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Title:
A STAR BLOCK COPOLYMER USED AS DRUG CARRIER FOR TREATMENT OF CANCER
Document Type and Number:
WIPO Patent Application WO/2021/225533
Kind Code:
A1
Abstract:
The invention relating to star block copolymer as drug carrier for the selective treatment of cancer having a hydrophobic core (10) comprising of a multiarm methyl metacrylate oligomer having a molecular weight less than or equal to 6000, a hydrophilic PAA shell(11) comprising of partially crosslinked PAA arms (12) where crosslinking is achieved by divalent cation, namely being one or a mixture of copper, magnesium, calcium, zinc, and iron, with a total molecular weight less than 35000 g/mol for yielding optimum particle size, has a very low critical micelle concentration and high drug loading capacity offering a potential for targeted and controlled delivery of anti-cancer drugs.

Inventors:
GENÇ GÖZDE (TR)
Application Number:
PCT/TR2020/050373
Publication Date:
November 11, 2021
Filing Date:
May 04, 2020
Export Citation:
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Assignee:
ILMOR KIMYA VE TEKSTIL SANAYI VE TICARET LTD SIRKETI (TR)
International Classes:
A61K47/32; C08F220/06; C08F293/00
Domestic Patent References:
WO2005035606A12005-04-21
Foreign References:
CN102961332A2013-03-13
Other References:
GENE, G. ET AL.: "Acrylic star block copolymers as hydrophobic drug carriers", 2009 14TH NATIONAL BIOMEDICAL ENGINEERING MEETING, May 2009 (2009-05-01), pages 1 - 4, XP031480019
TALELLI, M. ET AL.: "Core-crosslinked polymeric micelles: principles, preparation, biomedical applications and clinical translation", NANO TODAY, vol. 10, no. 1, 2015, pages 93 - 117, XP055872220
LI, X. ET AL.: "Amphiphilic multiarm star block copolymer-based multifunctional unimolecular micelles for cancer targeted drug delivery and MR imaging", BIOMATERIALS, vol. 32, no. 27, 2011, pages 6595 - 6605, XP028254249, DOI: 10.1016/j.biomaterials.2011.05.049
PRAMANIK, A. ET AL.: "An in-vivo study for targeted delivery of copper-organic complex to breast cancer using chitosan polymer nanoparticles", MATERIALS SCIENCE AND ENGINEERING: C, vol. 68, 2016, pages 327 - 337, XP029684611, DOI: 10.1016/j.msec.2016.05.014
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Claims:
CLAIMS

1. For the selective treatment of cancer;

- An amphiphilic multi-arm star block copolymer as drug carrier (1) characterized in that, it comprises a hydrophobic core (10) comprising of a multiarm methyl metacrylate oligomer, a hydrophilic PAA shell (11) with at least three partially crosslinked PAA arms (12) and modified with divalent cation, namely copper, more specifically with Cu(OH)2 used as crosslinker after drug loading. 2. The amphiphilic multi-arm star block copolymer as drug carrier (1) according to claim 1 characterized in that, it has maximum 10 ppm critical micelle concentration.

3. The amphiphilic multi-arm star block copolymer as drug carrier (1) according to claim 2 characterized in that, it is purified and loaded with hydrophobic drugs by dialysis method. 4. The amphiphilic multi-arm star block copolymer as drug carrier (1) according to claim 3, characterized in that, it remains in the circulatory system up to 24 hours without degrading and targeting tumors and then controlled release of the drug.

5. The amphiphilic multi-arm star block copolymer as drug carrier (1) according to claim 4, characterized in that, it has maximum 40000 g/mol of molecular weight.

6. The amphiphilic multi-arm star block copolymer as drug carrier (1) according to claim 5, characterized in that, it comprises PMMA core having a maximum molecular weight of 6000 g/mol.

7. The amphiphilic multi-arm star block copolymer as drug carrier (1) according to claim 6, characterized in that, it comprises a PMMA core diameter less than or equal to 5 nanometers in order to enable the elimination of polymethyl methacrylate residues through the urine.

8. The amphiphilic multi-arm star block copolymer as drug carrier (1) according to claim 7, characterized in that, it has hydrophobic core (10) is to have a particle size less than or equal to 5 nanometers.

9. The amphiphilic multi-arm star block copolymer as drug carrier (1) according to claim 8, characterized in that, it has hydrophobic core (10) is to have molecular weight less than or equal to 6000, more preferably less than 5000, in order to be kept less than 5 nanometers in size.

10. The amphiphilic multi-arm star block copolymer as drug carrier (1) according to claim 9, characterized in that, it has PMMA core which is optimum ratio of molecular weight of hydrophobic core (10) to total molecular weight of the molecule is between 0,3 and 0,4 .

11. The multi-arm star block copolymer as drug carrier (1) according to claim 10, characterized in that, it has molecular weight preferably less than or equal to 60000 g/mol.

12. The amphiphilic multi-arm star block copolymer as drug carrier (1) according to claim 11, characterized in that, it has molecular weight preferably less than or equal to 40000 g/mol.

13. The amphiphilic multi-arm star block copolymer as drug carrier (1) according to claim 12, characterized in that, it has molecular weight less than 35000 g/mol for yielding optimum particle size.

14. The amphiphilic multi-arm star block copolymer as drug carrier (1) according to claim 13, characterized in that, it has optimum total molecular weight is 16000- 24000 g/mol for a higher drug loading capacity.

15. The amphiphilic multi-arm star block copolymer as drug carrier (1) according to claim 14, characterized in that, it has molecular weight can be from two to ten times higher than molecular weight of the hydrophobic core (10) after hydrolysis.

16. Star block copolymers that are used as drug carriers for treatment of cancer, wherein obtaining them comprises the following steps;

- synthesizing a multi-arm methyl methacrylate core with molecular weight less than or equal to 6000 g/mol and having a particle size smaller than 5 nm.

- Synthesizing an multiarm block copolymer of poly(methyl methacrylate)-block- poly(t-butyl acrylate) (PMMA-b-PtBA) from the multi-arm methyl methacrylate core, having a total molecular weight less than or equal to 60000 g/mol,

- Hydrolyzing the multiarm block copolymer of poly(methyl methacrylate)-block- poly(t-butyl acrylate) to have multiarm block copolymer of poly(methyl methacrylate)-block-poly(acrylic acid) which has a particle size smaller than 100 nm in aqueous medium.

17. Star block copolymers proposed as drug carrier for treatment of cancer according to Claim 16, wherein the drug loading and surface modification process comprises the steps below;

- loading hydrophobic drugs to the star block copolymers using dialysis method, via accumulation of the drugs in the core of the star block copolymer due to hydrophobic - hydrophobic interaction.

- Partial crosslinking of the surface by linking -COO- groups of poly(acrylic acid) arms for both immobilization and controlled release of the drug.

- Partial crosslinking of the -COO- groups of poly(acrylic acid) arms by reacting with divalent cations such as Ca+2, Mg+2, Cu+2, Zn+2, Fe+2 or mixtures thereof.

- Partial crosslinking of the -COO- groups of poly(acrylic acid) arms by dissolving or dispersing divalent cation complexes in drug loading medium where the complexes being sulfates, nitrates, carbonates, chlorides, perchlorides or hydroxides.

- Partial crosslinking of the -COO- groups of poly(acrylic acid) arms by a divalent metal complex, and especially a copper complex such as Cu(OH)2, Cu(NO3)2, CuCO3, CuSO4 in order to provide targeting cancerous regions.

- Freeze drying to obtain drug loaded and partially crosslinked star-block- copolymer particles that may readily dissolve and form homogeneous aquoeus solutions of anti-cancer drugs.

18. Star block copolymers for treatment of cancer according to Claim 16 and Claim 17, wherein the use as a drug carrier to enable the controlled release of the drug in selective cancer treatments, comprises the steps below;

- Formation of a homogenous solution of the loaded hydrophobic drugs in water by the way of the water soluble poly(acrylic acid) arms,

- selectively, accumulation of the drug loaded molecules with molecular weight less than 40000 g/mol, and preferably with a molecular weight less than 35000 g/mol for yielding optimum particle size after hydrolysis, and ideally within the molecular weight range of 16000 - 24000 g/mol for higher drug loading capacity, within tumors rather than healty tissues, - remaining of the star block copolymers in the circulatory system up to 24 hours without degrading to provide the time to accumulate in the tumors,

- targeting cancer due to surface modification, namely partial crosslinking with Cu+2 complexes.

- enabling the controlled release of the drug it contains by the biodegradation of poly(acrylic acid) arms which may or may not have been partially crosslinked with divalent cations such as Ca+2, Mg+2, Cu+2 , Zn+2, Fe+2 and such, or mixtures thereof.

- discharging polymethyl methacrylate cores smaller than 5 nanometers which remain at the end of the degradation by urinary tract from the body.

Description:
A STAR BLOCK COPOLYMER USED AS DRUG CARRIER FOR

TREATMENT OF CANCER

RELATED TECHNICAL FIELD

The invention relates to the star block copolymers partially crosslinked with divalent cations used as drug carrying molecules in the selective treatments of cancer.

KNOWN STATE OF THE ART

At present, polymeric nanoparticles which can be used as a drug carrier in selective treatments of cancer are studied to improve conventional chemotherapy which can simply be described as intravenous introduction of toxic drugs to prevent growth of malign tumors and proliferation of cancerous cells. The primary objective of the drug carriers is to reduce detrimental side effects of chemotherapy. In other words, methods of targeted delivery has been considered to be helpful by delivering toxic drugs in cancerous regions and keep healthy tissues less affected. Another reason to develop drug carriers is to provide accurate dosing of hydrophobic anti-cancer drugs which are poorly soluble in aqueous medium.

On the state of art, polymeric structures such as poly(lactic acid)-poly(ethylene glycol) copolymers are one of the most common research topic on delivery of hydrophobic cancer drugs, disclosed in the patent US2015023875. In this method, hydrophobic poly lactic acid chains are formed and attached to polyethylene glycol molecules to form a biodegradable amphiphilic copolymer. Amphiphilic is a term used for molecules having both hydrophilic and hydrophobic structures and form micelle in aqueous medium. These block copolymers entrap hydrophobic molecules due to hydrophobic- hydrophobic interaction which makes them promising drug carriers. But they need a certain level of concentration to form micelle and entrap drug molecules which is called as critical micelle concentration (CMC). Critical micelle concentration of poly(lactic acid)-block-poly(ethyl ene glycol) copolymer nanoparticles (with molecular weight Mn≤5000 g/mol) is reported as 35 ppm in literature (Hagan et al., Langmuir, 1996, 12, 2153). On the other hand, since micellar structures are formed with weak bonds, there are risks involved in the prediction of their behavior in a medium with variable physical properties such as blood. For example, the pH, viscosity and electrolyte concentration of blood can change and affect the critical micelle concentration of the drug carrier. Because of this, the predicted targeting mechanism may not work perfectly at every metabolism. There need to be stable micelle forming behavior at low concentrations in the state of art to provide a reliable targeting mechanism.

In another known state of the art, in order for the drug carrying particles in the bloodstream to be specifically directed to the tumors, one of the methods used frequently is the conjugation method discussed in patent US2015157737, in which the nanoparticles are bonded to proteins, peptides or molecules in a nucleotide form that are thought to be present to a high level in cancerous regions. But these biological markers may be uptaken by healthy cells, depending on the condition of the body. For example, folate receptors increase on the surface of solid tumors (Zwicke et al, Nano Rev, 2012; 3: 101. But in case of cancer, body may also need supplements like folic acid more than normal condition. So the selectivity of these therapies depends and varies from person to person and requires personalization.

Another most important disadvantage of polylactic acid-polyethylene glycol copolymers, as discussed in patent CN104072707, is the fact that they are produced using the ROP (Ring Opening Polymerization) method and that toxic tin-containing catalysts are used.

One of the biggest difficulties in the use of star block copolymers in the drug industry is that metal catalysts are used during their synthesis and that it is rather difficult for this to be completely purified. That metal catalyst is generally stannous compound for polymerization of lactides and a copper compound for ATRP (Atom Transfer Radical Polymerization) that is a living polymerization technique for acrylic block copolymers. However, studies of the last decade show that a significant increase in intratumoral copper level is observed in many types of cancer. Many studies claim that copper is an angiogenic factor in tumors which means tumor uses copper to stimulate the proliferation and migration of endothelial cells for the formation of new vessels that are needed for the growth of the tumor (Wang et. al, Curr Med Chem. 2010; 17(25): 2685- 2698.) This approach enables the use of copper imparted within star block copolymer for selective anti-cancer therapies.

Recent study under investigation in the state of the art, is use of copper coordination complexes with or instead of platinum based drugs to overcome drug-resistance and provide a potent anti-cancer therapy with reduced risk to healthy cells (Denover et al., Metallomics. 2015 Nov;7(11): 1459-76. doi: 10.1039/c5mt00149h & Tisato et al., Med Res Rev. 2010 Jul;30(4):708-49. doi: 10.1002/med.20174.). In these studies it has been claimed that, copper coordination complexes bind the existing copper in tumor and enhance the affectivity of the therapy.

Another aspect is that Cu complexes fights cancer on its own. On a very recent study CuO particles are reported to inhibit pancreatic tumor cells re-growth (Benguigui et al, Scientific Reports, vol 9, Article number: 12613 (2019)).

Another important issue related to targeted delivery of drugs with polymeric nanoparticles is the mechanism of drug release. The main attempt for targeted drug release of anti-cancer drug release by using polymeric micelles is to use pH sensitive block copolymers as in US Patent 7659314B2. Amphiphilic block copolymers forming micelles around hydrophobic anti-cancer drugs and targeted by addition of folate moieties are expected to release the drug loaded to this polymeric micelle not in blood but only in acidic environment. General approach is to use the presence of acidic environment in cancer tumors where pH sensitive micelle readily degrades and releases drug content onto tumor. pH of blood varies between 7.35 and 7.45 and it has been discovered that surface of aggressive cancer tumors mostly show a slightly lower pH like 6.5-7.0 (Tannock et al., Cancer Research 49, 4, 173-4384 (1989)). This is still a very little difference to tailor a definite drug release mechanism.

Therefore there need to develop new selective cancer treatments with drug carriers having advantages like; high loading capacity, low and stable critical micelle concentration, easy elimination from the body with no accumulation in vital organs, reliable targeting mechanism and providing synergistic effect with the anticancer drugs of interest. OBJECT OF THE INVENTION

The object of this invention is to use partially crosslinked multi-arm star block copolymers to transport anti-cancer drugs that are loaded to this polymer with high drug loading capacity to the cancerous regions in the body for treatment purposes.

To target the cancerous region, the aim is to benefit from the tendency of cancer tumors to take up copper element. For this reason, it has been claimed that, crosslinking of poly(acrylic acid) arms is achieved by divalent cations, and namely by Cu +2 . Another object of this invention is to gain time for the drug to reach the cancerous regions where overgrowth of capilar vessels are observed and delay the start of the release of the drug up to 24 hours owing to the fact that poly(acrylic acid) arms degrade more slowly due to crosslinking. Although degree of crosslinking changes the rate of biodegradation and causes a delayed and/or controlled release of the drug partially crosslinked poly (aery lie acid) chains must eventually degrade.

Another object of this invention is elimination of poly methyl methacrylate cores from the body by urinary tract after the degradation of biodegradable outer shell of the drug loaded particles and not accumulate in essential organs like heart, liver, lungs and kidneys. In addition, using star block copolymers that are single molecules that work as micelle particles on their own is recommended to provide stable micelle forming behavior and very low critical micelle concentration in this invention. Therefore, in summary, by the invention, new selective cancer treatments with drug carriers having advantages like; high loading capacity low critical micelle concentration easy elimination from the body with no accumulation in vital organs reliable targeting mechanism and providing synergistic affect with the anticancer drugs of interest are obtained.

Star block copolymers that are used as drug carriers for treatment of cancer, wherein obtaining them comprises the following steps;

- synthesizing a multi-arm poly(methyl methacrylate) core with molecular weight less than or equal to 6000 g/mol and having a particle size smaller than 5 nm.

- Synthesizing an multiarm block copolymer of poly(methyl methacrylate)-block- poly(t-butyl acrylate) (PMMA-b-PtBA) from the multi-arm methyl methacrylate core, having a total molecular weight less than or equal to 60000 g/mol,

- Hydrolyzing the multiarm block copolymer of poly(methyl methacrylate)-block- poly(r-butyl acrylate) to have multiarm block copolymer of poly(methyl methacrylate)-block-poly(acrylic acid) which has a particle size smaller than 100 nm in aqueous medium.

Star block copolymers proposed as drug carrier for treatment of cancer, wherein the drug loading and surface modification process comprises the steps below;

- loading hydrophobic drugs to the star block copolymers using dialysis method, via accumulation of the drugs in the core of the star block copolymer due to hydrophobic - hydrophobic interaction.

- Partial crosslinking of the surface by linking -COO- groups of poly(acrylic acid) arms for both immobilization and controlled release of the drug.

- Partial crosslinking of the -COO- groups of poly(acrylic acid) arms by reacting with divalent cations such as Ca +2 , Mg +2 , Cu +2 , Zn +2 , Fe +2 or mixtures thereof.

- Partial crosslinking of the -COO- groups of poly(acrylic acid) arms by dissolving or dispersing divalent cation complexes in drug loading medium where the complexes being sulfates, nitrates, carbonates, chlorides, perchlorides or hydroxides.

- Partial crosslinking of the -COO- groups of poly(acrylic acid) arms by a divalent metal complex, and especially a copper complex such as Cu(OH) 2 , Cu(NO 3 ) 2 , CuCO 3 or CuSO 4 in order to provide targeting cancerous regions.

- Freeze drying to obtain drug loaded and partially crosslinked star-block- copolymer particles that may readily dissolve and form homogeneous aquoeus solutions of anti-cancer drugs.

Star block copolymers for treatment of cancer wherein the use as a drug carrier to enable the controlled release of the drug in selective cancer treatments, comprises the steps below;

- Formation of a homogenous solution of the loaded hydrophobic drugs in water by the way of the water soluble poly(acrylic acid) arms,

- selectively, accumulation of the dmg loaded molecules with molecular weight less than 40000 g/mol after hydrolysis, and ideally within the molecular weight range of 16000 - 24000 g/mol for higher drug loading capacity, within tumors rather than healty tissues.

- remaining of the star block copolymers in the circulatory system up to 24 hours without degrading to provide the time to accumulate in the tumors,

- targeting cancer due to surface modification, namely partial crosslinking with Cu +2 complexes.

- enabling the controlled release of the drug it contains by the biodegradation of poly(acrylic acid) arms which may or may not have been partially crosslinked with divalent cations such as Ca +2 , Mg +2 , Cu +2 , Zn +2 , Fe +2 and such, or mixtures thereof.

- discharging poly(methyl methacrylate) cores smaller than 5 nanometers which remain at the end of the degradation by urinary tract from the body.

Description of the Drawings Figure 1. A method of the obtaining multiarm star block copolymers.

Figure 2. A method of obtaining a drug loaded multiarm star block copolymers. Figure 3. A schematic view of drug loaded and surface modified nanoparticles.

DETAILED DESCRIPTION OF THE INVENTION

Description of the References in the Drawings

For a better understanding of the invention, the numbers in the figures are provided as follows:

1. Drug carrier

10. Hydrophobic Core

11. Hydrophilic PAA Shell

12. Partially crosslinked PAA Arms

100, 200 -Method

An amphiphilic multi-arm star block copolymer as drug carrier (1) for the selective treatment of cancer comprises a hydrophobic core (10) comprising of a multiarm methyl methacrylate oligomer, a hydrophilic PAA shell (11) with at least three partially crosslinked PAA arms (12) and modified with divalent cation, namely copper, more specifically with Cu(OH) 2 used as crosslinker after drug loading.

PMMA is poly(methyl methacrylate) and PAA is poly(acrylic acid).

Hydrophobic core (10) is a PMMA core.

Hydrophilic shell is PAA shell (11). Partially crosslinked PAA arms (12) constitute the outer shell of drug loaded multi-arm star block copolymer with surface modification through crosslinking with divalent cations. The invention relating to amphiphilic multi-arm star block copolymer having extremely low critical micelle concentration such as 10 ppm and remains in the circulatory system up to 24 hours without degrading and targeting tumors and then controlled release of the drug it contains in the selective treatments of cancer, has maximum 40000 g/mol of molecular weight, with PMMA core having a minimum molecular weight of 1000 g/mol and a maximum molecular weight of 6000 g/mol, with a hydrophobic core (10) diameter less than or equal to 5 nanometers in order to enable the elimination of poly(methyl methacrylate) residues through the urine.

In order for the star block copolymers of the invention to be obtained, first the multi- arm hydrophobic core (10) molecules are synthesized. Hydrophobic core (10) is to have a particle size less than or equal to 5 nanometers.

The star block copolymer of the invention may have three, four, six or more arms depending on the OH groups of the starting molecule. Hydrophobic core (10) is to have molecular weight less than or equal to 6000, more preferably less than 5000, in order to be kept less than 5 nanometers in size. That means, each arm that constitutes the hydrophobic core (10) has a repeating unit definitely less than 20 and more preferably between 10 and 12. The molecular weight of the hydrophobic core (10) may be optimized for a specific drug in order to obtain minimum core diameter and maximum drug content which also depends on hydrophobic core (10) ratio. Optimum ratio of molecular weight of hydrophobic core (10) to total molecular weight of the molecule is between 0,3 and 0,4 .

In order for the star shaped polymers to be synthesized, a suitable starting molecule is functionalized for the atom transfer radical polymerization with halogenation of OH endgroups of the starting molecule that constitutes the center of the core of multi-arm star block copolymer. Then the hydrophobic core (10) with a maximum molecular weight of 6000 g/mol is synthesized with ATRP (Atom Transfer Radical Polymerization) of methyl methacrylate using the halogenated form of the starting molecule. After that, the addition reaction of butyl acrylate arms is achieved by the living polymerization method (ATRP) and through this, multi-arm methyl methacrylate- block-butyl acrylate copolymers with a maximum molecular weight of 60000 g/mol are prepared. Afterwards, the butyl acrylate groups are transformed into acrylic acid with hydrolysis reaction. Thus, the amphiphilic star copolymers of the multi-arm poly(methyl methacrylate)-block-poly(acrylic acid) (PMMA-b-PAA) consisting of a hydrophobic core (10) and a hydrophilic PAA shell (11) are obtained.

A method (100) of obtaining the star block copolymers of the invention that are used as drug carriers for treatment of cancer comprises the following steps;

- Synthesizing a multi-arm hydrophobic core (10) (101),

- after that, conducting the addition reaction of butyl acrylate monomer with the living polymerization method and through this, obtaining multi-arm methyl methacrylate-block-butyl acrylate copolymers (102),

- obtaining the amphiphilic star copolymers of the multi-arm methyl methacrylate- block- acrylic acid by converting poly(butyl acrylate) arms into poly(acrylic arms) with hydrolysis reaction (103),

Molecular weight of hydrophobic PMMA core is preferably less than or equal to 6000 g/mol. Molecular weight of PMMA-b-PtBA star block copolymers is preferably less than or equal to 60000 g/mol. Preferred molecular weight of the amphiphilic PMMA-b- PAA star block copolymer is less than 40000 g/mol for a higher drug loading capacity. Optimum total molecular weight of the star-block-copolymer is 16000-24000 g/mol. Molecular weight of star block copolymers can be from two to ten times higher than molecular weight of the hydrophobic core (10) after hydrolysis. Hydrophobic core (10) ratio of the amphiphilic star block copolymer may have a value from 0.1 to 0.5 and can be decided considering drug molecular weight and drug-polymer interaction. Both of these factors affect drug loading capacity, biodegradation and drug release profile. Diagram of 101 st step: Synthesis of a multi-arm hydrophobic core (10) molecule from brominated starting molecule (being one of glycerine, triethanolamine, trihydroxybenzene, trimethylolpropane, di(trimethylolpropane), pentaerythritol, dipentaerythritol, or another multi-hydroxyl-functional molecule) by ATRP with addition of methyl methacrylate monomer as follows. Diagram of 202 nd step: Synthesis of the multi arm PMMA-block-PtBA copolymer by ATRP reaction as follow. Diagram of 203 rd step: Conversion of tBA groups into acrylic acid by hydrolysis reaction as follow:

The multi-arm star block copolymers comprising of hydrophobic poly(methyl methacrylate) core (10) and hydrophilic poly(acrylic acid) PAA shell (11) are purified and loaded with hydrophobic drugs by dialysis method (as in method 200). Due to the interaction of the loaded drugs with poly(methyl methacrylate) core, the drug accumulates at the center of the star block copolymer and due to hydrophilicity of the partially crosslinked PAA arms (12), hydrophobic drugs are prone to form homogenous aqueous solutions.

As long as the molecular weight of star block copolymer of the invention as a drug carrying molecule is in the range of 10000 g/mol - 60000 g/mol (before hydrolysis), the hydrodynamic radius of the hydrolysed and drug loaded amphiphilic molecule remains below 100 nm. With the appropriate particle size (between 10-100 nm) molecules can selectively reach to and accumulate in tumors more than healthy tissues,

Hydrodynamic radius of the particle in aqueous media depend on, molecular weight of the carrier, number of arms of star-block-copolymer, molecular weight ratio of hydrophobic core (10), type of the loaded drug which may interact with the hydrophilic chains besides pH end electrolyte concentration of medium. Molecular weight of the drug carrying molecule has been observed to be 15000-45000 g/mol yielding an average hydrodynamic particle size about 30 nm in buffer saline solutions for four-arm PMMA- block- PAA copolymers of that of molecular weight. The star block copolymers of the invention retain the drug in the circulatory up to 24 hours and therefore find enough time to accumulate in the tumors that function as a filter. When drug loaded particles are placed in aqueous medium, in standard buffer saline solution representing blood serum for instance, particles tend to agglomerate and form agglomerations with particle sizes 2-3 times greater than the original particle size. Due to this agglomeration, and due to interaction with the drug and off course because of surface modification provided by partial crosslinking with divalent cations, onset of biodegradation is delayed depending on the level of these three factors. Afterwards, due to biodegradation of partially crosslinked PAA arms (12), the surface, namely the hydrophilic PAA shell (11) of the nanoparticle starts to erode and enable the controlled release of the drug it contains. At the end of the degradation of the partially crosslinked PAA arms (12), the remaining non-degraded poly(methyl methacrylate) core (10) can be eliminated from the body through urinary tract since it is less than 5 nanometers. Polymethyl methacrylate is not biologically degraded but when it is synthesized with a particle size less than or equal to 5 nanometers, it can be eliminated from the body through the kidneys. For this reason, unlike polymethyl methacrylate, methyl methacrylate oligomer is a promising material to be used as drug carrier.

The carrier molecule of the invention is a covalently bonded system and the critical micelle concentration is extraordinarily low (10 ppm).

Owing to this invention, concentrated drug solutions can be prepared with a smaller amount of polymer and a controlled drug release as well as dose control may be provided. The size of the polymeric nanoparticles loaded with the drug can be measured precisely with particle size analyzer. Since the particles with the appropriate size that is claimed to be higher than 10 nm and less than 100 nm, tend to accumulate especially in tumors due to the abnormal growth of capillary vessels in tumors.

The use of star block copolymers of the invention in selective cancer treatments as a drug carrier, to enable the controlled release of the drug comprises the following steps as described in a method (200);

- loading the star block copolymers with a hydrophobic drug using dialysis method (201),

- the drugs accumulating in the core of the star block copolymer due to higher hydrophobicity of the core (202),

- divalent cations that be used for crosslinking -COOH moieties being one of Ca +2 , Mg +2 , Cu +2 , Zn +2 , Fe +2 and such elements in form of metal complexes (203),

- freeze drying to obtain drug loaded star-block copolymers ready to form homogeneous aqueous solutions owing to the water soluble acrylic acid arms

(204), where the requirements of the material to be used as intravenous drug carrier for selective cancer treatments are as follows;

- the drug loaded molecules yielding a hydrodynamic radius less than 100 nm, in order to stay in circulatory system without being eliminated by macrophage system,

- the drug loaded molecules yielding a hydrodynamic radius greater than 10 nm and passing through the capillary vessels that exhibits abnormal growth due to phenomena being called angiogenesis, and accumulating selectively in tumor cells,

- star block copolymers staying in the circulatory system without degrading up to 24 hours to provide the time to accumulate in the tumors that function as a filter, which is provided due to surface modification of PAA shell (11) through crosslinking with divalent cations, metal complexes used for crosslinking -COOH moieties being one of or a mixture of oxides Ca(OH) 2 , Mg(OH) 2 , Cu(OH) 2 , Fe(OH) 2 , Zn(OH) 2 and such, or carbonates CaCO 3 , MgCO 3 , CuCO 3 , FeCO 3 , ZnCO 3 and such, nitrates Ca(NCO 3 ) 2 , Mg(NO 3 ) 2 , Cu(NO 3 ) 2 , Fe(NO 3 ) 2 , Zn(NO 3 ) 2 , and such, or sulfates CaSO 4 , MgSO 4 , CuSO 4 , FeSO 4 , ZnSO 4 , and such, or chlorides CaCl 2 , MgCl 2 , CuCl 2 , FeCl 2 , ZnCl 2 , and such, or perchlorates Ca(ClO 4 ) 2 , Mg(ClO 4 ) 2 , Cu(ClO 4 ) 2 , Fe(ClO 4 ) 2 , Zn(ClO 4 ) 2 , and such. Degree of crosslinking depends on concentration of divalent cation and can be tuned accordingly.

Schematic view of 201 st step: Loading the star block copolymers with a hydrophobic drug using dialysis method as follow:

Diagram of 202 th step: The drugs accumulating in the core of the star block copolymer due to higher hydrophobicity of the core

Diagram of 203 rd step: Divalent cations that be used for crosslinking -COOH moieties being one of or a mixture of Ca +2 , Mg +2 , Cu +2 , Zn +2 , Fe +2 complexes

X: Cu, Mg, Ca, Zn, Fe Multiarm poly(methyl metacrylate) core : Diagram-1: Schematic presentation of drug loaded and partially crosslinked (with Cu +2 ) 4-arm PMMA-b-PAA copolymer.

The star block copolymer of the invention has a high drug carrying performance with impressive drug loading capacity. The star block copolymer proposed as drug carrier for selective chemotherapy with hydrophobic anti-cancer drugs like cisplatin, doxorubycin, paclitaxel, 5-fluorouracyl, methotrexate, docetaxel, etc.

The star block copolymer, or multiarm block copolymer of the invention has a hydrophobic core (10) consisting of a multiarm methyl methacrylate oligomer and a hydrophilic PAA shell (11) consisting of poly(acrylic acid) PAA arms, the overall structure having amphiphilic character.

The star block copolymer, or multiarm block copolymer of the invention can have three, four, six or more arms each having amphiphilic character by consisting of hydrophobic methyl methacrylate oligomers and poly(acrylic acid) chains. The starting material for the multi-arm block copolymer of the invention can be a molecule with more than or equal to three hydroxyl groups such as glycerine, triethanolamine, trihydroxybenzene, trimethylolpropane, di(trimethylolpropane), pentaerythritol, dipentaerythritol, or another multi-hydroxyl-functional molecule.

The hydrophobic core (10) obtained from hydroxyl terminated starting material by ATRP reaction that consists of methyl methacrylate oligomer arms having a number equal to the number of hydroxyl groups of the starting molecule. This hydrophobic core (10) has a hydrodynamic diameter less than or equal to 5 nm which corresponds to a molecular weight less than or equal to 6000.

The hydrophobic PMMA core (10) of the invention remains undegraded after the degredation of partially crosslinked PAA arms (12) and release of the drug, but still can be eliminated from the body since particles less than 5 nm size can be filtered out by urinary system.

The hydrophilic PAA shell (11) is obtained by continuing ATRP reaction with tertiary butyl acrylate monomer and building tBA arms on MMA chain-ends and then converting tBA branches to acrylic acid by hydrolysis reaction.

Then star block copolymer is loaded with anti-cancer drug by dialysis method. Both drug and carrier are dissolved in appropriate solvents and then mixed in solution to enable the drug molecules penetrate into hydrophobic core (10). Then unloaded drug and impurities remaining from ATRP reaction are washed out by dialysis that has a MWCO (Molecular Weight Cut-Off) selected according to molecular weight of star block copolymer used as drug carrier.

After purification of drug loaded particles, the surface modification is achieved in aqueous solution. The hydrophilic PAA shell (11) can be partially crosslinked by divalent cations, each binding two -COOH groups together. This divalent cation can be Ca +2 , Mg +2 , Cu +2 , Zn +2 , Fe +2 or other. Each divalent cation can be introduced by dissolving or dispersing a metal complex in aquoeus medium, metal complex being a nitrate, sulfate, carbonate or hydroxide.

Preferred metal cation is Cu +2 , and preferred form of copper complex is Cu(OH) 2 in aqueous solution, buffer saline solution or salt water with reduced pH that has to be adjusted according to type of drug. Degree of crosslinking, and in turn biodegradability of the polymer, depends on;

-pH of the solution,

-concentration of metal complex solubilized in the medium,

-molecular weight of poly(acrylic acid) chains. This way, three advantages are achieved with drug carrying molecules that are surface modified by crosslinking with copper (Cu) or other divalent cation. The first is that the molecule shows affinity to the cancer cells. The studies of the recent years show that there is an extraordinarily high level of copper accumulation in cancer tumors. Another is that PAA molecules in the outermost part of the molecule that normally dissolve rapidly in water and degrade, degrade slower with the partial crosslinking. Degradation rate of the partially crosslinked PAA arms (12) varies according to the type of cation as well as degree of crosslinking. Thus, after partial crosslinking with one or a mixture of divalent cations, the start of the drug release can be delayed up to 24 hours. This provides the time needed for the drug carrying molecule targeted to cancerous region and reduce the effect of anti-cancer drug on healty cells.

EMBODIMENT 1 Three arm initiator is synthesized by bromination of trimethylolpropane, from 13.4 g trimethylolpropane dissolved in THF (Tetrahydrofuran). 10 ml 4-dimethyl amino pyridine solution in THF is to be used as ligand. 67 ml TEA (Triethyleneamine) and 60 ml 2-BIB (2-Bromo isobutyryl bromide) is added at 0°C and the vessel is mixed at room temperature during the reaction. The product of the reaction is purified by extraction and dried under vacuum to be used as starting molecule for the hydrophobic core. Three-arm brominated trimethylolpropane is then used as initiator for synthesis of three- arm hydrophobic core from methyl methacrylate monomer by Atom Transfer Radical Polymerization (ATRP). 0.726 g brominated trimethylol propane is reacted with 100 ml methyl methacrylate in anisole. 0.78 ml N,N,N’,N’,N-pentamethyl diethylenetriamine is used as ligand and 0.37 g CuCl 2 is added as catalyst. The mixture of reactants were degassed by freeze and thaw cycles and reacted at 70°C for 10 minutes. Molecular weight of the product is determined by Gel Permeation Chromatography to assure maximum 5000 g/mol molecular weight of the hydrophobic core. Reaction is terminated by rapid cooling and elimination of catalyst by passing through activated alumina column.

Purified three-arm PMMA is then dissolved in 326 ml of tertiary-butyl aciylate diluted with anisole. 7,76 ml N,N,N’,N’,N-pentamethyl diethylenetriamine and 0.54 g CuBr is added. The mixture is degassed to remove oxygen and reacted at 90°C for 4 to 12 hours depending on desired molecular weight, rate of mixing and the purity of reactants. Reaction is terminated by cooling and elimination of catalyst by passing the reaction solution from activated alumina column. The star block copolymer of poly(methyl methacrylate)-block-poly(r-butyl acrylate) is purified and dried prior to hydrolization reaction. Hydrolization of poly(methyl methacrylate)-block-poly(t-butyl acrylate) is achieved by addition of 20 ml trifluoroacetic acid solution in dichloromethane by mixing at room temperature for 24 hours. The final product of three-arm poly(methyl methacrylate)-block-poly(acrylic acid) copolymer is filtered, washed with deionized water and dried to be used for drug loading.

EMBODIMENT 2

Four arm initiator is synthesized by bromination of di(trimethylolpropane). from 18.4 g di(trimethylolpropane) dissolved in THF (Tetrahydrofuran). After 10 ml 4-dimethyl amino pyridine solution diluted in THF and 67 ml TEA (Triethyleneamine), 60 ml 2- BIB (2-Bromo isobutyiyl bromide) is added at 0°C. The reaction is carried at room temperature until all hydroxyl groups are replaced with bromine. The product of the reaction is purified by extraction and dried under vacuum to be used as starting molecule for polymerization.

Four-arm brominated initiator synthesized as described above is used for synthesis of four-arm hydrophobic core from methyl methacrylate monomer by Atom Transfer Radical Polymerization (ATRP). 0.795 g brominated di(trimethylolpropane) is reacted with 100 ml methyl methacrylate in anisole. 0.78 ml N,N,N’,N’,N-pentamethyl diethylenetriamine as ligand and 0.37 g CuCl 2 is added as catalyst. The mixture of reactants were degassed by freeze and thaw cycles and reacted at 70°C for about 12 minutes. Molecular weight of the product is determined by Gel Permeation Chromatography to assure maximum 6000 g/mol molecular weight of the hydrophobic core. Reaction is terminated by rapid cooling and elimination of catalyst by passing through activated alumina column.

Purified four-arm PMMA is also dissolved in 326 ml of tertiary-butyl acrylate diluted with anisole. 7.76 ml N,N,N’,N’,N-pentanethyldiethylenetriamine and 0,54 g CuBr is added. The mixture is degassed to remove oxygen and reacted as described in Embodiment 1. Hydrolysis of poly(methyl methacrylate)-block-poly(t-butyl acrylate) is implemented as in Embodiment 1. The final product of four-arm poly(methyl methacrylate)-block-poly(acrylic acid) copolymer is filtered, washed with deionized water and dried to be used for drug loading.

EMBODIMENT 3

Star block copolymer with three or four arms, produced as described in Embodiment 1 and Embodiment 2 respectively, is used for hydrophobic drug delivery with model drug, 5-Fluoruracil, for instance.

1 g star block copolymer with 4-arms and 30000 g/mol molecular weight is dissolved in 10 ml ethanol. The model drug, 5-Flurouracil is dissolved in 100-500 ml of 1% HCl solution. Polymer solution is added and drug loading in aqueous medium is performed for 2 hours by mixing at 15°C. The excess drug and acid is eliminated from drug loaded star block copolymers by dialysis method. The amount of drug dissolved in lading medium is 0.2 g for maximum drug loading yield (87%) and 1.0 g for achieving maximum loading capacity (20,4%).

At the end of loading process 0.045 g Cu(OH) 2 is dissolved in aqueous solution of drug loaded star block copolymers for a partial crosslinking of 4% of the acrylic acid moieties.

The solution is transferred into dialysis bag with appropriate molecular weight cut-off (1000 g/mol) and placed in deionized water at 4°C. Dialysis water is refreshed frequently for an efficient purification and the duration of process is decided by measuring the concentrations of impurities by UV-spectrophotometer. The content of dialysis bag is freeze-dried to obtain drug loaded particles.

Critical micelle concentration of the drug loaded multiarm poly(methyl methacrylate)- block-poly(acrylic acid) copolymer is determined as 10 ppm by using pyrene as florescent probe. The invention is not limited to the above exemplary embodiments, and a person skilled in the art can readily put forward embodiments of the invention. These are considered within the scope of the invention as claimed by the accompanying claims.