 nm to about 160 nm. For a specific polymer, the average particle size of the nanoparticle may be about 108 + 1 nm. For another specific polymer, the average particle size of the nanoparticle may be about 104 + 2 nm while for a third specific polymer the average particle size of the nanoparticle may be about 154 + 2 nm.

The polymer may comprise a drug linked with a pH sensitive group. The particle size of the resultant (loaded) nanoparticle formed from this polymer may be in the range of about 550 to about 600 nm. In one embodiment, the average particle size of this nanoparticle may be 574.1 + 3.6 nm.

The polydispersity of the polymer may be in the range selected from the group consisting of about 1.0 to about 2.0, about 1.0 to about 1.1, about 1.0 to about 1.2, about 1.0 to about 1.3, about 1.0 to about 1.4, about 1.0 to about 1.5, about 1.0 to about 1.6, about 1.0 to about 1.7, about 1.0 to about 1.8, about 1.0 to about 1.9, about 1.1 to about 2.0, about 1.2 to about 2.0, about 1.3 to about 2.0, about 1.4 to about 2.0, about 1.5 to about 2.0, about 1.6 to about 2.0, about 1.7 to about 2.0, about 1.8 to about 2.0 and about 1.9 to about 2.0. For a specific polymer, the polydispersity may be about 1.22. Due to the narrow polydispersity of the polymer, better biodistribution of the core contents can be achieved so that almost all of the core-shell nanoparticles can reach the target site (such as a tumour tissue) in order to release the core contents in a targeted manner to the desired sites. This may aid in minimizing any side effects. This is in comparison to larger particles that are too big and can get entrapped in some particular organ, such as lung or liver, without reaching the target site.

The molecular weight of the polymer may be in the range selected from the group consisting of about 20 kDa to about 200 kDa, about 20 kDa to about 40 kDa, about 20 kDa to about 60 kDa, about 20 kDa to about 80 kDa, about 20 kDa to about 100 kDa, about 20 kDa to about 120 kDa, about 20 kDa to about 140 kDa, about 20 kDa to about 160 kDa, about 20 kDa to about 180 kDa, about 40 kDa to about 200 kDa, about 60 kDa to about 200 kDa, about 80 kDa to about 200 kDa, about 100 kDa to about 200 kDa, about 120 kDa to about 200 kDa, about 140 kDa to about 200 kDa, about 160 kDa to about 200 kDa, about 180 kDa to about 200 kDa, about 34 kDa to about 35 kDa, about 29 kDa to about 30 kDa and about 67 kDa to about 68 kDa. The molecular weight of a specific polymer may be about 34.1 kDa. The molecular weight of another ^" specific polymer may be about 29.7 kDa while the molecular weight of a third specific polymer maybe about 67.4 kDa.

The number average molecular weight of the polymer may be in the range selected from the group consisting of about 20 kDa to about 200 kDa, about 20 kDa to about 40 kDa, about 20 kDa to about 60 kDa, about 20 kDa to about 80 kDa, about 20 kDa to about 100 kDa, about 20 kDa to about 120 kDa, about 20 kDa to about 140 kDa, about 20 kDa to about 160 kDa, about _. 20 kDa to about 180 kDa, about 40 kDa to about 200 kDa, about 60 kDa to about 200 kDa, about 80 kDa to about 200 kDa, about 100 kDa to about 200 kDa, about 120 kDa to about 200 kDa, about 140 kDa to about 200 kDa, about 160 kDa to about 200 kDa, about 180 kDa to about 200 kDa and about 37 kDa to about 38 kDa. The molecular number of a specific polymer may be about 28.0 kDa. The molecular number of another specific polymer may be about 24.0 kDa while the molecular number of a third specific polymer maybe about 37.4 kDa.

The nanoparticle may be stable at a temperature of about 20°C to about 45°C for months. The nanoparticle may maintain ^' high stability in neutral pH environments and may release completely in weakly acidic environments via hydrolysis in the long term.

Brief Description of Drawings

The accompanying drawings illustrate a disclosed embodiment and serve to explain the principles of the disclosed embodiment. It is to be understood, however, that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the invention.

Fig. 1 (a) is a reaction scheme of the three-step procedure to synthesize the initiator, 2 , 2 , 5-trimethyl-3- ( 1-phenylethoxy ) -4-phenyl-3-azahexane (TPPA) , in Example 1.

Fig. 1(b) is a reaction scheme of the procedure to synthesize poly (VBA-TEGSt ) -NHN=DOX having a pH-sensitive hydrazone bond and linking doxorubicin with poly (TEGSt-co- VBA) in Example 5. Fig. 2 shows the H NMR spectrum of Compound (1) obtained in Step 1 of Example 1.

Fig. 3 shows the ¹ H NMR spectrum of Compound (2) obtained in Step 2 of Example 1.

Fig. ^' 4 shows the ¹ H NMR spectrum of Compound (3) (i.e. TPPA) obtained in Step 3 of Example 1.

Fig. 5 shows the ¹ H NMR spectrum of TEGSt obtained in Example 2.

Fig. 6 shows the ¹ H NMR spectrum of DOXSt obtained in Example 3.

Fig. 7 shows the ¹³ C NMR spectrum of DOXSt obtained in Example 3.

Fig. 8 shows a schematic diagram of the arrangement of the monomers of the four polymers A to D synthesized in Example .

Fig. 9 shows the ^λ Ε NMR spectra of Polymer A (poly (TEGSt-co-VBA) obtained in Example 4.

Fig. 10 shows the ¹ H NMR spectra of Polymer B (poly (TEGSt-co-VBA) -DOX) obtained in Example 4.

Fig. 11 shows the ½ NMR spectra of Polymer C (poly (TEGSt-co-VBA-co-DOXSt ) obtained in Example 4.

Fig. 12 shows the ¹ H NMR spectra of Polymer D (poly (TEGSt-co-DOXSt-co-VBA) ) obtained in Example 4.

Fig. 13 shows the gel permeation chromatography (GPC) curves of Polymers A to D obtained in Example 4.

Fig. 14 shows the graph of the percentage release of doxorubicin at different pH values of poly ( VBA-TEGSt ) - NHN=DOX obtained in Example 5.

Fig. 15 shows the particle size distribution of poly (TEGST-VBA) =NNH-DOX obtained in Example 5 as measured by dynamic light scattering (DLS) . Figs. 16(a), (b) and (c) show the transmission electron microscopy (TEM) images as well as photographs and particle size distributions as measured by DLS of the magnetite nanocube (MN) -loaded Polymers B, C and D (referred to as B-MN, C-MN .and D-MN respectively) obtained in Example 7.

Fig. 17 shows the thermal gravimetric analysis (TGA) of B-MN obtained in Example 7.

Figs. 18(a), (b) and (c) show graphs of the particle sizes of Polymers B, C and D compared against B-MN, C-MN and D-MN respectively referred to in Example 7.

Fig. 19(a) shows the X-ray diffraction (XRD) graph of the magnetite nanoparticles (top curve) as compared with B- MN (bottom curve) obtained in Example 7.

Fig. 19(b) shows the fluorescence of HepG2 cells of Example 8 after 15 min of incubation with application of a magnetic field (right) as compared to without application of a magnetic field (left) .

Fig. 20 shows the intensity of the toxicity .of Polymers A and B loaded with MNs (i.e. MN-P (TEGSt-co-VBA) and MN-P (TEGSt-co-VBA) -DOX respectively) against polymer concentration referred to in Example 8.

Fig. 21 shows the bio-distributions of doxorubicin and iron in the tumor, heart, kidney, liver, lung and spleen in balb/C mice referred to in Example 9 tracked via red fluorescence and Prussian staining. The fluorescence images are shown in the first and third columns, while the Prussian staining images are shown in the second and fourth columns . Examples

Non-limiting examples of the invention will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

In the following examples, tri (ethylene glycol )monomethyl ether (> 97%), 4-vinyl benzoic acid (VBA) , N, N' -dicyclohexylcarbodiimide (DCC) , triethylene amine, tert-nitrobutane, isobutyraldehyde, ammonium chloride, zinc powder, bromobenzene, brine, Na ₂ S0 ₄ , NH ₄ C1, NH ₄ OH, Cu(OAc) ₂ , NaHC0 ₃ , anisole and silica gel used for flash column chromatography were purchased from Merck & Co (New Jersey, United States of America (USA) ) . 4-vinylbenzyl chloride (90%), sodium hydride (60% dispersion in mineral oil), N- hydroxysuccinimide (NHS), magnesium turnings, [Ν,Ν'- bis (2, 5-di-tert-butylsalicylidene) -1, 2- cyclohexanediaminato] -manganese (III) chloride, di-tert- butyl peroxide, sodium borohydride and solvents for nuclear magnetic resonance (NMR) measurement (i.e. deuterated chloroform (CDC1 ₃ ) and deuterated dimethyl sulfoxide (d ₆ - DMSO) ) were purchased from Sigma-Aldrich Co (Missouri, USA) . Doxorubicin*HCl was purchased from Xingcheng Chempharm Co. Ltd. (China). All other solvents were purchased from -J. T. Baker (New Jersey, USA) . All chemical reagents were used without further purification.

Example 1

Synthesis of Initiator

The initiator, 2 , 2 , 5-trimethyl-3- ( 1-phenylethoxy) -4- phenyl-3-azahexane (TPPA) , was synthesized via a three-step procedure. The three-step procedure is detailed in the following in accordance with the reaction scheme illustrated in Fig. 1(a).

Step 1: Preparation of Compound (1)

A mixture of tert-nitrobutane (15.468 g, 150 mmol) , isobutyraldehyde (13.778 mL, 150 mmol), ammonium chloride (8.826 g, 165 mmol) and 150 mL of water was cooled to 0°C. 150 mL of Et ₂ 0 was then added to partially dissolve the crystallized tert-nitrobutane. Zinc powder (39.234 g, 600 mmol) was added and stirred at room temperature for 8 h. The mixture was filtered, and the residue was washed with 135 mL of methanol.

The residue was then partitioned with CH ₂ C1 ₂ and the organic layer was washed with brine, dried over Na ₂ S0 ₄ , and concentrated under vacuum. The colorless liquid (15.179 g, 944 mmol, 70.8%), Compound (1), was used directly for the next reaction, step 2, without further purification.

Compound (1) was characterized by ¹ H NMR spectroscopy (Bruker AVANCE 400 spectrometer, Bruker, Germany) at 400 MHz using the solvent CDCI ₃ to determine the chemical shifts expressed in parts per million (δ) values using residual protons in the solvent as the internal standard and the spectrum is shown in Fig. 2. As seen from Fig. 2, a peak from 6.58 ppm to 6.57 ppm represents (d, 1H) , a peak from 3.18 ppm to 3.06 ppm represents (m, 1H) , a peak at 1.42 ppm represents (s, 9H) and a peak from 1.05 to 1.03 represents (d, 6H) .

Step 2: Preparation of Compound (2)

A solution of -3.5M of Grignard reagent was freshly prepared from bromobenzene. Compound (1) (3.50 g, 24.5 mmol) was dissolved in 26.5 mL of tetrahydrofuran (THF) to obtain a mixture and cooled to 0°C under argon atmosphere.

The Grignard reagent was then added dropwise to the mixture and stirred at room temperature overnight. The excess Grignard reagent was quenched via the addition of 10 mL of saturated NH ₄ C1 solution at 0°C. 30 mL of water was then added until all the solids were dissolved. The aqueous layer was then extracted with 30 mL of Et ₂ 0 thrice, washed with 50 mL of brine, dried with Na2 S0 ₄ , filtered and concentrated to obtain a residue. The residue was then treated with a mixture of 200 mL of methanol, 15 mL of NH ₄ OH and Cu(OAc) 2 (0.459 g, 0.23 mmol) to give a pale yellow solution.

A stream of air was bubbled through the pale yellow solution until it became a dark blue solution in 30-45 min. The dark blue solution was concentrated and the residue was dissolved in a mixture of 200 mL of CHC1 ₃ , 50 mL of NaHS0 ₄ and 200 mL of H2O . The mixture was then partitioned between CHCI ₃ and water. The organic layer was extracted with 100 mL of chloroform thrice, washed with NaHCC>3, dried over a2 S0 _{4 /} filtered and concentrated under vacuum to obtain a crude product .

The crude product was purified by flash column chromatography on silica gel using 1:19 of ethyl acetate/hexane . The desired fractions were evaporated to obtain the product, Compound (2), as an orange oil (3.011 g, 13.7 mmol, 56.1%) .

Compound (2) was characterized by ^Χ Η NMR spectroscopy at 400 MHz using the solvent CDC1 ₃ to determine δ values and the spectrum is shown in Fig. 3. As seen from Fig. 3, a peak from 7.70 ppm to 7.33 ppm represents (m, 5H) , a peak from 3.68 ppm to 3.61 ppm represents (m, 1H) , a peak from 2.13 ppm to 2.07 ppm represents (m, 1H) , a peak from 1.46 ppm to 1.32 ppm represents (d, 9H) and a peak from 1.11 ppm to 0.92 ppm represents (m, 6H) .

Step- 3: Preparation of TPPA (Compound (3))

To a solution of styrene (2.09 mL, 18.3 mmol) and Compound (2) (2.00 g, 9.14 mmol) in 80 mL of 1:1 of toluene/ethanol mixture, [N, N' -bis ( 2, 5-di-tert- butylsalicylidene ) -1 , 2-cyclohexanediaminato] manganese ( III ) chloride (1.16 g, 1.83 mmol), di-tert butyl peroxide (1.44 ml, 13.7 mmol) and sodium borohydride (1.04 g, 27.4 mmol) were added under argon atmosphere.

The reaction was stirred at room temperature for 12 h, evaporated to dryness and partitioned between CH ₂ CI ₂ and water. The combined organic layer was then dried using Na ₂ S0 ₄ and evaporated to dryness. The crude product was then purified using column chromatography (1:19 of dichloromethane/hexane ) to give a viscous liquid, TPPA (2.97 g, 9.14 mmol, 69.4%) .

The TPPA synthesized was characterized by ¹ H NMR spectroscopy at 400 MHz using the solvent CDC1 ₃ to determine δ values and the spectrum is shown in Fig. 4. As seen from Fig. 4, a peak from 7.46 ppm to 7.16 ppm represents (m, 10H) , a peak from 4.94 ppm to 4.89 ppm represents (qq, 1H) , a peak from 4.22 ppm to 4.02 ppm represents (m, 1H) , a peak from 3.44 ppm to 3.29 ppm represents (dd, 1H) , a peak from 2.37 ppm to 2.30 ppm represents (m, 1H) , a peak from 1.64 ppm to 1.63 ppm represents (d, 1H) , a peak from 1.561 ppm to 1.54 ppm represents (d, 3H) , a peak from 1.321 ppm to 1.31 ppm represents (d, 1H) , peaks at 1.05 ppm and 0.78 ppm represent (2s, 9H, mixture of isomers), a peak from 0.93 ppm to 0.89 ppm represents (dd, 2H) , a peak from 0.566 ppm to 0.54 ppm represents (d, 1H) and a peak from 0.238 ppm to 0.21 ppm represents (d, 1H) .

Example 2

Synthesis of 4-Vinylbenzyl methoxy triethylene glycol ether

(TEGSt)

20 mL of anhydrous THF was added into tri (ethylene glycol) monomethyl ether (4.0 g, 24.4 mmol) that was dried overnight at 50°C under vacuum in a three-necked flask. Thereafter, sodium hydride (1.5 g, ^' 37.4 mmol) was added to the flask. The system was stirred and allowed to react for 1 h at room temperature under argon atmosphere.

4-vinylbenzyl chloride (2.4 mL, 17.0 mmol) in 15 mL of anhydrous THF was added dropwise into the flask. The mixture was then allowed to react for 20 h. Thereafter, the mixture was neutralized with IN of HC1. The mixture was washed with 25 mL of diethyl ether four times.

The organic extracts were collected and dried using anhydrous Na ₂ S0 ₄ . The solvent was removed by using a rotoevaporator and purified by column chromatography (1:1 of ethyl acetate/hexane) to give 2.2 g of pure product, TEGSt, as a light yellow liquid at 46% yield.

The TEGSt synthesized was characterized by ^X H NMR spectroscopy at 400 MHz using the solvent CDC1 ₃ to determine δ values and the spectrum is shown in Fig. 5. As seen from Fig. 5, a peak at 7.36 ppm represents (d, 2H) , a peak at 7.287 ppm represent (d, 2H) , a peak at 6.70 ppm represents (dd, 1H) , a peak at 5.72 ppm represents (dd, 1H) , a peak at 5.21 ppm represents (dd, 1H) and a peak at 4.54 ppm represents (s, 2H) . Example 3

Synthesis of 4-vinylbenzoic doxorubicin amide (DOXSt)

4-vinyl benzoic acid (296.32 mg, 2 mmol) was added to a two-neck flask containing 20 mL of anhydrous DMSO. DCC (495.2 mg, 2.4 mmol), NHS (276.22 mg, 2.4, mmol) and 1.5 mL of triethylamine were added sequentially. The reaction was allowed to stir for 4 h before adding doxorubicin*HCl (1.162 g, 2.0 mmol) .

The reaction was allowed to react for another 24 h under argon atmosphere and aluminum foil. Thereafter, the solution was filtered and precipitated in ether to harvest the dark red powder. The crude product was re-dissolved in dichloromethane and filtered to remove the unreacted doxorubicin residue. The solution was evaporated to obtain a red powder, DOXSt, for purification using silica gel column chromatography (100:5 of dichloromethane (DCM) /methanol) .

The red powder product was synthesized was characterized by ½ NMR spectroscopy at 400 Hz using the solvent d ₆ -DMSO to determine δ values, ¹³ C NMR spectroscopy at 400 Hz using dg-DMSO to determine δ values and time-of- flight mass spectrometry (MS-TOF) using electrospray ionization (ESI) to determine mass-to-charge (m/z) ratio.

The ¹ H NMR spectrum is shown in Fig. 6. As seen from Fig. 6, a peak at 7.83 ppm represents (m, 2H) , a peak at 7.74 ppm represents (d 2H) , a peak at 7.57 ppm represents (m, 1H) , a peak at 7.48 ppm represents (dd, 2H) , a peak at 6.73 ppm represents (dd, 1H) , a peak at 5.88 ppm represents (dd, 1H) , a peak at 5.32 ppm represents (dd,lH), a peak at 5.23 ppm represents (t, 1H) , a peak at 4.90 ppm represents (t, 1H), a peak at 4.57 ppm represents (s, 2H) , a peak at 4.20 ppm represents (m, 2H) , a peak at 3.90 ppm represents (s, 3H) , a peak at 3.52 ppm represents (m, 1H) , a peak at 2.87 ppm represents (m, 2H) , a peak at 2.19 ppm represents (m, 2H) , a peak at 2.05 ppm represents (m, 2H) and a peak at 1.12 ppm represents (d, 3H) .

The ¹³ C NMR spectrum is shown in Fig. 7. As seen from Fig. 7, the peak values are 213.0, 185.7, 185.6, 164.9, 159.9, 154.8, 153.2, 138.9, 135.0, 134.4, 133.7, 133.0,

132.7, 126.9, 125.1, 119.0, 118.8, 118.2, 115.5, 110.0,

109.8, 99.7, 74.1, 69.2, 67.0, 65.9, 62.8, 55.7, 45.1, 31.1, 28.5 and 16.2.

In the MS-TOF spectrum (not shown), the peak representing [M-H] ^~ calculated for C36H35NO12 is 672.6 while the actual value obtained by mass spectrometry is 672.2.

Example 4

Synthesis of Polymers

Four polymers A to D were synthesized in this example in accordance with the schematic diagram in Fig. 8. As seen in Fig. 8, the monomers, used were 4-Vinylbenzoic acid (VBA) , TEGSt and DOXSt and the arrangement of the monomers differ in each of polymers A to D.

VBA was used as the monomer for building the block to bind the magnetite nanocubes and to randomly conjugate doxorubicin. TEGSt synthesized from Example 2 was employed in building the hydrophilic segment and protecting against entrapment by the RES. DOXSt synthesized from Example 3 was designed to conjugate doxorubicin.

The polymers synthesized were characterized by ¹ H NMR spectroscopy and gel permeation chromatography (GPC) . The ¹ H NMR spectra of Polymer A (poly (TEGSt-co-VBA) , Polymer B (poly (TEGSt-co-VBA) -DOX) , Polymer C (poly (TEGSt-co-VBA-co- DOXSt) and Polymer D (poly (TEGSt-co-DOXSt-co-VBA) ) are shown in Figs. 9 to 12 respectively. The GPC curves of Polymers A to D are shown in Fig. 13. Further, the loading of each monomer unit into each polymer synthesized is detailed in Table 1 further below.

Polymer A

Polymer A, made up of the monomers TEGSt and VBA, was synthesized here. This diblock polymer was synthesized via a two-step procedure using a living polymerization reaction, nitroxide-mediated radical polymerization (NMRP) .

In the first step of NMRP, TEGSt (1.23 g, 4 mmol) was added into a 10-mL ampule bottle with a stirrer bar and was allowed to dissolve in 2 mL of anisole added with the TPPA obtained from Example 1 (13 mg, 0.043 mmol). The solution in the ampule bottle was degassed by freeze-pump- backfilling with nitrogen for 6 times and sealed with a blow torch. After warming to room temperature, the ampule was placed in a 130°C oil bath and stirred overnight.

• Next, the ampule was opened and the solution was diluted with tetrahydrofuran (THF) and precipitated in hexane. The solid product was re-dissolved in THF and re- precipitated in hexane to remove the unreacted monomer completely. The semi-solid product was dried overnight in a vacuum oven at 50 °C to result in a yield of 1.0 g of TEGSt polymer product or about 81% yield.

In the second step of NMRP, the TEGSt polymer product obtained ^' was then dissolved in 5 ml of anisole and applied as a macroinitiator . The macroinitiator was dissolved in anisole and added to a 10-mL ampule bottle with a stirrer bar. Thereafter, VBA (300 mg, 2 mmol) was added. The solution in the ampule bottle was degassed by freeze-pump- refilling of nitrogen for 6 times and sealed with a blow torch. The ampule was warmed to room temperature, placed in a 130°C bath and stirred overnight. It was then unsealed and the solution was diluted with THF and precipitated with ether twice to obtain 0.52 g of a light yellow solid , product, a copolymer product of TEGSt and VBA. The copolymer product is evidenced by appearance of a broad peak at 7.8 ppm in the ¹ H NMR spectrum of polymer A shown in Fig. 9 indicated the successful initialization of VBA by macroinitiator (poly (TEGSt )) .

The molar percentages of the two polymer blocks, i.e. the TEGSt polymer product and the copolymer product of TEGSt and VBA, were estimated by analyzing the ^X H NMR spectrum shown in Fig. 9. Specifically, the ratio of the (CH)2-C¾- peak at δ 4.47 (equivalent to A _T EGst/2) which was attributed to TEGSt, was compared with the aromatic peak at δ 6-8 (equivalent to A _T EGst + Δ _Υ ΒΑ) which was attributed to the copolymer of TEGSt and VBA, to estimate the molar percentages of the two polymer blocks. The content of TEGSt and VBA calculated from the ¹ H NMR spectrum in Fig. 9 was 62 mol% and 38 mol%, respectively. About 96% of the monomers were incorporated into the polymer.

Upon calculation, the molar ratio of TEGSt to VBA monomers in the polymer was concluded to . be equal to ^ EGst / ^VBA as seen in Fig. 9.

To obtain a narrow polydispersity, a relatively lower monomer conversion rate as compared to prior literature was intentionally used. The polydispersity for polymer A was 1.22, which was much narrower than that of polymers obtained by other non-living polymerization methods (usually higher than 1.5). However, the GPC curve in Fig. 13 of the Polymer A synthesized shows a shoulder peak, which is common for the NMRP reaction with high monomer to initiator ratio and high monomer conversion.

Polymer B

Here, DOX was , introduced by conjugation onto Polymer A directly by partial reaction of the carboxylic acid groups with DOX via amidation to obtain polymer B, with DOX randomly distributed on the VBA segment as shown in Fig. 8. Conjugation of DOX onto Polymer A, i.e. poly (TEGSt-co-VBA) , was synthesized as follows.

0.52 g of poly (TEGSt-co-VBA) having 56.74 mol% of VBA monomer unit (i.e. 1.4 mmol of VBA monomer unit estimated by ^X H NMR) was dissolved in 10 mL of DMF and added with DCC (148.35 mg, 0.72 mmol), NHS (83.3 mg, 0.72 mmol) and 0.5 mL of triethylamine sequentially. Doxorubicin *HC1 (417.7 mg,

0.72 mmol) was added after 4 h and allowed to react overnight.

Thereafter, the solution was filtered and dialyzed against DMSO for 2 days, followed ^' by deionized water for another 2 days. A solution with some red precipitate was harvested and freeze dried to obtain 0.7 g of red solid,

1. e. a copolymer of TEGSt, VBA and DOX.

30 wt% of doxorubicin conjugation in the polymer was determined via UV measurement (UV machine was obtained from Hitachi with model number 2801) at 485 nm. The resulting polymer B consisted of randomly distributed DOX on the VBA block.

The ¹ H NMR spectrum of Polymer B is shown in Fig. 10 and the GPC curve is shown in Fig. 13.

A narrow polydispersity of 1.2 and a high doxorubicin loading of 30 wt% were achieved, as shown in Table 1 further below. Polymer C

Here, DOX was introduced by a second method. Specifically, DOX was introduced by polymerizing DOXSt to obtain Polymer C, i.e. poly (TEGSt-co-VBA-co-DOXSt ) , as seen in Fig . 8.

200 mg of VBA was added to a 10-mL ampule containing 1 g of poly(TEGSt) in 2mL of anisole and stirred with a magnetic stirrer bar. The ampule was degassed by a freeze- pump-nitrogen gas refilling procedure for 6 times and sealed with a blow torch. After reaction at 130°C overnight, the solution was precipitated twice in hexane . 1 g of dry product, i.e. poly (TEGSt-co-VBA) was obtained.

Next, 200 mg of DOXSt was added to a 10-mL ampule containing poly (TEGSt-co-VBA) dissolved in 6 mL of anisole, degassed the same way as described above, and sealed. The system was allowed to react overnight at 130°C. The solution was diluted by DMSO, dialyzed against DMSO for one day and dialyzed against deionized water for another day. The dialysis tubing molecular weight cut-off. (MWCO) ^■ used was 3500 Da. A product, i.e. poly (TEGSt-co-VBA-co-DOXSt ) , weighing 0.90 g was harvested by freeze drying.

VBA was initiated successfully as shown in Fig. 11. From Fig. 11, the TEGSt incorporated into Polymer C is 72 mol%, the VBA incorporated was 24.4 mol% and the DOXSt incorporated was 3.6 mol% or 8.7 wt% . This is described in Table 1 further below.

Polymer D

In this example, DOX was introduced in the same way as for Polymer C, except that Polymer D carried DOXSt and VBA in a reversed manner as compared to Polymer C. As seen in Fig. 8, DOX was introduced by polymerizing DOXSt to obtain Polymer D, i.e. poly (TEGSt-co-DOXSt-co-VBA) .

200 mg of DOXSt was added to a 10-mL ampule with 1 g of poly(TEGSt) dissolved in 6 mL of anisole. After degassing with the freeze-pump-nitrogen gas refilling procedure for 6 times as described above and sealing, the reaction was allowed to take place overnight at 130 °C. The product was diluted, dialyzed against DMSO for one day and dialyzed against deionized water for another day. The dialysis tubing MWCO used was 3500 Da. The product was freeze-dried and redissolved in 6 mL of anisole before it was added to a 10-mL ampule. "

200 mg of VBA was added to the ampule. The ampule was degassed in the same manner as described earlier and sealed It was allowed to react at 130°C overnight. The solution was precipitated in ether twice and dried in a vacuum oven to obtain 0.84 g of the product, Polymer D.

Polymer D was characterized by ¹ H NMR spectroscopy as seen in Fig. 12. Referring, .to Fig. 12 and Table 1 below, the ¹ H NMR spectrum showed that there were about 90 mol% of TEGSt, 5 mol% or 12.2 wt% of DOXSt and 5 mol% of VBA in Polymer D. Further, it can be seen from Fig. 12 that the DOXSt loading was much lower than the monomer added at a ratio of TEGSt to DOXSt of 1 g to 0.2 g.

Two shoulders were observed in the GPC curve shown in Fig. 13 for this polymer. Accordingly, Polymer D has a much broader polydispersity of 1.88 than the other three polymers .

These results- indicated that only a limited amount of DOXSt was introduced into the polymer by this method and that DOXSt significantly blocked further polymerization. The molecular weights (MWs) and doxorubicin loadings of Polymers A to D obtained from the above examples as measured by ultraviolet (UV) spectroscopy are summarized in Table 1 below.

Table 1

Polymer Mn Mw Mw/Mn Doxorubicin Loading

(kDa) (kDa) (wt%)

A 30.5 37.1 1.22 —

B 28.0 34.1 1.22 30.0

C 24.0 29.7 1.24 8.7

D 37.4 67.4 1.80 12.2

The MWs of the polymers were determined by GPC (using Waters 2690, Waters Corporation, Massachusetts, USA)- with a differential refractometer detector (Waters 410). 10 mg of polymer was dissolved in 5 mL of THF and filtered. The mobile phase was THF with a flow rate- of 2 mL/min. Weight and number average molecular weights (Mw and Mn) were calculated from a calibration curve using a series of polystyrene standards (Polymer Laboratories, Inc., Massachusetts, USA) with molecular weights ranging from 1,300 to 30,000 Da.

Example 5

Synthesis of pH-sensitive material (poly (VBA-TEGSt) - HN=DOX)

A pH-sensitive material, poly (VBA-TEGSt) -NHN=D0X having a pH-sensitive hydrazone bond, linking doxorubicin with poly (TEGSt-co-VBA) was synthesized in this example. The procedure is detailed in the following in accordance with the reaction scheme illustrated in Fig. 1(b). 581 mg (1.0 mmol) doxorubicin was allowed to react with 524.9 mg (5.0 mmol) hydrazine · 2HCL in 20 ml DMF overnight under protection of aluminum foil. 3 drops of trifluoroacetic acid (TFA) was added as catalyst. Next, DMF was removed by rotavapor and dried further in vacuum. Afterwards, the solid was washed by 10 mL of deionized (DI) water for four times carefully. The residue was dried again by vacuum.

Doxorubicin linked with hydrazone was conjugated to poly (TEGSt-co-VBA) as follows. 0.52 g of poly (TEGSt-co-VBA) with 56.74 mol% of VBA monomer units (1.44 mmol of VBA monomer units estimated by ¹ H NMR) was dissolved in 10 mL of DMF and added with DCC (148.35 mg, 0.72 mmol), NHS (83.3 mg, 0.72 mmol) and 0.5 mL of triethylamine sequentially. Hydrazone linked doxorubicin (454 mg, 0.72 mmol) was added after 4 h and allowed to react overnight. Thereafter, the solution was filtered, dialyzed against DMSO for 2 days and dialyzed against DI water for another 2 days.

The solution with some red precipitate was harvested and freeze dried to obtain 0.6 g of red solid.

24 wt% of doxorubicin conjugation in the polymer was determined via UV measurement (Hitachi, 2801) at 485 nm.

The percentage release of doxorubicin at different pH values of 4.6 and 7.4 was charted and the graph is shown in Fig. 14. The pH value of 4.6 was achieved using 0.2 M of sodium acetate buffer and the pH value of 7.4 was achieved using phosphate buffered saline (PBS).

The particle size of the nanoparticles obtained after dialysis was measured by dynamic light scattering (DLS) using ZetaPALS (from Brookhaven Instruments Corporation, New York, USA) equipped with a He-Ne laser beam at 658 nm having a scattering angle of 90° at different temperatures. Each DLS measurement in the examples was repeated 5 times and an average value was obtained from the five measurements.

The particle size of poly (TEGST-VBA) =NNH-DOX measured by DLS is shown in Fig. 15. As seen in Fig. 15, the average particle size of the polymer is about 574.1 + 3.6 nm with a polydispersity of 0.225. The relative intensity (or Rel . Int.) is 89.94, the cumulative intensity (or Cum. Int.) is 34.16 and the diameter (or diam. ) is 232.54 nm.

Example 6

Synthesis of Fe ₃ 0 ₄ Magnetite Nanocubes

Magnetite nanoparticles were synthesized by thermal decomposition of iron oleate complex in this example.

2.7 g of FeCl ₃ '6H ₂ 0 (10 mmol) and 9.125 g of sodium oleate (30 mmol) were mixed in a solution of 20 mL of ethanol, 15 mL of deionized water and 35 mL of hexane. After refluxing at 70°C for 4 h, the upper portion of the reddish brown hexane solution was separated and washed three times with 10 mL of deionized water in a separatory funnel. After evaporating hexane, a dark reddish brown, oily iron oleate complex was obtained.

9 g of the as-prepared complex (10 mmol) was dissolved in 25 g of 1-octadecene with the addition of 1.52 g of sodium oleate (5 mmol) . The mixture was heated to 320°C at a ramp-up of 3-5°C/min for 30 min under argon. The resulting black nanocrystals solution was cooled to room temperature and 2-propanol was used to precipitate the magnetite nanocubes (MNs).

After centrifugation, the MNs were washed with hexane and ethanol three times and then re-dispersed in hexane or toluene. Example 7

Synthesis and Fabrication of Magnetite Nanocube-Loaded

Polymeric Nanoparticles

The polymers obtained from Example 4 were used in the co-delivery of cancer drug doxorubicin (DOX) via the reaction of carboxylic acid groups with magnetite (Fe ₃ 0 ₄ ) nanocubes. Reaction of Fe ₃ 0 ₄ nanocubes with Polymers B, C and D was achieved via a three-step dialysis procedure.

For example, 45 mg of polymer B was dissolved in 15 mL of toluene by heating to 80°C, followed by the addition of 300 μΐ· of Fe30 ₄ nanocubes dispersed in 20 mg/mL of toluene. The solution was added to a dialysis tubing from Spectra Pro ^® (Spectrum Laboratories, Inc., California, USA) with, a MWCO of 3500 Da, and dialyzed against toluene for 24 h. During dialysis, the toluene was changed four times to allow for the exchange of polymer with the oleic acid on the surface of Fe ₃ 0 ₄ nanocubes.

Thereafter, the tubing was dialyzed against ^' DMSO for another 24 h. During dialysis, DMSO was changed more than six times to remove toluene and the oleic acid residue completely .

Lastly, the solvent was dialyzed against deionized water for 1 day to remove DMSO completely..

During the three-step dialysis, no obvious precipitation was observed. 50 mL of solution was harvested for the magnetite nanocube (MN) -loaded polymeric nanoparticles.

For the preparation of the nanoparticles without Fe ₃ 0 ₄ MNs, 45 mg of polymer B was dissolved in 15 mL of DMSO, and dialyzed against deionized water using the same dialysis tubing for 3 days. 70 mL of solution was harvested. 30 mL of the solution filtered with 0.45-μια syringe filter paper was freeze-dried to obtain solid samples for the concentration estimation and Fe ₃ 0 ₄ loading level via thermogravimetric analysis (TGA) .

TGA using PerkinElmer TGA 7 (from PerkinElmer, Massachusetts, USA) was performed by examining the solid samples obtained by freeze drying under air flow, with a ramp of 5°C/min between room temperature and 500°C.

The particle morphologies were determined using transmission electron microscopy (TEM) . Specifically, a drop of solution of nanoparticles was placed on a carbon- coated copper grid and air dried overnight. TEM studies were performed using ■ a FEI Tecnai™ G ² F20 electron microscope at 200 kV (from FEI Company, Oregon, USA) .

The particle morphologies and sizes of Polymers B, C and D were compared after they were loaded with magnetic nanocubes, referred to as B-MN, C-MN and D-MN respectively herein. TEM images, photographs and particle size distributions demonstrated by DLS of the MN-loaded Polymers B, C and D are shown in Figs. 16(a), (b) and (c) respectively. Figs. 16(a), (b) and (c) show that the size, size distribution and morphology of the MN-loaded polymeric particles were dependent on the polymer structures. The TEM image in Fig. 16(a) showed that polymer B bound MNs tightly, forming a core of MNs with a polymeric shell. Such a core of MNs was not formed with polymer C as seen in Fig. 16(b), possibly due to the position of carboxylic acid groups on this polymer. With polymer D, several MNs were found to aggregate to ^' form one polymeric nanoparticle as seen in Fig . 16(c).

The thermal gravimetric analysis (TGA) of MN-loaded Polymer B (B-MN) as shown in Fig. 17 evidenced that B-MN contained 28.0 wt% of MNs. This example provided an excellent loading of Fe ₃ 0 ₄ of up to 50 wt% in aqueous phase, which was much higher than that achieved by prior methods.

Polymer B has a relatively narrower polydispersity of 0.12, as compared to polymer C having a polydispersity of 0.28 and polymer D having a polydispersity of 0.28. The average particle sizes were 108 ± 1 nm, 104 ± 2 nm and 154 ± 2 nm for B-MN, C- N and D-MN, respectively, at 20 °C.

The particle sizes of Polymers B, C and D are compared against B-MN, C-MN and D-MN and the graphs are shown in Figs. 18(a), (b) and (c) respectively. As can be seen from Fig. 18(c), the particle size of Polymer D decreased when loaded with MNs. However, it can be seen from Figs. 18(a), (b) and (c) that the MN-loaded polymeric nanoparticles have excellent stability from 20°C to 45°C.

It can be concluded that the positions of doxorubicin and binding groups for MN on the polymer played an important role in the morphology and structure of the nanoparticles. Polymer B having doxorubicin randomly conjugated onto carboxylic acid groups gave rise to a core- shell structure when loaded with MNs.

X-ray diffraction (XRD) patterns were obtained by freeze drying the solution of nanoparticles and Fe3C>4 nanoparticles without polymer and measured with a PANalytical X' Pert PRO Diffractometer (from PANalytical, Netherlands) equipped with a multichannel X' celerator detector also from PANalytical, using Cu K radiation (where λ = 1.540598 A, 40 kV, 40 mA) in the 2Θ range of 20- 85°:

The magnetite nanoparticles in B-MN were analyzed by XRD and shown in Fig. 19(a) . As seen in Fig. 19(a), the crystalline structure of the Ns (top curve) did not change after loading into Polymer B (bottom curve) .

Example 8

Study of Cellular Uptake of B-MN With and Without the

Presence of Magnet

The effect of magnetic field on the cellular uptake of Polymer B loaded with magnetite nanocubes (B-MN) was studied in this example to investigate the coordinated movement of doxorubicin with MN. Specifically, the cellular uptake of B-MN with and without the presence of a magnet (sintered NdFeB magnet N52) was examined.

HepG2 cells were used in this example and were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM of L- glutamine, 100 U/mL of penicillin and 100 g/mL of streptomycin at 37 °C under an atmosphere with 5% C0 ₂ using a 75-mL plastic flask. To subculture the confluent cells, the medium was removed first and washed with 5 ml of PBS buffer, and the cells were detached by 1 mL of trypsin. 1/5 of the cells was passed to the next flask.

Two 5-cm Petri dish were seeded with 50,000 HepG2 cells in 2 ml/dish of DMEM for 24 h. 2 ml of B-MN solution was then added to each dish. One dish was placed above a magnet, and the other dish was not placed above any magnet. The two dishes were both kept in the incubator for 15 min and then their culture media were removed.

They were rinsed and each refilled with 1 ml of phosphate buffered saline (PBS) . The Petri dishes were observed under fluorescence microscope directly with green light excitation. As seen in Fig. 19(b), after 15 min of incubation, the fluorescence of cells was stronger under the application of a magnetic field (right) as compared to without application of a magnetic field (left) . When _. a magnetic field was applied below the cell culture plate, the B-MN particles would settle faster onto the bottom of the plate and thus, be taken up more rapidly by the cells. This result revealed that there was coordinated uptake of doxorubicin and MN by the cells.

The toxicity of Polymers A and B loaded with MNs (i.e. MN-P (TEGSt-co-VBA) and MN-P ( TEGSt-co-VBA) -DOX respectively) was measured and the results are shown in Fig. 20. As can be seen in Fig. 20, MN-P (TEGSt-co-VBA) -DOX had a lower toxicity intensity as compared to MN-P (TEGSt-co-VBA) carrying no doxorubicin. Accordingly, it can be concluded that Polymer B with conjugation of doxorubicin effectively targeted and reduced tumour growth, thereby leading to lower toxicity.

Example 9

Bio-Distribution Study of Doxorubicin and MNs in Tumor- Bearing Animals

Balb/C mice of from about 20-30 g were purchased from Singapore Animal Center and used for the in vivo study.

4T1 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS, 2 mM of L-glutamine, 100 unit/mL of penicillin and 100 μg mL of streptomycin at 37 °C under an atmosphere with 5% CO ₂ . The 4T1 cells were subcultured as above.

To harvest 4T1 cells for subcutaneous seeding in mice, the cells were rinsed with PBS and detached by trypsin. Thereafter, the medium containing the cells was neutralized by RMPI 1640 medium with 10% FBS and centrifuged at 1000 rpm for 5 min. The cells were then washed with PBS twice to remove the FBS completely. They were dispersed evenly in free RMPI 1640 medium without FBS and injected subcutaneously near the belly- of the mice.

200 ]i and 1x106 cells were injected per animal. After 2 weeks, the tumor grew to about 200 mg . These tumor- bearing mice were employed in the in vivo studies.

200 μΙ_ of B-MN was injected into each mouse through the tail vein. After 24 h, the tumor, heart, liver, spleen, lung and kidney of the mouse were harvested and immersed in 4% paraformaldehyde.

For the bio-distribution analysis, the samples were rinsed with deionized water, covered with ^' Jung tissue freezing medium and dissected into slices of 10—12 μπι thickness using Leica CM3050S cryostat (L ^' eica Microsystems GmbH, Germany) . Two sequential intersectional slices of the samples were sent for fluorescence imaging and Prussian blue iron staining for the distributions of doxorubicin- and MNs, respectively.

For doxorubicin imaging, one slice of sample was hydrated in deionized water and then dehydrated quickly with alcohol and xylene before mounting onto glass slides for fluorescence imaging with green light excitation.

The other slice of sample was stained with Sigma- Aldrich Accustain iron staining kit (HT20) according to the vendor's protocols. Briefly, the sample was hydrated in deionized water, placed in the working iron staining solution for 10 min and rinsed with deionized water. It was then stained in the working pararosaniline solution for 3-5 min and rinsed with deionized water again. The sample was next dehydrated rapidly with alcohol and xylene and mounted for microscopy imaging.

Coordinated bio-distribution of doxorubicin and iron was achieved in the organs of the mice injected with MN-B nanoparticles . The bio-distributions of doxorubicin and iron in the tumor, heart, kidney, liver, lung and spleen in balb/C mice were tracked via red fluorescence and Prussian staining respectively and shown in Fig. 21. Referring to Fig. 21, the fluorescence images are shown in the first and third columns, while the Prussian staining images are shown in the second and fourth columns. The strongest doxorubicin fluorescence and blue iron staining were both observed in the tumor and lung. The distribution of doxorubicin indicated that MN-B nanoparticles. have strong passive targeting, ability simply due -to their stable and well- protected structure.

Thus, it is critical to maintain the stability of the nanoparticles loaded with both anti-cancer drugs and MRI agents, such as magnetite nanocubes, and this was successfully achieved by chemical binding by the methods demonstrated in the examples to realize the same bio- distribution of doxorubicin and MNs in vivo.

Applications

The disclosed core-shell nanoparticle avails its internal core space to the containment of compounds such as the drug and magnetic nanoparticles via chemical binding. In addition, the drug and magnetic nanoparticles within this core structure may be further protected by an outer shell structure such that leakages of the core contents cargo due to breakages may be substantially minimized. In the core-shell nanoparticle, the availability of cleavable bonds between the core contents and the polymeric shell may facilitate the release of the core contents (for instance, therapeutic drugs) at a locality of a disease site .

The core-shell nanoparticle may be advantageous over the indiscriminate targeting of cells (such as in dendrimer-based carriers) by making available the option of magnetically-guiding the carrier to the site targeted for drug-release possible.

The magnetic nanoparticle encapsulated in the core may be non-toxic, biocompatible iron-oxide nanoparticles, which would eventually break down to form blood hemoglobin.

The core-shell nanoparticle may display strong passive and/or active targeting of tumors and a coordinated bio- distribution of its released cargo- compounds. The core- shell nanoparticle may allow for real-time monitoring of the drug distribution at the target tissue. Hence, ^■ the effect of therapeutics may be monitored as the disease progresses.

The core-shell nanoparticle may be used in medicine. Firstly, the core-shell nanoparticle may provide a synergistic approach to controlled drug therapy, when combined with a suitable technique (e.g. magnetic resonance imaging) in guiding and tracking the contained magnetic nanoparticles. Such drug therapy can be in the form of administered chemotherapeutics for use in the treatment of cancer. Moreover, the core-shell nanoparticle will also be applicable in hyperthermia techniques of treating cancer. In such a method, hysteretic heating of magnetic nanoparticles with alternating magnetic frequencies can be used to treat cancer with little damage to normal tissues as it avoids the marrow suppression that results from many drugs or high levels of radiation.

It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.