WO2012089768A1

WO2012089768A1 - System for the release of a therapeutic agent, pharmaceutical compositions containing it, the preparation and medical use thereof

Info

Publication number: WO2012089768A1
Application number: PCT/EP2011/074150
Authority: WO
Inventors: Laura VIVERO SANCHEZ; Judith SENDRA CUADAL; Hanna PARKKOLA; Joaquín QUEROL SASTRE; Marc Ramis Castelltort
Original assignee: Endor Nanotechnologies, S.L.
Priority date: 2010-12-28
Filing date: 2011-12-28
Publication date: 2012-07-05
Also published as: ES2392528B1; ES2392528A1

Abstract

The present invention describes a new system for the selective and controlled release of a therapeutic agent comprising: (i) a carrier comprising: a) a metal nanoparticle and b) a hydrophilic HA coating bound to the metal nanoparticle through at least a first ligand, and (ii) at least one therapeutic agent bound to the nanoparticle, to the HA or encapsulated in the HA coating. The invention also describes pharmaceutical compositions containing the system for release, their use in the treatment of diseases such as cancer and a method for obtaining them.

Description

SYSTEM FOR THE RELEASE OF A THERAPEUTIC AGENT. PHARMACEUTICAL COMPOSITIONS CONTAINING IT, THE PREPARATION AND MEDICAL USE

THEREOF

Field of the Invention

The present invention relates to a new system for the selective and controlled release of a therapeutic agent, comprising a metal nanoparticle, hyaluronic acid and a therapeutic agent. The invention also relates to compositions containing it and to the use of said system and said compositions in therapeutic treatments.

Background of the Invention

Therapeutic agents administered to the human or animal body are distributed throughout the organism according to their physicochemical properties. The therapeutic agents used today in general do not have specific properties for acting selectively on the selected cells targeted for treatment. These agents therefore randomly affect the cells of the organism, including both the selected cells targeted for application as well as different types of cells not targeted for medical application, causing unwanted side effects.

Today, the development of new systems for the controlled and selective release of therapeutic agents is a technological and scientific field in which there is a great deal of activity. The objective of a system for release is to make the desired amount of the therapeutic agent reach the selected cells to obtain a desired therapeutic effect, thereby minimizing the unwanted exposure of the rest of the organism to the agent. In order for the necessary amount to be the minimum amount possible and at the same time therapeutically effective, it is desirable for the system for release to improve the effectiveness of the therapeutic agent.

In particular, many therapeutic agents used today, for example in oncological treatments, are specific at the molecular level but not at the cellular level. Therefore, in some cases only a small fraction of the agent reaches the tumor, whereas the rest acts in other tissues of the organism or, in other cases, it is rapidly eliminated from the organism before it can produce any effect in the target tissues, or in contrast, it has a high circulation time and can act in other tissues of the organism causing unwanted effects.

A property that systems for the release of therapeutic agents must have is remaining the necessary time in the circulatory system to passively and with the highest probability possible access the target cells. However, if the system for release is not suitably protected, the phagocytic cells of the mononuclear phagocyte system (MPS) can rapidly eliminate it.

One of the most widely used techniques for protecting the system for release against the MPS is to coat it with a type of hydrophilic agent that provides it with an aqueous coating that protects it. The components making up the system for release, including the therapeutic agent, are protected and less susceptible to being internalized by the cells of the MPS, which results in a longer blood circulation time. An example of a widely used hydrophilic agent is the polyethylene glycol (PEG).

Furthermore, a system for the release of a therapeutic agent must exert a selective action on the target cells. In this sense, in order to direct the system for release to a target cell of the organism it is necessary to have molecules with an affinity for said target cell. Specifically, some monoclonal antibodies recognizing tumor markers are used in the case of tumors due to their capacity to be selectively bound to tumor cells (for example Herceptin (trastuzumab) is a widely used antibody in breast cancer). Binding the antibody that performs vector functions to the system for release can provide it with selectivity but it is still necessary to protect the system for release to prevent it from being internalized by cells of the MPS.

Therefore, in order for a system for release to be effective it is desirable for it to have this dual protection and selectivity functionality. Specifically, the system for release must be made up of a hydrophilic agent (for example, PEG) to remain in the blood for the necessary time and a vector molecule conferring selectivity (for example, a monoclonal antibody) and selectively releasing the agent on the target cells. In many cases, the hydrophilic agent and the vector molecule interfere in their functions such that both the hydrophilic capacity and the selectivity of the system for release are lost. This effect limits the application of these systems and in many cases it is necessary to choose between a high blood circulation time or optimal selectivity on the target cells.

One way to solve this problem is to have a system for release made up of a single molecule that performs the protection and selectivity functions. Said molecule must keep the system with the agent protected while it is circulating throughout the organism and until it is released in the target cells; it must reach the target cells in the shortest circulation time possible such that the system for release does not circulate more time than that necessary, causing unwanted side effects and its accumulation in other organs. Furthermore, said molecule must have optimal affinity and selectivity in order to be internalized into target cells such that it can perform vector functions.

Finally, the release of the therapeutic agent by the system for release is a decisive factor for determining the effectiveness of the system. The objective in this case is for the system to stably conserve the agent until reaching the target cell and once there, to release it without the molecular structure of the agent being altered.

Diverse strategies have been designed in the last decade for controlling the kinetics and release of agents. One of the most widespread strategies is the use of liposomes synthesized from biodegradable materials which store the therapeutic agent therein and, as they degrade, gradually release the agent. In order for a system for release made up of liposomes to be effective it must also include a hydrophilic agent protecting against the MPS on the surface of the liposome and a vector molecule which preferably directs the agent to the cells targeted for treatment. Such systems for release have several drawbacks such as the progressive uncontrolled degradation of the liposome in biological medium, making the agent be gradually released as the liposome degrades, compromising the arrival and the release of the agent in the target cell. Another factor to take into account in such systems is the particle size. In this sense, synthesized liposomes usually have a size comprised between hundreds of nanometers and microns which considerably reduce their capacity to be internalized at the cellular level and therefore to provide the intracellular release of the agent.

Both liposomes and other nano-structured materials (polymers, dendrimers, micelles, carbon nanotubes, etc.) are object of study as candidate systems for the release of agents for different medical applications.

An emergent alternative within nano-systems for release are inorganic nanoparticles (NP), general gold nanoparticles (Au-NP). Au-NPs are used today as a structure on which systems for the release of agents are designed (Chem Soc Rev. 2009 Jun; 38(6) :1759-82). The surface of the Au-NP is a very versatile platform to which a wide range of molecules can be bound by means of chemical bonds with stability similar to a covalent bond. By conjugating different molecules to the same NP, multifunctional conjugates are developed which can perform the function of a system for the selective release of agents.

Different studies using Au-NPs as systems for release are currently being conducted (Nanomed. 2009 Jun; 4(4):401 -10). The surface of the Au-NP is modified by binding hydrophilic agents, such as PEG for example (J Appl Toxicol. 2009 Nov 9) for protecting the Au-NP from the action of the MPS (Au-NP-Protector). Vector molecules, such as monoclonal antibodies, directing the Au-NP-Protector preferably to the target cells (Au-NP-Protector-Vector), are also bound on the surface of the

Au-NP-Protector. Finally, the surface of the Au-NP-Protector-Vector system is modified with a therapeutic agent (Au-NP-Protector-Vector-Agent). Therefore, to obtain the effects of protection and directionality and the medical effect of the system for release, it is necessary to conjugate at least three different types of molecules (Protector-Vector-Agent) to the surface of the Au-NP. This design of nano-systems has several drawbacks that reduce their efficacy as a system for the release of agents: it increases the likelihood that the molecules will interfere with one another and lose some of their properties; the space available of the agent on the surface of the Au-NP is reduced due to using three different types of molecules (Protector-Vector-Agent).

Therefore, in view of the foregoing there continues to be a need in the state of the art to provide new systems for selective and controlled release based on metal nanoparticles which overcome the aforementioned drawbacks at least in part and are capable of effectively exercising the aforementioned desirable functions in a system for release.

Brief Description of the Drawings

Figure 1 depicts a system for the release of the therapeutic agent cisplatin consisting of a metal nanoparticle to which hyaluronic acid (HA) is bound through a ligand molecule (type 1 ) and cisplatin through a ligand (type 2).

Figure 2 shows the results of the internalization of a carrier (EDS) having a gold nanoparticle and HA of 30-50 KDa and the viewing thereof within Panc-1 cells by means of electron microscopy (TEM) at 24 h.

Figure 3 shows the internalization of a carrier (EDS) consisting of a gold nanoparticle and HA by means of inductively coupled plasma source mass spectrometry (ICP-MS); the amount of intracellular gold (ng/100,000 cells) is observed.

Figure 4 shows images of the study of the internalization in Panc-1 cells of the carrier (EDS) (consisting of gold nanoparticle and HA 30-50 KDa) labeled with fluorophore by means of confocal microscopy, and the cores of the cells are observed in a); the cores and the EDS carrier around them are observed in b); and the cores and the CD44 target receptor of EDS are observed in c).

Figure 5 depicts the accumulation of gold (ppm) of an EDS carrier with a gold nanoparticle and HA of different sizes: (i) EDS (HA 30-50 KDa), (ii) EDS (HA 15-30 KDa), (iii) EDS (HA 8-15 KDa) and (iv) EDS (HA 5 KDa) in a tumor obtained from human colon tumor cells implanted in a murine model.

Figure 6 is a graph which shows the gold concentration in the blood of mice at different times of an EDS carrier with a gold nanoparticle and HA of different sizes: (i) EDS (HA 30-50 KDa), (ii) EDS (HA 15-30 KDa), (iii) EDS (HA 8-15 KDa) and (iv) EDS (HA 5 KDa).

Figure 7 comparatively shows the proportion between the amount of an EDS carrier (with a gold nanoparticle and HA 30-50 KDa) accumulated in the tumor and the amount in blood and the same proportion in the tumor with respect to blood for a carrier consisting of polyethylene glycol bound to a gold nanoparticle (PEG-Gold NP).

Figure 8 shows the UV-vis spectrum of the system for release of the invention EDS001 . The absorbance is represented on the y-axis and the wavelength (nm) is represented on the x-axis.

Figure 9 shows the electron microscopy (TEM) image of the system for release EDS001

Figure 10 shows the size distribution by dynamic light scattering (DLS) intensity of the system for release EDS001 ; the intensity (%) is represented on the y- axis and the size is represented on the x-axis.

Figure 1 1 shows the z-potential distribution of the system for release EDS001 where the total count (photons counted per second) is represented on the y-axis and the potential in mV is represented on the x-axis.

Figure 12 shows the results of an in vitro viability study for 72 hours (with human lung tumor cells) of a system for release EDS001 conducted using different concentrations thereof and measuring the activity of the enzyme hexosaminidase. The study was conducted in a case with a system consisting of nanoparticles having a size of 4 nm and in another case of nanoparticles having a size of 12 nm, HA between 30-50 kDa and cisplatin in both cases. The x-axis shows the percentage of treatment added to the cells.

Figure 13 shows the UV-vis spectrum of the system for release of the system of the invention EDS002 where the absorbance is represented on the y-axis and the wavelength (nm) is represented on the x-axis.

Figure 14 shows the electron microscopy (TEM) image of the system for release EDS002.

Figure 15 shows the size distribution by dynamic light scattering (DLS) intensity of the system for release EDS001 ; the intensity (%) is represented on the y- axis and the size is represented on the x-axis.

Figure 16: shows the zeta-potential distribution of the system for release EDS002 where the total count (photons counted per second) is represented on the y- axis and the potential in mV is represented on the x-axis.

Figure 17 shows the results of an in vitro viability study (with human lung tumor cells, where A549 is the name of the tumor line) of a system for release EDS002 conducted using different concentrations thereof and measuring the activity of the enzyme hexosaminidase; the study was conducted with a system consisting of nanoparticles having a size of 12 nm, HA between 30-50 kDa and encapsulated cisplatin. The y-axis represents the viability percentage and the x-axis represents the concentration (μΜ) of cisplatin in the treatment.

Description of the Invention

In one aspect, the present invention relates to a new system for the selective and controlled release of a therapeutic agent comprising:

(i) a carrier comprising :

a) a metal nanoparticle and

b) an HA coating bound to the metal nanoparticle through at least a first ligand, and

(ii) at least one therapeutic agent bound to the carrier according to one of the following alternatives:

1 ) bound to the metal nanoparticle by means of a second ligand;

2) bound to the HA coating by means of a third ligand, or

3) encapsulated in the HA coating,

with the proviso that when the therapeutic agent is bound by means of a second ligand to the metal nanoparticle said agent is different from a protein, a peptide or an inhibitor of HA hydrolysis.

As it is used herein, the term ligand relates to a molecule capable of forming at least two chemical bonds, thus binding at least two elements of the system for release to one another. As it is used herein, a first ligand (type 1 ) relates to a molecule forming at least one chemical bond with the HA and another chemical bond with the metal nanoparticle, therefore binding both elements. The first type of bond can be an amide, ester, ether type bond, and the second is through at least one thioether (-S-) functional group. As it is used in the present invention, second ligand (type 2) relates to a molecule forming at least one chemical bond with a therapeutic agent and another chemical bond with the metal nanoparticle. The second ligands (type 2) which are bound to the nanoparticle generally do so through at least one-S- functional group as do the first ligands (type 1 ). As it is used in the present invention, third ligand (type 3) relates to a molecule forming at least one chemical bond with a therapeutic agent and another chemical bond with the HA. The third ligands (type 3) generally bind to the HA by means of at least one ester, amide, or ether type bond, etc. The chemical bond between the type 2 ligand or the type 3 ligand and the therapeutic agent may vary depending in each case on their chemical structure.

The therapeutic agent can also be encapsulated in the HA coating. In that case the agent is bound to the coating by means of at least one chemical bond which can be of different types: covalent, hydrogen bridge, ionic, van der Waals force, etc. In this sense, cisplatin can be encapsulated in HA by means of the formation of an ion complex as disclosed in J Pharm Sci. 2008 Mar;97(3): 1268-76. Another example is the encapsulation of docetaxel in a hydrophilic mesh formed from HA as disclosed in Biomaterials. 2009 Oct;30(30):6076-85. When the agent is encapsulated in the HA coating, it is selectively released in the target cells when the HA chains are degraded by means of the action of selective enzymes (hyaluronidases located in the extracellular matrix or within the target cells) within the target cell.

The first, second and third ligands, also referred to as type 1 , type 2 and type 3 ligands, respectively, are different molecules.

Specifically, the nature and stability of type 2 ligand molecules which bind the agent to the metal nanoparticle or of the type 3 ligand which binds the agent to the HA, allow the agent to remain bound and not be gradually released from the system for release while moving throughout the organism and they favor said release taking place once the system for release has been internalized into the target cell, for example, by means of a pH change. Therefore, type 2 ligand or type 3 ligand molecules can have quite varied chemical structures variables depending, for example, on the type of therapeutic agent that is to be bound, and they can be readily designed by the person skilled in the art in each case, for releasing said agent selectively in the target cell. A particular case of a target site is the interior of cells expressing CD44. Another particular site is cells which overexpress CD44. The system for release of the present invention acts selectively on the cells in which the CD44 receptor is expressed or overexpressed due to the vector function carried out by HA. The system is then internalized by said cells and the agent is released in the target cell by means of different mechanisms, such as for example by means of the effect of the aforementioned pH change, or changes in the redox potential or in the concentration of specific enzymes, etc. The release takes place synchronously.

The choice for each particular embodiment of the type 2 ligand can be made by the person skilled in the art in a simple manner depending on the therapeutic agent that is selected and on the release mechanism that is chosen. The literature discloses cases of agents bound to gold nanoparticles by means of ligands (type 2) (Adv Drug Deliv Rev. 2008 Aug 17;60(1 1 ):1307-15). The mechanisms of action of these type 2 ligands can be classified into different types: (1 ) by photoregulation (J Am Chem Soc. 2009 Apr 29;131 (16):5728-9) (2) infrared radiation (J Mater Sci

Mater Med. 2009 Oct;20(10):2091 -103) (3) redox potential (Bioconjug Chem. 2008 Jul;19(7):1342-5) (4) enzymatic concentration (J Am Chem Soc. 2009 Jan 14;131 (1 ):66-8.) or (5) pH (J Biomed Mater Res A. 2008 Jun 1 ;85(3):787-96). The description of said mechanisms is included herein by reference. The type 2 ligands are commercially available compounds or they can be selected and prepared according to standard methods.

The choice in each particular case of the type 3 ligand can be made by the person skilled in the art in a simple manner depending on the therapeutic agent that is selected and on the release mechanism that is chosen. The literature discloses examples of agents bound to HA by means of type 3 ligands (Mol Pharm. 2008 Jul-

Aug;5(4):474-86). One example is the binding of paclitaxel to the carboxylate group of HA by means of a type 3 ligand (Biomacromolecules. 2000 Summer;1 (2):208-18). Another example is the binding of the amide group of doxorubicin to the carboxylate group of HA by means of a type 3 ligand (Biotechnol. Bioeng. 2008, 99, 442^454). The description of said ligands is included herein by reference.

The type 1 ligands are conventional molecules that are commercially available or can be obtained by means of synthesis methods known by a person skilled in the art. Some type 1 ligands as well as methods for binding them to HA are described in detail in patent application WO2009087254 the content of which is incorporated herein by reference. In a particular embodiment the ligand has formula H₂N-(CH₂)₂-SH.

The metal nanoparticle of the system for release of the invention can be a) a nanoparticle or b) a core-shell particle. In the context of the present invention the nanoparticle has a homogenous composition of one or more materials selected from the group consisting of Au, Ag, Pt, Co, Fe, oxides of Au, Ag, Pt, Co, Fe, Ti0₂ and their mixtures. The core-shell particle consists of at least two different parts: a core and a shell which can be independently made up of one or more of the same mentioned materials. The size of the metal nanoparticle may vary within a broad range. The size is typically comprised between 2 and 100 nm. In a particular embodiment the size is comprised between 4 and 12 nm. The metal nanoparticle can have any shape, without limitations. In a particular embodiment it has a shape selected from spherical, bar-shaped, cylindrical, tubular, cube-shaped, triangular and star-shaped.

In another particular embodiment the metal nanoparticle is a core-shell particle in which the core is of superparamagnetic Co and the shell is Au. In a preferred embodiment the metal nanoparticle is a gold nanoparticle, and more preferably of a size comprised between 5 and 30 nm, even more preferably between 4 and 12 nm.

The HA of the coating is made up of hyaluronic acid (HA) chains which can have the same or different molecular weights. Furthermore the chains can be cross- linked. The molecular weight of an HA chain can vary between broad ranges. In a particular embodiment said molecular weight is comprised between 0.5 KDa and a maximum molecular weight determined by the biological nature of HA. More particularly the molecular weight of the HA fragments that can be used for putting the invention into practice is comprised between 1 and 500 KDa, preferably between 5 and 50 KDa, and more preferably between 30 and 50 KDa. These fragments, hereinafter also referred to as HA oligomers, can be prepared for example by enzymatic hydrolysis of HA obtained from a natural source or they can be commercially acquired. The HA oligomers are derivatized with the type 1 ligands according to methods well known by a person skilled in the art. Alternatively the HA oligomers derivatized with a type 1 ligand can be formed in an also conventional manner by first derivatizing HA with a type 1 ligand, and then hydrolyzing the resulting product for example by means of enzymatic hydrolysis. Preparation methods are described in the literature, for example in application WO2009087254.

In the context of the present invention, a therapeutic agent relates to any compound or substance which is used for the treatment and/or prevention of a disease or condition or an unwanted physiological process of the human or animal body. More particularly, the agent of the present invention can be, among others, any chemical compound, pharmaceutical agent, drug, biological factor, fragment of a biological molecule, such as for example of an antibody, of a protein, of a lipid, of a nucleic acid or of a carbohydrate, nucleic acid, antibody, protein, lipid, nutrient, cofactor, nutraceutical, anesthesic, detection agent, or an agent having an effect on the body or any combination thereof. Several non-limiting examples of therapeutic agents which can be used according to the present invention are: biological factors including, for example, cytokines, growth factors, active macromolecule fragments, neurochemical compounds, cell communication molecules and hormones. Furthermore, it can use any pharmaceutical agent among those just mentioned, for example anti-inflammatory agents, antibodies, antibiotics, analgesics, angiogenic and antiangiogenic agents, COX-2 inhibitors, chemotherapeutic agents, immunotherapeutic agents, nucleic acid-based materials.

The system of the invention can include one or more different therapeutic agents. The treatment and/or the prevention of a specific disease or condition or unwanted physiological process of a specific area of a human or animal organism with more than one different therapeutic agent is thus possible.

In a particular embodiment the therapeutic agent is any antitumor agent used in the therapeutic treatment of cancer or of a tumor. Examples of said agents are: bevacizumab, G-CSF, cisplatin, RGD peptide, AFM, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin, busulfan, mannosulfan, treosulfan, ThioTEPA, cyclophosphamide, estramustine, uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, streptozotocin, dacarbazine, temozolomide, actinomicyn, bleomycin, mitomycin, plicamycin, aminopterin, methotrexate, pemetrexed, raltitrexed, cladribine, clofarabine, fludarabine, mercaptopurine, pentostatin, thioguanine, carmofur, cytarabine, decitabine, fluorouracil, floxuridine, gemcitabine, capecitabine, enocitabine, sapacitabine, camptothecin, topotecan, irinotecan, rubitecan, belotecan, etoposide, teniposide, mitoxantrone, pixantrone, daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, pirarubicin, zorubicin, amrubicin, vinblastine, vincristine, vinorelbine, vindesine, docetaxel, larotaxel, paclitaxel, ixabepilone, tiazofurin, pegfilgrastim, interferon B1 a, glatiramer, PEGinterferon alpha-2a, rituximab, transtuzumab, imatinib, cetuximab, erlotinib, bortezomib, anastrozole, bicalutamide, leuprolide, goserelin, leuprolide, letrozole, exemestane, triptorelin, fulvestrant, leuprolide and their mixtures. In a preferred embodiment said agent is selected from cisplatin, oxaliplatin, carboplatin, doxorubicin, paclitaxel and fluorouracil, more preferably cisplatin.

In a preferred embodiment the system for release of the present invention comprises a gold nanoparticle of a mean diameter size selected between 4 and 12 nm, an HA coating made up of oligomers having a mean molecular weight of between 30-50 KDa bound through a type 1 ligand to the gold nanoparticle and cisplatin as the agent bound to the nanoparticle through a type 2 ligand. In a more preferred embodiment, the type 1 ligand has the formula -NH-(CH₂)₂-S- and the type 2 ligand has the formula -S-(CH₂)₂-N[CH₂-COO^"]₂.

In another preferred embodiment the system for release of the present invention comprises a) a gold nanoparticle, more preferably of a diameter size between 5-30 nm, more preferably 12 nm, b) an HA coating consisting of oligomers having a mean molecular weight of between 30-50 KDa bound through at least one type 1 ligand and c) cisplatin encapsulated in the HA coating. In a more preferred embodiment the type 1 ligand has the formula -NH-(CH₂)₂-S-.

Another object of the present invention relates to a composition, hereinafter pharmaceutical composition of the invention, comprising at least one system for the selective and controlled release according to the invention and at least one pharmaceutically acceptable excipient. By way of illustration, the excipient can be for example one or several selected from fats, beeswax, semisolid or liquid polyols, natural or hydrogenated oils, etc.; water (for example, distilled water, particularly distilled water for injection, etc.), physiological saline, alcohol (for example, ethanol), glycerol, polyols, aqueous glucose solution, mannitol, vegetable oils, etc.; additives such as amplifying agent, disintegrant, binder, lubricant, wetting agent, stabilizer, emulsifier, dispersant, preservative, sweetener, coloring, flavoring agent, diluent, buffer substance, solvent or solubilizing agent, chemical product for achieving the storage effect, salt for modifying the osmotic pressure, coating agent or antioxidant and the like.

With respect to each preparation of the pharmaceutical composition and to each system for release of the invention, different forms of preparation can be selected for the administration through any possible administration route: oral, parenteral, intravenous, topical, buccal, nasal, rectal, etc. In this sense the excipients and their amounts can be readily selected by the person skilled in the art in each case. Illustrative examples of pharmaceutical preparations are tablets, sugar-coated tablets, capsules, granules or pellets, solutions, suspensions, syrups, and reconstitutable dry preparations, intramuscular, intravenous or subcutaneous injections, preparations for drip or intravenous infusion, etc.

The preparation can be prepared by a person having normal skill in the art according to standard pharmaceutical techniques such as those described in the Spanish or European Pharmacopoeias or similar texts.

In a particular embodiment the pharmaceutical composition of the invention comprises more than one different system for release, each one comprising a different therapeutic agent. The system for release of the invention can optionally be used in combination with any other agent useful for the treatment of a disease or condition in each case, preferably cancer. In this sense in a particular embodiment the pharmaceutical composition of the invention comprises in addition to at least one system for release of the invention, at least one free therapeutic agent, in the sense that it is not bound to a carrier such as the one of the present invention. Said therapeutic agent can be the same as or different from the one bound to the system of those defined above. In the case of the treatment of cancer the system for release of the invention can be administered in combination with radiotherapy. Radiation therapy itself means a normal method in the field of the treatment of cancer. For radiation therapy different radiations can be used, such as X rays, γ rays, neutron rays, electron beam, proton beam; and radiation sources. The system for release of the invention combined with radiation therapy can enhance the therapeutic effect of the agent released in the treatment of cancer.

In a preferred embodiment the pharmaceutical composition of the invention comprises an amount of the system for release of the invention capable of releasing a therapeutically effective amount of at least the therapeutic agent selected in each case. In a more preferred embodiment said therapeutic agent is an antitumor agent, more preferably one of those mentioned above.

The system for release of the invention and another therapeutic agent where appropriate can be administered in combination at different times or at the same time as separate preparations or as a single preparation. Therefore, the present invention must be interpreted such that it includes all the methods for the administration of the combination of the system for release of the invention and any other agent useful for the disease in each case at the same time or at different times, and such that it includes each and every one of the possible combinations of the systems for release of the invention with each and every one of the pharmaceutical agents useful for the treatment of the disease in each case. In a preferred embodiment said disease is cancer.

In an additional aspect the invention relates to the system for release of the present invention or to a pharmaceutical composition which comprises it for use in the treatment and/or prevention of a disease or condition. In a preferred embodiment said use is for the treatment of cancer. The system for release can be used in combination with another therapeutic agent as described above, preferably with another therapeutic agent the latter being an antitumor agent.

As it is used herein, the term "cancer" includes different sarcomas and carcinomas and it includes solid cancer and hematopoietic cancer. As it is used herein, solid cancer includes, for example, a brain tumor, cervicocerebral cancer, esophageal cancer, thyroid cancer, small cell cancer, non-small cell cancer, breast cancer, lung cancer, stomach cancer, gall bladder/bile duct cancer, liver cancer, pancreatic cancer, colon cancer, rectal cancer, ovarian cancer, choriocarcinoma, uterine cancer, cervical cancer, pelvic renal/ureter cancer, bladder cancer, prostate cancer, penile cancer, testicular cancer, fetal cancer, Wilms' tumor, skin cancer, malignant melanoma, neuroblastoma, osteosarcoma, Ewing's tumor, alveolar soft part sarcoma. On the other hand, hematopoietic cancer includes, for example, acute leukemia, chronic lymphatic leukemia, chronic myelocytic leukemia, polycythemia vera, malignant lymphoma, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma. Finally, the term "cancer" also includes the cancerous stem cells responsible for the recurrence and metastasis of tumors. As it is mentioned herein, the term "treatment of cancer" means that an anticarcinogenic agent is administered to a case of cancer to inhibit the growth of cancer cells where appropriate. Preferably, the treatment results in cancer growth regression, or reducing the size of a detectable cancer. More preferably, the treatment results in the complete disappearance of the cancer.

In another aspect the invention relates to a method for the treatment and/or prevention of a disease or condition in a patient in need of said treatment comprising the administration of a prophylactic or therapeutically effective amount of a system for the release of a therapeutic agent to a patient in need of said treatment. In a preferred embodiment said condition or disease is cancer. Prophylactic or therapeutically effective amount is understood as that amount sufficient for producing a benefit for a patient and inhibiting the growth of the cancer cells in the particular event that the disease is cancer. The treatment with the system for release of the invention can be optionally combined with another treatment with another therapeutic agent as mentioned above.

In the method according to the invention, the preferred therapeutic unit may vary according to, for example, the administration route of the system for release of the invention, the type of system for release used; the type, administration route and dosage of the other therapeutic agent, preferably an antitumor agent, used in combination; and the type of cells to be treated, the patient's condition, and the like. The optimal treatment under given conditions can be determined by a person skilled in the art. In the method according to the invention, the therapeutic unit for the system for release of the invention may vary according to, specifically, the type of system used, the type of therapeutic agent, the application frequency and the specific site to be treated, disease severity, patient age, doctor's diagnosis, or the like.

In another aspect the invention relates to a method of preparing the system for release of the invention. The method comprises preparing the metal nanoparticles which can be done using standard methods known by a person skilled in the art.

For preparing the system for release comprising the encapsulated therapeutic agent, an aqueous suspension of metal nanoparticles is prepared and the HA oligomers derivatized with a type 1 ligand, previously obtained as described above, are added and the therapeutic agent is selected. The resulting solution is typically incubated at room temperature or higher under stirring and in the dark. The system for release according to the invention is purified by means of ultrafiltration membranes (MWCO 100 KDa) for removing the excess reagents not conjugated to the nanoparticles.

For preparing the system for release comprising the agent bound to the carrier the method varies depending on whether the agent binds to the HA (i) or to the metal nanoparticle (ii).

In the first case an aqueous solution containing HA derivatized with a type 1 ligand previously obtained as described above is typically prepared. Then the agent binds with the HA by means of a type 3 ligand binding a functional group (-OH, - NHCOCH₃ or -COOH) of each HA oligomer and a functional group of the selected therapeutic agent. The resulting solution is then incubated at room temperature or higher under stirring and in the dark if necessary. Finally, the incubated solution is added to an aqueous solution of metal nanoparticles. The system for release according to invention is purified by means of ultrafiltration membranes (MWCO 100

KDa) for removing excess reagents not conjugated to the metal nanoparticles.

In the second case a type 2 ligand as explained above selected depending on the therapeutic agent, is acquired or prepared. The type 2 ligand is incorporated into a solution of metal nanoparticles and the resulting mixture is kept under stirring for a specific time in each case. The mixture is generally maintained at room temperature.

HA oligomers derivatized with a type 1 ligand and obtained as described above and the therapeutic agent are then added to the resulting mixture. The reaction is generally kept at room temperature under stirring for a time to be determined in each case, the reaction being stopped by reducing the temperature. The resulting system for release is purified by means of ultrafiltration membranes (MWCO 100 KDa) for removing the excess reagents.

The inventors of the present invention have verified the usefulness and the advantages of the carrier of the system for release of the invention in obtaining a system for the release of a therapeutic agent. In this sense in another aspect the invention relates to the use of a carrier comprising:

a) a metal nanoparticle and

b) an HA coating bound to the metal nanoparticle through a first ligand, in obtaining the system for the release of a therapeutic agent of the present invention.

The usefulness of the carrier and the advantages derived from using it in producing a system for the release of a therapeutic agent have been demonstrated in a series of studies conducted by the inventors. On one hand experiments which have determined the internalization of the carrier (EDS) by tumor cells using various techniques have been conducted. These studies (see Examples 1 .4 and Figures 2, 3 and 4) show the entry of the carrier consisting of a metal nanoparticle and HA in the tumor cells. Figure 3 particularly shows that the degree of internalization of the EDS carrier is greater in the CD44 positive cell line (Panc-1 ) than in HepG2. This evidence suggests that the internalization of the EDS carrier of the system of the invention is mediated by the specific interaction between HA and CD44. These studies have shown that the internalization varies depending on the size of the HA oligomer of the carrier, generally being greater when the EDS has HA oligomers between 30-50 KDa than when the oligomers are smaller in size, and being greater in Panc-1 cells than in HepG2 cells. Figure 3 furthermore shows a comparison of the degree of internalization of an EDS carrier of the system of the invention and of gold nanoparticles bound to polyethylene glycol (PEG) showing that the degree of internalization of the carrier of the system of the invention into the Panc-1 cells (15,000 ng/100,000 cells) is much greater than that of the internalization of Au-NP bound to PEG (2,000 ng/100,000 cells).

In Figure 4 the cores of the cells are observed in a); the cores and the EDS carrier around them are observed in b); and the cores and the CD44 target receptor of the EDS are observed in c).

The inventors have further studied the degree of accumulation of the EDS carrier of the system of the invention in a tumor obtained from human colon tumor cells implanted in a murine model (Example 1 .5). The results of the study are shown in Figure 5 where it is shown that the carrier penetrates into the tumor cells and that the greater accumulation surprisingly occurs for carriers having HA oligomers of a greater molecular weight, between 30-50 KDa.

In addition to penetrating into the cells a carrier must generally have a suitable blood elimination profile. It should be pointed out that the blood circulation time is also a key characteristic in the onset of unwanted side effects. Longer time in the blood entails a higher probability that the system for release reaches non-target tissues and the agent causes unwanted side effects. This is why the carrier must optimize the blood circulation time of the agent for favoring its accumulation in the tumor but preventing the agent from being longer than necessary in the blood to prevent side effects.

The inventors have conducted a study of the elimination of the carrier in blood at different times (Figure 6) showing that the carrier is eliminated over time. The results obtained show that those carriers having HA of 30-50 kDa are eliminated more slowly than carriers with other HA molecular weights, allowing the blood circulation of the system for the time sufficient to reach the target tissue. Nonetheless, the circulation time is less than that obtained with other hydrophilic agents which, due to the high circulation time, cause unwanted side effects.

On the other hand, the inventors have also studied the relationship between the amount of EDS carrier that accumulates in the tumor and the amount present in blood for determining exactly the amount of carrier that has been trapped within the tumor and is not circulating in the blood. Figure 7 shows the results of a comparative study between the EDS carrier with the gold nanoparticle and HA of between 30-50 KDa and gold nanoparticles bound to polyethylene glycol (PEG) showing that the relationship between the amount of carrier accumulated in the tumor with respect to the amount in blood is much greater in the case of the EDS of the system of the present invention. This data shows that there is a specific interaction between HA and CD44 which favors the selective accumulation of the EDS carrier in tumor cells which overexpress CD44.

Therefore and in view of the results explained, the usefulness of the EDS carrier in obtaining a system for the release of a therapeutic agent is demonstrated.

The inventors of the present invention have also conducted viability or cytotoxicity studies of the systems for release of the invention for both EDS001 (Example 2.4) and EDS002 (Example 3.2), showing its efficacy.

For the EDS001 study, the inventors used two different systems for release obtained as described in the examples (Example 2), differing from one another in the size of the gold nanoparticle, in this case 4 nm and 12 nm, respectively. Different dilutions (100, 25, 6.3 and 1 .6) of a 100% solution of the EDS001 system (solution obtained in Example 2) were used. The results of this in vitro study are shown in Figure 12 where the different dilutions to which the solutions of the EDS001 system obtained in Example 2 were subjected are depicted. The data show how the capacity of inducing apoptosis in lung tumor cells is greater the higher the concentration of the EDS001 system in the medium and the greater the size of the gold nanoparticle (12 nm) in this case as well.

In the second study the inventors used the system for release described in

Example 3. Solutions of the EDS002 system at different concentrations (using cisplatin as the standard agent) were used. The results of this in vitro study are shown in Figure 17 where it is observed how the toxicity of cisplatin (Cis) increases the higher its concentration, and how at the same concentration of free Cis as encapsulated Cis in the EDS002 system for release according to the invention, cisplatin produces greater toxicity in cells when it is transported by the EDS002 system for release than when it is free and is released into the medium unconjugated.

Therefore and in view of the explanation, it is demonstrated that the system for release of the present invention satisfactorily complies with the functional requirements necessary for a system to be effective, in addition to being simpler than other systems of the state of the art.

One of the main advantages of the system for release of the present invention resides in it high hydrophilic capacity due to the presence of the HA coating acting as a hydrophilic agent creating an aqueous layer around the system and protecting it against the action of the MPS (mononuclear phagocyte system). The hydrophilic capacity of HA is related to its molecular weight and to the capacity of the different HA oligomers of creating bonds with one another, a phenomenon known as cross-linking. Conjugation to the metal nanoparticle improves cross-linking between the different HA oligomers because they are bound to the metal nanoparticle and therefore cannot separate from one another. Furthermore, the arrangement of HA around the metal nanoparticle makes degradation thereof difficult due to the action of hyaluronidase. Therefore the HA conjugated to the metal nanoparticle is stable and hydrophilic and satisfactorily complies with the objective of protecting the system for release against the action of the MPS. The system for release thus remains in the circulatory system longer, which enables the system with the therapeutic agent to reach the target cells.

Another advantage of the system for release of the invention resides on its capacity of selectively transporting a therapeutic agent to a specific area of the organism, enhancing the activity thereof mostly in said area and at the same time reducing the side effects of the agent in the remaining areas of the organism. Said property is also based on HA, which in addition to protecting the system for release against the MPS as mentioned above, serves to selectively vectorize it to some proteins of the organism. A particular objective of the system of the release of the invention is to transport the agent to the CD44 receptor based on the affinity of HA (ligand) for said receptor. CD44 is a transmembrane glycoprotein involved in the adhesion between cells and different components of the extracellular matrix, including HA (Curr Pharm Des. 2009; 15(12):1309-17). Different studies have demonstrated that the CD44 receptor is present in epithelial, neuronal, hematopoietic cells, and also especially in carcinoma, melanoma, lymphoma, pancreatic, breast, colon, ovarian and lung cancer cells. In particular, certain tumors overexpress the levels of

CD44 (Semin Cancer Biol. 2008; 18(4):244-50). In fact, many tumors are characterized by the production and accumulation of HA around the CD44 receptor and neoplastic cells usually exhibit high affinity to HA. CD44 is also highly expressed in tumor stem cells (Proc Natl Acad Sci USA. 2003 Apr 1 ;100(7):3983-8). As a result, the system for release of the invention can be directed to any type of tissue or cells of the human or animal organism having CD44. It has further been seen that another very interesting property of the HA-CD44 affinity is that the CD44 transmembrane receptor is responsible for cellular internalization of HA (Matrix Biol. 2002; 21 (1 ):15-23). As described, HA is the molecule of the system for release of the invention responsible for internalization of the metal nanoparticles and of the therapeutic agent into cells expressing CD44.

The elimination of the system for release of the present invention is determined by the HA coating; once the agent is released intracellular^, the metal nanoparticle conjugated with HA oligomers, the ligand used for conjugating the HA to the nanoparticle and where appropriate the ligand or ligand fragment used for binding the therapeutic agent to the carrier, remains. HA is a compound endogenously generated by the human body which is present in the extracellular matrix of tissues and performs various functions in different biological processes of the organism. Its elimination is perfectly defined and carried out naturally by the organism due to the presence of endogenous HA which must be continuously eliminated. Therefore, the processes for removing the system for release of the organism will be similar to those followed by endogenous HA.

When the agent is encapsulated in the HA coating, it is released into in the target cells when the HA chains degrade by means of the selective action of enzymes

(hyaluronidases).

In summary, the system of the invention has a sole feature, which is the use of a single molecule (HA) with the dual functionality of (1 ) being responsible for protecting the agent against the MPS system, maintaining the agent in the blood for an optimal time (pharmacokinetics of the optimized agent); and (2) being responsible for releasing the agent selectively in target cells by means of the affinity of the ligand HA to the CD44 receptor. The stability of the HA-NP bond as well as the stability of the NP itself in biological medium assure that the system for release does not lose its HA coating the entire time it remains in the organism. In the same manner, the coating of the system for release with HA reduces the cytotoxicity of the compound in healthy tissues (Nucl Med Biol. 2009 Jul; 36(5):525-33).

Illustrative examples of the present invention provided for better understanding of the invention are presented below and they should not be considered as a limitation to the scope thereof. Examples

The techniques and apparatuses used for physicochemical characterization were: UV-vis absorption spectrum (with UV-2501 -PC spectrophotometer, UV-VIS Shimadzu), transmission electron microscopy (TEM) (JEOL 1010 at 80KW), dynamic light scattering (DLS) (Malvern Nano-Zetasizer), inductively coupled plasma source mass spectrometry (ICP-MS) (Perkin Elmer ELAN 6000) z-potential (Z-Pot) (Malvern Nano-Zetasizer) HPLC-MS and NMR.

Example 1 : Obtaining an EDS nanosvstem

1 .1 Synthesis of gold nanoparticles of 4 nm and 12 nm (Au-NP)

a) Synthesis of gold nanoparticles of 4 nm

An aqueous solution (200 mL) of sodium citrate (25 mM) and HAuCU (25 mM) was stirred at room temperature. 6 mL of an aqueous solution (100 mM) of cold sodium borohydride were then added. The reduction took place instantly and a colloidal solution of gold nanoparticles was formed, which experienced an indicative color change from yellow to deep red. Finally, the colloidal solution was concentrated

10 times.

b) Synthesis of gold nanoparticles of 12 nm

An aqueous solution (150 mL) of sodium citrate (2.2 mM) was heated to boiling under vigorous stirring. 1 mL of an aqueous solution (25 mM) of HAuCU was then added to the boiling solution. The reduction took place in approximately 3 minutes and a solution of gold nanoparticles was formed, which experienced an indicative color change of purplish to deep red. Finally the reactor was removed from the heat source and was left to cool to room temperature. Finally, the colloidal solution was concentrated 10 times.

1.2 Derivatization of hyaluronic acid (30-50 KDa)

0.57 g of cystamine dihydrochloride in 250 ml of a reaction buffer (which was prepared from 1.5 g of H₃B0₃ and 5.85 g of NaCI in 250 ml of water adjusting the pH to 8.5 with 1 M NaOH) were added to 1 g of HA of 30-50 KDa. The solution was adjusted to a temperature of 45^eC and 3.15 g of sodium cyanoborohydride were added. The reaction was carried out under controlled argon gas atmosphere and was left for 5 days. Finally, 12.5 g of dithiothreitol (DTT) were added and it was left for 1 hour.

Once the HA derivatized (thiolation), the product resulting from the HA-ligand bond (HA-SH) was purified by means of dialysis for extracting the excess DTT and cystamine dihydrochloride using membranes with a molecular weight cutoff of 3.5 KDa by standard protocol. The final dry HA-SH product was obtained and lyophilized. 1.3 Conjugation of Au-NP and 30-50 KDa HA-SH

Two different EDS nanosystems were obtained:

(i) an EDS made up of gold nanoparticles (Au-NP) of 4 nm obtained in

Example 1 .1 with derivatized hyaluronic acid HA-SH 30-50 KDa obtained according to Example 1 .2 and

(ii) an EDS made up of gold nanoparticles (Au-NP) of 12 nm obtained in Example 1 .1 with derivatized hyaluronic acid HA-SH 30-50 KDa obtained according to Example 1 .2.

To that end 5 mg/mL of HA-SH 30-50 KDa (Example 1 .2) were added to a colloidal solution of 5 mL of gold nanoparticles of 4 nm and 12 nm respectively (Example 1 .1 ) and the resulting mixture was maintained in each case for 30 minutes at room temperature. The purification was done by ultrafiltration membranes (MWCO 100 KDa) for removing the excess HA-SH 30-50 KDa not conjugated to the gold nanoparticles.

The EDS nanosystems obtained were characterized by means of UV-vis, transmission electron microscopy (TEM), and z-potential (Z-Pot) as described in patent application WO 2009087254.

1 .4 Study of CD44-EDS internalization

Different HA-SH sizes (5, 8-15, 15-30 and 30-50 KDa) bound to gold nanoparticles were analyzed for this study. For the analysis of cellular internalization of the EDS system three different techniques were performed:

1 .4.1 Study of EDS internalization by means of Transmission Electron Microscopy (TEM)

Human pancreatic tumor cells (Panc-1 ) were treated at a 30% concentration of the EDS obtained in Example 1 .3 using different HA-SH sizes in this study. After 24 hours, the cells were washed and fixed. They were then processed according to standard protocol for the viewing thereof by means of TEM (Figure 2).

1 .4.2 Study of EDS internalization by means of Inductively Coupled Plasma Source

Mass Spectrometry (ICP-MS)

Two different types of tumor cells were used for this analysis: some with a high expression of CD44 (Panc-1 ) and others with a low expression of CD44 (HepG2). Both types were treated at a 30% concentration of the EDS obtained in Example 1 .3 using different HA-SH sizes in this study. After 24 hours of incubation, the treatment supernatants were collected and the cells were washed with PBS-Tween 20 (0.1 %) for removing the non-internalized EDS adhered to the cell membrane. The cells were then collected with PBS. All the samples were processed according to standard protocol for quantification by ICP-MS (Figure 3), and the results were normalized by the number of cells present.

1.4.3 Study of EDS internalization by means of Confocal Microscopy

A suitable fluorophore was adhered to the EDS system for this analysis (Biomaterials. 2008 Dec; 29(35):4709-18): EDS-Hylite. Pancreatic tumor cells (Panc- 1 ) were treated at a concentration of 30% of the EDS-Hylite obtained in Example 1 .3 using different HA-SH sizes in this study. After 24 hours of incubation, the cells were washed with PBS, fixed with 10% formalin for 15 minutes and the non-specific interaction of the antibodies was blocked with 1 % PBS-BSA. The level of CD44 was analyzed by means of incubation with anti-CD44 (Cell Signaling, 156-3C1 1 ) and FITC-labeled anti-mouse (antibodies against mouse immunoglobulins labeled with the fluorescent marker FITC (fluorescein isothiocyanate)) (Sigma, F9384). The cell cores were stained with 4',6-diamidino-2-phenylindole (DAPI). The internalization of EDS- Hylite and CD44 were viewed by means of confocal microscopy (Figure 4).

1.5 ln-vivo study of gold nanoparticles - HA

The study was conducted in an animal model using mouse strains. Human colon tumor cells (HCT-1 16) were subcutaneously implanted in the animal's back and were left to grow until forming a tumor of a specific size (at least 5 mm). Once the tumor with a sufficient size was formed, a solution of EDS with a gold concentration of 10 mg/Kg (with different HA-SH sizes) was intravenously injected in each mouse. After 24 hours of treatment, the mice were sacrificed following established protocols and the gold concentration in the tumors was studied (Figure 5). The gold concentration in the blood at different times and the tumor/blood gold ratio were analyzed (Figure 6).

Example 2. Obtaining a system for release: EDS001

A system for controlled release according to the invention made up of Au-NP obtained in Example 1 .1 , HA-SH 30-50 KDa obtained according to Example 1 .2, and the therapeutic agent cisplatin bound by means of a type 2 ligand (L2) to the Au-NP was obtained. For that purpose the following steps were performed:

2.1 Synthesis of Ligand L2 L2 of formula [CH2-COO-]2-N-(CH2)2-S-S-(CH2)2-N-[CH2-COO-]2 was obtained from a solution of cystamine dihydrochloride of formula CINH₃-(CH₂)₂-S-S- (CH₂)₂ -NH₃CI (2,25 g) in 200 ml of ethanol and 20 ml of triethylamine, to which ethyl bromoacetate (6.6 ml) and potassium iodide (1 .04 mg) were added. After 6 hours of stirring at room temperature, the resulting insoluble solid was filtered. The filtrate was rotoevapo rated and the intermediate product of formula [CH₂-COOEt]₂-N- (CH₂)₂-S-S-(CH₂)₂-N- [CH₂-COOEt]₂ was obtained, which was purified by flash chromatography (1 :1 CH₂CI₂: AcEt) and was characterized by means of NMR (CDCI₃, 400 MHz).

The product was then dissolved in methanol (MeOH) (10 ml) and NaOH (5 ml) under stirring over night, H₂0 (5 ml) was added and it was acidified to pH 3 with 1 M HCI. Finally, it was left at 4^eC and a precipitate corresponding to the product L2 was formed, which was filtered, purified with EtOH/H₂0 and characterized by means of NMR (DMSO, 400 MHz).

2.3 Obtaining the EDS001 system according to present invention by conjugation with gold nanoparticles, HA-SH and cisplatin bound to the gold nanoparticles by means of a ligand L2

The gold nanoparticles (of 4 nm and 12 nm) obtained according to Example 1 .1 were conjugated with the HA-SH oligomers (30-50 KDa) obtained in Example 1 .2, with the ligand L2 obtained in Example 1 .3 and with cisplatin (Cis) as the therapeutic agent. To that end:

The ligand L2 (392 μΙ of a stock solution of 2 mg/ml) was added to a colloidal solution of gold nanoparticles (5 ml of a 31 .1 nM solution of nanoparticles), the pH was adjusted to 1 1 and the reaction was carried out at room temperature and under stirring for 30 minutes. It was specifically carried out by means of the initial addition of

0.41 mM of L2 to a colloidal solution of 5 mL of gold nanoparticles of 4 nm and to a solution of gold nanoparticles of 12 nm in two different embodiments for 30 minutes at room temperature in each case.

Next, HA-SH 30-50 KDa (Example 1 .2) and Cis were simultaneously added in each case at different proportions and concentrations (for example, 620 μΙ of a 2 mg/ml solution = 0.83 mM) of Cis in 1 ml of solution (2.5 mg/ml of HA-SH 30-50KDa =12.5 mM). Each reaction was carried out at room temperature for 12 hours. The purification (and adjustment of pH to 7) was done by ultrafiltration membranes (MWCO 100 KDa) for removing the excess L2, HA-SH and Cis not conjugated to the gold nanoparticles. Finally, EDS001 (system shown schematically in Figure 1 ) was purified by means of ultrafiltration membranes and re-dissolved in biological medium at pH 7.

The resulting EDS001 system was characterized by means of UV-vis (Figure 8), transmission electron microscopy (TEM) (Figure 9), dynamic light scattering (DLS) (Figure 10), inductively coupled plasma source mass spectrometry (ICP-MS) and z- potential (Z-Pot) (Figure 1 1 ). The ICP-MS results (μς/g) were as follows:

Pt Au

11.73 281.5

2.4 EDS001 cellular viability study

Lung tumor cells (A549) were used for the viability study of the EDS001 system of the invention (with gold nanoparticles of 4 nm and 12 nm). The cells were seeded in 96-well plates in complete medium for 24 hours. The treatment was then added.

Complete medium was used as a negative control and complete medium with 1% Tween 20 as a positive control for this study. To study the cellular effect of EDS001 , the stock solution obtained in Example 2.3 was used, from which three different dilutions (1 :4, 1 :16 and 1 :64) were made. This treatment was maintained for 72 hours after which time the cells were processed for the study of hexosaminidase activity. To that end, the treatment medium which contained the cells was removed and 60 μΙ_ of substrate solution (7.5 mM of p-nitrophenol-N-acetyl-beta-D- glucosaminide and 0.1 M of sodium citrate at pH 5.0 at 50% in a 0.5% solution of Triton X-100 in water) were added in each well. After 3 hours of incubation, 90 μΙ_ of developing solution (50 mM of glycine at pH 10.4 and 5 mM EDTA) were added in each well. The absorbance of each sample-well was then measured at 410 nm (Figure 12).

Example 3. Obtaining a system for release: EDS002

A system for controlled release according to invention made up of Au-NP obtained in Example 1 .1 of 4 and 12 nm in size, HA-SH 30-50 KDa obtained according to Example 1 .2, and the encapsulated therapeutic agent cisplatin was obtained. To that end the following steps were carried out:

3.1 Conjugation of gold nanoparticles, HA-SH and cisplatin (EDS002) 77.5 mg of HA-SH 30-50 KDa and then cisplatin (150 μΙ of a stock solution of 2 mg/ml) were added in approximately 3 ml of aqueous solution. The resulting solution was incubated overnight at 40±5 ^eC under stirring and in the dark. 51 .7 ml of a solution of gold nanoparticles at a concentration of 9.33 nM were then added and the resulting mixture was maintained under stirring for 30 minutes. The system for release EDS002 was purified by means of ultrafiltration membranes (MWCO 100 KDa) for removing the excess HA-SH and Cis not conjugated to the gold nanoparticles.

The EDS002 system was characterized by means of UV-VIS (Figure 13), transmission electron microscopy (TEM) (Figure 14), dynamic light scattering (DLS) (Figure 15), inductively coupled plasma source mass spectrometry (ICP-MS) and z- potential (Z-Pot) (Figure 16). The ICP-MS results (μς/g) were as follows:

Pt Au

23.1 770

3.2 EDS002 cellular viability study

Lung tumor cells (A549) were used for the viability study of the EDS002 system (with gold nanoparticles of 12 nm). The cells were seeded in 96-well plates in complete medium for 24 hours. The treatment was then added.

Complete medium was used as a negative control and complete medium with 1% Tween 20 as a positive control for this study. To study the cellular effect of the EDS002, the stock solution obtained in Example 3.2 was used, from which four different dilutions (1 :1 , 1 :4, 1 :16 and 1 :64) were oberserved. This treatment was maintained for 72 hours after which time the cells were processed for the study of hexosaminidase activity. To that end, the treatment medium which contained the cells was removed and 60 μΙ_ of substrate solution (7.5 mM of p-nitrophenol-N-acetyl-beta- D-glucosaminide and 0.1 M of sodium citrate at pH 5.0 at 50% in a 0.5% solution of Triton X-100 in water) were added. After 3 hours of incubation, 90 μΙ_ of developing solution (50 mM of glycine at pH 10.4 and 5 mM EDTA) were added in each well. The absorbance of each sample-well was then measured at 410 nm (Figure 17).

Claims

1 . - System for the selective and controlled release of a therapeutic agent comprising:

(i) a carrier comprising :

a) a metal nanoparticle and

1 ) bound to the metal nanoparticle by means of a second ligand;

2) bound to the HA coating by means of a third ligand, or

3) encapsulated in the HA coating,

2. - System for the release of a therapeutic agent according to claim 1 , wherein the metal nanoparticle is selected from the group consisting of nanoparticles and core-shell particles, and wherein the nanoparticle, the core and the shell can be independently made up of a material selected from the group consisting of the metals

Au, Ag, Pt, Co, Fe, their oxides, Ti0₂ and their mixtures.

3. - System for the release of a therapeutic agent according to claim 2, wherein the metal nanoparticle is a core-shell particle in which the core is superparamagnetic Co and the shell is Au.

4.- System for the release of a therapeutic agent according to claim 2, wherein the metal nanoparticle is a gold nanoparticle, preferably of a size comprised between 5 and 30 nm, more preferably between 4 and 12 nm.

5. - System for the release of a therapeutic agent according to any one of claims 1 to 4, wherein the HA coating is made up of HA oligomers having a molecular weight comprised between 1 and 500 KDa, preferably between 5 and 50 KDa, and more preferably between 30 and 50 KDa.

6. - System for the release of a therapeutic agent according to any one of the preceding claims, wherein the therapeutic agent is selected from the group made up of chemical compounds, pharmaceutical agents, drugs, biological factors, fragments of antibodies, proteins, lipids, nucleic acids and carbohydrates, nucleic acids, antibodies, proteins, lipids, nutrients, cofactors, nutraceuticals, anesthesics, detection agents or a combination thereof.

7. - System for the release of a therapeutic agent according to claim 6, wherein the therapeutic agent is a pharmaceutical agent selected from the group consisting of anti-inflammatory agents, antibodies, antibiotics, analgesics, angiogenic and antiangiogenic agents, COX-2 inhibitors, chemotherapeutic agents, immunotherapeutic agents, nucleic acid-based materials and their combinations.

8. - System for the release of a therapeutic agent according to any one of claims 1 to 5, wherein the therapeutic agent is selected from the group made up of cytokines, growth factors, active macromolecule fragments, neurochemical compounds, cell communication molecules, hormones and mixtures.

9. - System for the release of a therapeutic agent according to any one of claims 6 to 8, wherein the therapeutic agent is an antitumor agent.

10.- System for release according to claim 9, wherein said antitumor agent is selected from the group consisting of bevacizumab, G-CSF, cisplatin, RGD peptide, AFM, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin, busulfan, mannosulfan, treosulfan, ThioTEPA, cyclophosphamide, estramustine, uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, streptozotocin, dacarbazine, temozolomide, actinomicyn, bleomycin, mitomycin, plicamycin, aminopterin, methotrexate, pemetrexed, raltitrexed, cladribine, clofarabine, fludarabine, mercaptopurine, pentostatin, thioguanine, carmofur, cytarabine, decitabine, fluorouracil, floxuridine, gemcitabine, capecitabine, enocitabine, sapacitabine, camptothecin, topotecan, irinotecan, rubitecan, belotecan, etoposide, teniposide, mitoxantrone, pixantrone, daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, pirarubicin, zorubicin, amrubicin, vinblastine, vincristine, vinorelbine, vindesine, docetaxel, larotaxel, paclitaxel, ixabepilone, tiazofurin, pegfilgrastim, interferon B1 a, glatiramer, PEGinterferon alpha-2a, rituximab, transtuzumab, imatinib, cetuximab, eriotinib, bortezomib, anastrozole, bicalutamide, leuprolide, goserelin, leuprolide, letrozole, exemestane, triptorelin, fulvestrant, leuprolide and their mixtures.

1 1 .- System for release according to claim 10, wherein said antitumor agent is selected from the group consisting of cisplatin, oxaplatin, carboplatin, doxorubicin, paclitaxel and fluorouracil, more preferably cisplatin.

12. - System for release according to any one of the preceding claims comprising a gold nanoparticle of a mean diameter size selected between 4 and 12 nm, an HA coating made up of oligomers having a mean molecular weight of between 30-50 KDa bound through at least a first ligand to the gold nanoparticle and cisplatin bound to the gold nanoparticle through a second ligand.

13. - System for release according to any one of claims 1 to 1 1 , comprising a gold nanoparticle of a mean diameter size of 12 nm, an HA coating made up of oligomers having a mean molecular weight of between 30-50 KDa bound through a first ligand to the gold nanoparticle and cisplatin encapsulated in the HA coating.

14. Therapeutic pharmaceutical composition comprising at least one system for release according to any one of the preceding claims.

15. - System for the release of a therapeutic agent according to any one of claims 1 to 13 for use in medical or preventive treatment.

16. - System for the release of a therapeutic agent according to claim 15 for use in the treatment of cancer.

17. - Use of a carrier comprising:

a) a metal nanoparticle and

b) a hydrophilic HA coating bound to the metal nanoparticle through at least a first ligand for obtaining the system for release according to any one of claims 1 to 13.