ES2386712A1 - DEVELOPMENT AND USE OF POLYMER NANOPARTICLES THAT INCLUDE POLI (EPSILÓN-CAPROLACTONA) AND DOXORRUBICINA. - Google Patents

Abstract

Development and use of polymeric nanoparticles comprising poly ({ep} -caprolactone) and doxorubicin. # The present invention relates to polymeric nanoparticles with an average size of less than 200 nm, comprising one or more biodegradable polymers, one or more active principles and at least one surfactant, where at least one biodegradable polymer is poly ({ep} -caprolactone) and where at least one active ingredient is doxorubicin, and when using said nanoparticles for the preparation of medicaments for the treatment of cancer, preferably cancer of breast

Description

DEVELOPMENT AND USE OF POLLMERIC NANOPARTICLES THAT UNDERSTAND POLL (c-CAPROLACTONA) and DOXORRUBICINE

The present invention relates to polymeric nanoparticles with an average size of less than 200 nm, comprising one or more biodegradable polymers, one or more active ingredients and at least one surfactant, where at least one biodegradable polymer is poly (E-caprolactone) and where at least one active ingredient is doxorubicin, and the use of said nanoparticles for the preparation of medicaments for the treatment of cancer, preferably breast cancer. Therefore, the present invention is encompassed in the field of pharmaceutical art.

STATE OF THE TECHNIQUE

Doxorubicin is an anticancer drug belonging to the group of nucleoside analogues, whose antitumor efficacy has been widely demonstrated in the treatment of a wide variety of solid tumors. However, despite its great therapeutic efficacy, this chemotherapeutic agent has numerous drawbacks that seriously condition a wide clinical use. Specifically, the development of resistance by cancer cells, and a very poor pharmacokinetic profile (very short plasma half-life, due to its rapid metabolism). All this determines that large doses are necessary to obtain an adequate therapeutic effect, which is associated with the occurrence of severe adverse effects (Grosse PY, Bresolle F, Rouanet P, Joulia JM, Pinguet F. Int. J. Pharm. 180 (1999 ) 215).

The enormous need to find new therapeutic strategies for the treatment of cancer has determined the development of biodegradable colloids for the selective transport of drugs to tumor tissue.

For example, N. Chiannilkulchai et al. (Ooxorubicin-Loaded Nanoparticles: Increased Efficiency in Murine Hepatíc Metastases. Sel. Cancer Ther. 5 (1989) 1) develop poly (isohexylcyanoacrylate) particles of a size of 250 nm, whereby said nanoparticles may have problems in extravasation in the tumor mass (see Maeda et al., Eur. J. Pharm. Biopharm. 71 (2008) 409; And Decuzzi et al., Pharm. Res. 26 (2009) 235). In addition, the enormous toxicity of poly (alkylcyanoacrylates) in vivo has been described in the literature. This work does not include any type of in vitro preclinical study conducted on cell cultures and the only data provided in relation to the association of the nanoparticle-doxorubicin is that of the modification of the number of metastases that the cells can cause but not with the effect of this compound on the tumor cell itself even with the volume of the primary tumor mass.

On the other hand, Loredana Serpe (Conventional Chemotherapeutic Orug Nanoparticles for Cancer Treatment. Nanotechnologies for the Life Sciences, vol. 6, Nanomaterials for Cancer Therapy. 2006 Wiley-Vch Verlay GmbH and Co. KGaA, Weinheim) describes the use of nanoparticles of: copolymers of poly (D, Llactide) and polyethylene glycol (PEG); copolymers of PEG and poly (isobutyl-2-cyanoacrylate); and methoxyPEG and hexadecylcyanoacrylate copolymers. This document refers to the use of caprolactone as a drug transporter. However, the biodegradable polymer has been tested with tamoxifen and not with doxorubicin (see reference Talk and Amiji.

Cellular uptake and concentrates of tamoxifen upon administration in poly (epsilon-caprolactone) nanoparticles, AAPS Pharm. Sci. 5 (2003) E3; And also the reference Shenoy and Amiji. Poly (e thylen e oxide) -modified poly (epsilon-caprolactone) nanoparticles for targeted delívery of tamoxifen in breast cancer, Int. J. Pharm. 293 (2005) 261).

Other works that describe polymeric nanoparticles are the article entitled

Toxicological studies of doxorubicin bound to polysorbate 80-coated poly (butyl

cyanoacrylate) nanoparticles in healthy rats and rats with intracranial glioblastoma (Toxicol. Lett. 126 (2002) 131), where poly (butylcyanoacrylate) nanoparticles coated with polysorbate 80 and loaded with doxorubicin are described. Its average size is 270 ± 30 nm. In the article entitled In vitro and in vivo anti-tumor activities of nanoparticles based on doxorubicin-PLGA conjugates (J. Control. Release 68 (2000) 419) polyparticles of poly (D, L-lactide-co-glycolide) are described (PLGA) loaded with doxorubicin, its average size being 200 nm. Negative preclinical results with stealth nanospheres-encapsulated doxorubicin in an orthotopic murine brain tumor model (J. Control. Release 100 (2004) 29) describe nanoparticles of poly (hexylcyanoacrylate) coated with PEG and loaded with doxorubicin. They have a loading capacity (drug loading) of doxorubicin between only 5.7 and 9.4%. In addition, its average size is between 150 and 200 nm. Due to the average sizes, these nanoparticles have the same drawbacks as those described by N. Chiannilkulchai et al.

Proceedings of the 3rd International Conference on the Oevelopment of BME (Vietnam, 11-14 January 2010, 158-161) describes a work entitled Ooxorubicin Oelívery by Copolymeric Nanoparticle for Treatment of Breast Cancer, which describes a complex methodology for preparing a copolymer of methoxy-PEG and poly (E-caprolactone). Specifically, in the case of this work it is necessary to neutralize doxorubicin hydrochloride with triethylamine in order to incorporate it into this copolymer. This forces, once the synthesis is finished, to eliminate triethylamine by dialysis for 24 hours.

In the work entitled Folate-decorated poly (lactide-co-glycolide) -vitamin E TPGS nanoparticles for targeted drug delívery (Biomaterials 28 (2007) 1889) describes nanoparticles of PLGA loaded with doxorubicin and whose surface has been modified with folate remains and of vitamin E. In addition to being a complex synthesis, the doxorubicin vehiculization obtained by this strategy is low and the drug load is very low having a load never exceeding 3.97% (in the case of this polymer). Finally, particle size (::::: 360 nm) would result in the same problems as described in N. Chiannilkulchai et al.

The nanoparticles described in the work entitled Nano-Sízed Mícel / es of Block Gopolymers of Methoxy Poly (ethylene glycol) -PoIY (E-caprolactone) -Graft-2Hydroxyethyl Gel / ulose for Ooxorubicin Oelívery (J. Nanosci. Nanotechnol. 8 (2008 ) 2362) are composed of a copolymer of methoxy-PEG, poly (Ecaprolactone) and hydroxyethylcellulose loaded with doxorubicin. Its average size is much larger (340.7 nm).

Doxorubicin associated with nanoparticles has also been studied to improve its biological characteristics in tumor-like cells by Shuaia et al. (Mícel / ar carríers based on block copolymers of POIY (E-caprolactone) and poly (ethylene glycol) for doxorubícín delívery (J. Controlled Rel. 98 (2004) 415)). This study is conducted in the MCF-7 line of breast cancer. The results of the authors demonstrate that through the use of the copolymer poly (E-caprolactone) and PEG there is an increase in the entry of doxorubicin inside the tumor cell. However, Shuaia et al. indicates that most of the drug is located in the cytoplasm. According to these authors, the inhibitory dose (IC5o) of the nanoparticle bound doxorubin (0.033-0.048 11M) is higher than that determined for free doxorubicin (0.001 11M).

Cuong et al. use poly (phospho-caprolactone) polyphosphoester copolymers associated with doxorubicin for the treatment of tumor cells (Ooxorubicin-Loaded Nanosízed Mícel / es of a Star-Shaped Poly (varepsilon-Gaprolactone) -Polyphosphoester Block Bre-polymer Gan Treatment for Treatment Gan-polymer for Treatment J. Biomater. Sci. Polym. Ed., 2010, doi: 10.1163 / 09205061 OX51 0533). In this same situation are studies conducted with PEG-b-poly (2-hydroxyethylmethacrylate-g-poly (Ecaprolactone) (Zhang et al. Amphiphilic Toothbrushlike Copolymers Based on Poly (ethylene glycol) and POIY (E-caprolactone) as Drug Carriers with Enhanced Properties (Biomacromolecules 11 (2010) 1331) as they continue to use copolymers for tests with doxorubicin and in urinary bladder carcinoma cells and not in ovarian carcinoma cells. Maximum accumulation of doxorubicin in the cell nucleus in culture after treatment with the nanoparticle-drug complex occurs at 24 hours, measured by fluorocytometry.In addition, as indicated by the author, the concentration of doxorubicin bound to the nanoparticles with which the IC50 is reached was relatively higher than the dose of free doxorubicin with which the same effect is achieved.

DESCRIPTION OF THE INVENTION

The present invention relates to polymeric nanoparticles comprising poly (E-caprolactone) and doxorubicin, hereinafter the nanoparticles of the invention. The present inventors have discovered that surprisingly the nanoparticles of the invention behave as an excellent doxorubicin transport system. The nanoparticles of the invention allow a high vehiculization of the anticancer agent, achieve a very high extravasation in the tumor mass and, in turn, a more controlled release of it. In addition, studies of in vitro antitumor efficacy have demonstrated a very significant increase in the antitumor activity of this drug when it is vehiculized through the nanoparticles of the invention. One of the reasons for the effectiveness of nanoparticles against cancer is the large amount of real drug (in this case, doxorubicin) that is incorporated into the tumor cell interior once the drug is administered, a determination that is quantitative thanks to the application of fluorocytometry technique. In addition, the results demonstrate an easy exchange of drugs towards the nucleus where this agent has its essential action. Another advantage associated with the invention is that a

controlled and prolonged release of the active substance, reducing the effects

Side effects associated with treatment.

In addition, the nanoparticles of the invention modulate IC5o, that is the concentration of drug that is capable of reducing the number of tumor cells by 50% when applied to a cell culture, of doxorubicin in breast cancer cells and help the incorporation of this agent into the tumor cell. These results are advantageous compared to other similar systems, allowing to reduce the amount necessary to reach the IC50 in the MCF-7 line up to more than 5 times in some embodiments. Even the nanoparticles of the invention allow a high loading capacity of doxorubicin (drug loadíng) of up to in some cases 18.7 ± 0.9%.

Therefore, a first aspect of the invention relates to a polymeric nanoparticle with an average size of less than 200 nm, preferably less than 190 nm, comprising one or more biodegradable polymers, one or more active ingredients and at least one surfactant, where at least one biodegradable polymer is poly (E-caprolactone) and where at least one active ingredient is doxorubicin.

A second aspect of the invention relates to a process for the synthesis of a polymeric nanoparticle with an average size of less than 200 nm, preferably less than 190 nm, comprising one or more biodegradable polymers, one or more active ingredients and at least one surfactant, where at least one biodegradable polymer is poly (E-caprolactone) and where at least one active ingredient is doxorubicin. The polymeric nanoparticles of the invention are preferably synthesized by the interfacial arrangement method of the polymer, consisting of:

i) dissolving poly (E-caprolactone) in an organic solvent,

preferably dichloromethane; ii) the addition of the solution obtained in (i) on a mixture comprising an anti-solvent and a surfactant; iii) the isolation of the obtained particle; where the active substance doxorubicin is incorporated by association surface on the nanoparticle obtained after step (iii) or by association within the polymer matrix during step (i).

A third aspect of the invention relates to a pharmaceutical composition. comprising the nanoparticles of the invention and an excipient pharmaceutically acceptable. One of the advantages of the compositions of the present invention is that the toxicity of the drug is reduced by preventing its effects on non-cancerous tissues.

A fourth aspect of the invention relates to the therapeutic use of the nanoparticle of the invention or of the pharmaceutical composition comprising the nanoparticle of the invention according to the first and / or second aspects or any of your particular realizations of these aspects.

Detailed description of the invention

Different methods have been described in the literature for the preparation of nanoparticles that include drugs such as, for example, the continuous emulsion polymerization method in the aqueous phase, the continuous emulsion polymerization method in the organic phase, the interfacial polymerization, the arrangement Interfacial polymer with solvent evaporation, macromolecule desolvation and dialysis methods (Marty et al. Nanoparticles -a new colloidal drug delivery system (Pharm Acta Helv 53 (1978) 17)). As mentioned previously, the nanoparticles of the invention are preferably synthesized by the interfacial arrangement method of the polymer. The interfacial arrangement of polymers is a process for the preparation of biodegradable nanoparticles, or nanospheres. In this method, the biodegradable polymer is first dissolved in an organic solvent, for example acetone or dichloromethane. The resulting organic solution is poured under stirring into the water containing surfactant, such as a polaxamer, with which the aqueous phase immediately becomes a milky solution, indicating the formation of nanoparticles. The organic solvent is removed under reduced pressure. The colloidal suspension formed is concentrated to the desired volume.

The use of the interfacial arrangement of polymers for the synthesis of the nanoparticles of the invention entails several advantages. In addition to being a simple and fast synthesis, it allows nanoparticles with very relevant characteristics to be obtained such as: a very small size colloidal system with very low average sizes, and also very low polydispersions (reaching average diameters of up to 95 ± 7 nm). The process allows a much higher drug load, which among other advantages allows the amount of polymeric material administered to be smaller. All this allows: i) that the mass to be administered from the transport system is not very high to obtain a significant antitumor effect (e.g., with only 200 mg of biodegradable polymer, the vehicleization of a 64.1 ± 2.9 is achieved % of the amount of drug used), compared to what happens with other colloids previously proposed in other works; and, in addition, ii) an intimate and very prolonged contact between the anticancer molecule and the tumor cells.

Specifically, the nanoparticle synthesis process of the invention of poly (E-caprolactone) is based on the interfacial arrangement method of polymers.

In a particular embodiment of the first aspect of the invention, the polymeric nanoparticle is obtained by the process comprising the following stages: i) dissolving poly (E-caprolactone) in an organic solvent, preferably dichloromethane; ii) the addition of the solution obtained in (i) on a mixture comprising an anti-solvent and a surfactant; iii) the isolation of the obtained particle; where the active substance doxorubicin is incorporated by association surface on the nanoparticle obtained after step (iii) or by association within the polymer matrix during step (i).

The superficial association consists in the incorporation by adsorption of the doxorubicin antitumor drug on the surface of nanoparticles previously obtained polymers.

In the association within the polymer matrix, the chemotherapeutic agent It is incorporated into the nanoparticles by absorption. This process is very similar with the only difference that the antisolvent also comprises doxorubicin, normally dissolved at a molar concentration of drug up to 0.01.

In a preferred embodiment the method further comprises a step of evaporation of the organic solvent, preferably under reduced pressure.

Step (iii) can be carried out by centrifugation, preferably at less than 30,000 rpm, more preferably from 5,000 and 15,000 r.p.m.

The procedure can also comprise consecutive stages of redispersion of polymeric nanoparticles and new centrifugation, until

that the conductivity of the supernatant is less than or equal to 10 ¡JS / cm. The conductivity of the supernatant obtained was determined at 25.0 ± 0.5 oC using a Crison micropH 2001 conductivity meter (Spain). The redispersion of the sediment obtained after centrifugation of the nanoparticles can be carried out by adding double distilled water to this sediment and exposing this mixture (at 25.0 ± 0.5 OC) to ultrasound (450 W, Branson 5200E4 ultrasonic bath, USA.).

Step (ii) of the process is preferably performed sequentially.

Preferred solvents used in step (ii) are water or mixtures of water and an organic solvent, preferably a C1-C4 alcohol. More preferably the antisolvent is water.

The temperature at which the association is carried out inside the polymer matrix during step (i) is preferably below 60 ° C, more preferably below 35 ° C, and even more preferably at approximately 25 ° C. The surface adsorption procedure usually lasts 24 hours. Incorporation in matrix (absorption) can be done in 1 hour (noticeably shorter time). It is interesting to note that the drug absorption procedure inside the nanoparticles allows to obtain a greater doxorubicin vehiculization.

Good results have been obtained with nanoparticles having an average particle size between 65 and 150 nm, more preferably between 75 and 125 nm, even more preferably between 85 and 115 nm, and still more preferably about 95 nm.

Preferably the surfactant and the poly (E-caprolactone) are not covalently bound, that is the surfactant and the poly (E-caprolactone) are two different and independent chemical compounds.

The size of the biodegradable poly (E-caprolactone) polymer is not restricted, but good results are obtained when it has a molecular weight of less than 100,000 Daltons, preferably between 5,000 and 45,000 Daltons, and even more preferably between 7,000 and 20,000 Daltons, and even more preferably it is about 10,000 Dantons.

Poly (E-caprolactone) and doxorubicin are in a sufficient ratio to remain associated. The ratio sufficient to remain associated with poly (Ecaprolactone) and doxorubicin normally ranges from 1000: 1 to 1:50 plp respectively, more preferably it is a ratio between 1: 5 and 1:15 p / p.

In a preferred embodiment, the polymeric nanoparticle comprises a biodegradable polymer consisting of poly (E-caprolactone), that is, exclusively caprolactone polymers and never copolymers are used. Preferably it comprises only as a biodegradable polymer poly (Ecaprolactone).

In another preferred embodiment the polymeric nanoparticle comprises only doxorubicin as the active ingredient, and more preferably the nanoparticle further comprises a biodegradable polymer consisting of poly (E-caprolactone).

In another preferred embodiment the polymeric nanoparticle comprises only a biodegradable polymer consisting of poly (E-caprolactone).

The amount of doxorubicin in the polymeric nanoparticle of the invention it is preferably between 0.1% and 25% w / w, more preferably between 15% and 20% w / w.

The amount of poly (E-caprolactone) in the polymer nanoparticle of the invention is preferably between 70% and 99.8% w / w, more preferably between 79% and 84.5% w / w.

The amount of surfactant in the polymeric nanoparticle of the invention is preferably between 0.1% and 2% w / w, more preferably between 0.5% Yun 1% p / p.

Another embodiment of the present invention refers to a nanoparticle polymer with an average size between 65 and 150 nm, which comprises 70% Y 99.8% w / w poly (E-caprolactone), between 0.1% Y 25% w / w of doxorubicin, and between 0.1% and 2% w / w surfactant, where the Surfactant and poly (E-caprolactone) are not covalently bound.

Another embodiment of the present invention refers to a nanoparticle polymeric obtainable by the process comprising the following steps: i) dissolving poly (E-caprolactone) in an organic solvent, preferably dichloromethane; ii) the addition of the solution obtained in (i) on a mixture comprising an anti-solvent, where the anti-solvent is water or mixtures of water and a organic solvent, preferably a C1-C4 alcohol, and a surfactant; iii) the isolation of the obtained particle; where it is incorporated where doxorubicin is incorporated by association in the interior of the polymer matrix during step (i) is carried out at temperature below 60 ° C.

The pharmaceutical composition of the invention comprises: - a polymeric nanoparticle as described in the invention; preferably a nanoparticle obtained by the process of the described invention; -at least a pharmaceutically acceptable excipient.

In a particular embodiment of the present invention, the pharmaceutical compositions of the present invention comprise at least one pharmaceutically acceptable excipient that is a diluent. Preferably, the diluent is water. In another embodiment the diluent is microcrystalline cellulose, lactose or any combination thereof.

In another particular embodiment, the pharmaceutical composition of the invention is particularly adapted for parental use, preferably for intravenous, intraarterial, intratumoral, intramuscular or subcutaneous use. Parenteral compositions preferably comprise water. Even more preferably the nanoparticles of the invention are dispersed in an aqueous solution.

In another embodiment the pharmaceutical composition of the invention is in oral solid form, preferably in tablets or capsules. In this case, the pharmaceutical composition preferably further comprises microcrystalline cellulose, lactose or any combination thereof.

Another embodiment of the present invention refers to a pharmaceutical composition for parental use comprising: a polymeric nanoparticle with an average size between 65 and 150 nm, comprising between 70% and 99.8% w / w poly (E-caprolactone), between 0.1% and 25% w / w doxorubicin, and between 0.1% and 2% w / w surfactant; and a pharmaceutically acceptable excipient.

Both the polymeric nanoparticle of the invention and the pharmaceutical composition of the invention are useful in therapeutic methods. Preferably, they are useful for the preparation of a medicament for the treatment of cancer, preferably Hodgkin and non-Hodgkin lymphoma, breast cancer, ovarian cancer, testicular cancer, acute leukemia, soft tissue sarcoma, lung cancer, cancer of urinary bladder, gastric cancer, thyroid cancer, hepatocarcinoma, Wilms tumor or neuroblastoma, among others, and more preferably breast cancer.

Definitions:

The method used for measuring the particle size of the nanoparticles of the invention is photon correlation spectroscopy (PCS: Photon Correlation Spectroscopy).

The term biodegradable polymer refers to an organic macromolecule (consisting of small molecules called monomers) that is capable of being destroyed (degraded) in vivo under certain physiological conditions. In this way, it is metabolized and eliminated from the body. This destruction occurs mainly under the action of certain enzymes

or enzyme systems, e.g. eg, phospholipase A2, phospholipase C specific for phosphatidylinositol, transglutaminase, alkaline phosphatase, metalloproteinase, esterases, etc. Some biodegradable polymers are also sensitive to slightly acidic pHs (e.g., pH ::::: 6.6 characteristic of tumor interstitium) or temperature.

As used herein, the term "organic solvent" refers to any organic compound, or mixtures of organic compounds, capable of dissolving poly (E-caprolactone). Representative examples include alcohols, hydrocarbons, halogenated compounds, ethers and acetone. Particular examples of organic solvents are ethyl acetate, acetone, acetonitrile (MeCN), benzene, chloroform, methylene chloride, dichloromethane (DCM), 1: 1 mixtures of dichloromethane: chloroform, dimethylformamide (DMF), dimethylsulfoxide (DMSO), 1 , 4-dioxane, protic polar solvents, diethyl ether, tetrahydrofuran (THF), toluene and (ethanol, methanol, n-butanol, npropanol and isopropanol (lPA)). Preferably the organic solvent is dichloromethane.

The term "anti-solvent" refers to a liquid that, when mixed with a solvent in which a solute dissolves, that is, poly (E-caprolactone), reduces the ability of the organic solvent to dissolve the solute. Thus, when an antisolvent is mixed with a solution of a solute in an organic solvent, the solubility of the solute can be reduced to the point where it precipitates in the solution. The solvent must be sufficiently miscible with the organic solvent. It will be appreciated that miscibility can be controlled by varying one or more parameters, within which the solvency between the system and the solvent can be kept at a sufficiently low temperature so that the two liquids are not particularly miscible (during storage , for example), and as the temperature rises the two liquids become miscible and so the nanoparticles can be formed. The preferred antisolvent is an aqueous solution, although acidified aqueous solutions, such as a 2% (v / v) acetic acid solution in water, may also be preset.

The term "C1-C4 alcohol" refers to a linear or branched alkyl chain, which comprises 1 to 4 carbon atoms and one or more hydroxyl groups. Examples of "C1-C4 alcohol" are methanol, ethanol, 1-propanol, 2propanol, n-butanol, isobutanol or tert-butanol, among others.

The term "surfactant" agent, or surfactant, refers to surface active agents that have a structure characterized by a hydrophilic polar group and a hydrophobic tail. These active surface molecules generally have a long chain with at least eight carbon atoms. Surfactants are effective in modifying the properties of the interface, such as interfacial tension, at very low concentrations. Surfactants are also characterized by being able to modify surface thermodynamic properties (eg, hydrophobia), light scattering properties and solute solubilization. Specifically, an increase in surfactant concentration tends to significantly reduce surface tension, until a breakpoint known as the critical micellar concentration (CMC) is reached.

Any effective surfactant can be used in the practice of the present invention, including anionic, nonionic and cationic surfactants. Specific surfactants useful for the present invention are polysorbate 20, polysorbate 60, polysorbate 80, polyvinyl alcohol of high or low molecular weight, polyvinyl pyrrolidone, phosphatidyl choline, lecithins, poloxamers, solutol HS15, sodium glycocholate, sodium taurocholate, sodium tacholactodeoxychoicodium , cholesteryl hemisilocinate and tyloxapol, preferably polysorbate 80 .. The most preferred surfactants are polaxamers, preferably a copolymer of poly (propylene oxide) and poly (ethylene oxide), and more preferably pluronic® F-68.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Figure 1. (a) SEM photomicrograph of the nanoparticles of poly (E-caprolactone) with matrix incorporated doxorubicin prepared according to the procedure described in the examples. Inserted figure: detail of a nanoparticle. Bar length: 100 nm. (b) Efficiency of doxorubicin vehiculization (%) of poly (E-caprolactone) nanoparticles prepared according to the procedure described in the examples based on the concentration of drug in the equilibrium. Doxorubicin vehiculization was performed by surface adsorption (or) or by incorporation into the polymer matrix (absorption,.). (c) Release of adsorbed (or) or absorbed (.) doxorubicin from poly (E-caprolactone) nanoparticles in an incubation medium (phosphate buffer, pH = 7.4 ± 0.1) and at 37.0 ± 0.5 ° C.

Figure 2. Representative image of fluorescence microscopy of MCF-7 breast cancer cells exposed to doxorubicin in which the incorporation of this drug to the cytoplasm and nucleus of tumor cells can be observed when associated with the nanoparticle (polymer of E-caprolactone)

(A) prepared according to the procedure described in the examples of matrix absorption and when administered freely (B). As can be seen in the image, the intensity of fluorescence was clearly higher in cells exposed to polymer-associated doxorubicin than in cells exposed to the free drug.

Figure 3. A. Analysis by FASCcan of the presence of doxorubicin inside MCF-7 cells after treatment with free doxorubicin (DOX) and doxorubicin associated with E-caprolactone nanoparticle (DOX + POL) prepared according to the procedure described in the Examples of matrix absorption for 0.5 (a and b), 1.5 (c and d), 2 (e and f) and 4 hours (gyh). B. Graphical representation of the modulation of the mean fluorescence intensity emitted by doxorubicin in MCF-7 cells treated with

free doxorubicin or doxorubicin associated with an E-caprolactone nanoparticle prepared according to the procedure described in the examples of matrix absorption.

EXAMPLES

The following abbreviations have been used in the examples: IC50: 50 inhibitory dose; MTT: 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazole bromide; PCS: photon correlation spectroscopy; p / p: weight / weight; p / v: weight / volume; r.p.m: revolutions per minute. SEM: scanning electron microscopy.

Procedure for obtaining nanoparticles of poly (e-caprolactone) by the method of interfacial arrangement of polymers

Poly (E-caprolactone) nanoparticles were prepared by the polymer interfacial arrangement method (Arias et al., Colloids Surf. B: Biointerfaces 75 (2010) 204) A 2% (w / v) solution of biodegradable polymer in dichloromethane by mechanical stirring. This organic phase is added sequentially to 50 mL of an aqueous solution containing 1 gram of pluronic® F-68 surfactant [poloxamer: copolymer of poly (propylene oxide) and poly (ethylene oxide)]. Under these conditions instantaneous formation of the nanoparticles occurs. The removal of the organic solvent is carried out by evaporation at 37.0 ± 0.5 ° C and under reduced pressure, using a rotary evaporator. The aqueous suspension of poly (E-caprolactone) nanoparticles is finally subjected to repeated centrifugation cycles (10,700 rpm for 30 minutes) and redispersion in double-distilled water until the conductivity of the supernatant is :: ;; 10 ¡JS / cm. The characterization of the morphology (shape and size) of the nanoparticles is performed by photon correlation spectroscopy (PCS). The

nanoparticles obtained have an average size of 95 ± 7 nm). The load of

drug is 64.1 ± 2.9%)

The quantification of the amount of vehiculized drug was performed using a previously validated spectrophotometric procedure (to demonstrate its linearity, precision and accuracy). The measured wavelength was the maximum absorbance (481 nm). Two strategies were developed for the incorporation of the antitumor drug doxorubicin in polymeric nanoparticles. The first of these, surface adsorption, consists in bringing the nanoparticles already formed into contact with the antitumor drug for 24 hours at room temperature. On the contrary, the second of the strategies, absorption inside the polymer matrix, involves the incorporation of the chemotherapeutic molecule into the organic phase containing the dissolved biodegradable polymer, before its addition to the aqueous surfactant solution. The study of the release of doxorubicin from the nanoparticles was carried out using the dialysis method, and its quantification was carried out using the aforementioned spectrophotometric method. All experiments were performed in triplicate.

The particle size was determined in triplicate and at room temperature by photon correlation spectroscopy (PCS), and was confirmed by scanning electron microscopy (SEM). The amount of vehiculized drug was determined using a previously validated spectrophotometric analytical technique (A = 481 nm). The incorporation of doxorubicin in the poly (E-caprolactone) nanoparticles was tested by two methods of vehiculization. The first of these consists in the adsorption of the antitumor drug on the surface of the nanoparticles. In particular, a suspension of polymer nanoparticles (2% concentration (w / v)) is incubated with increasing concentrations of doxorubicin (up to 0.01 M), for 24 hours at 25.0 ± 0.5 ° C and under stirring mechanical (50 rpm). The second methodology (matrix absorption) is very similar to the procedure for preparing the poly (E-caprolactone) nanoparticles described above, with the only difference that an appropriate amount of drug (up to 0.01 M) is incorporated into the Organic polymer solution before adding to the aqueous surfactant solution. As for the in vitro drug release, the method used was dialysis, using a phosphate buffer of pH = 7.4 ± 0.1 as the release medium (Arias JL et al., J. Drug Target. 17 (2009) 586). For this, 12 hours before the experiment, dialysis bags were introduced into the release medium at 25.0 ± 0.5 ° C. The characteristics of these bags (pore size: 2000 Daltons, Spectrum® Spectra / Por® 6 dialysis membrane tubing, USA) mean that the polymeric nanoparticles are retained inside and that only the active substance is able to diffuse towards The means of release. After those 12 hours, 2 mL of the nanoparticle suspension (containing 3 mg / mL of doxorubicin) was placed in the dialysis bags, the ends of which were closed with tweezers. The bags (with the suspension inside) were deposited in a beaker containing 100 mL of dissolution medium under mechanical agitation (200 rpm). The temperature was maintained at 37.0 ± 0.5 ° C throughout the release test (performed in triplicate). From this moment, and at predetermined intervals (0.25, 0.5, 0.75, 1, 1.5, 2, 3, 6, 12, 24, 48, 72 and 96 hours), 2 mL were taken of means to analyze its content by means of the previously validated spectrophotometric technique (A = 481 nm). An equal volume of release medium (heated to 37.0 ± 0.5 ° C) was added to the beaker after each sampling.

In Figure 1 (a) it can be seen that the poly (Ecaprolactone) nanoparticles obtained are stable, of spherical morphology and with a size distribution within the colloidal range (mean size: 95 ± 7 nm).

The amount of vehiculized doxorubicin is much higher when the matrix absorption method is used (vehiculization efficiency ::::: 64%), compared to the surface adsorption method (vehiculization efficiency ::::: 26%) . As can be seen in Figure 1b, there is a beneficial effect of the amount of drug used on the vehiculization capacity. The release profile of doxorubicin from poly (E-caprolactone) nanoparticles is biphasic (Figure 1 C): in the first stage the release occurs quickly (up to ::::: 37% after 6 hours, probably consequence of the release of the drug located at the superficial level), while the remaining amount is released very slowly in the next 90 hours. The release of the vehicularized antitumor by surface adsorption is much faster, being total after less than 12 hours. This may be due to a very poor physical adsorption of this hydrophilic drug on the hydrophobic surface of the polymer.

In vitro assays have been performed using the human breast cancer line MCF-7. In this line, the toxicity of the nanoparticles and the IC50 of free doxorubicin and poly (Ecaprolactone) encapsulated doxorubicin was determined. The study was performed at 72 hours and growth inhibition was determined using the MTT assay (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazole bromide). The study of incorporation of doxorubicin into the cancer cell was performed using flow cytometry and fluorescence microscopy after incubation of MCF-7 for 0.5, 1, 1.5, 2 and 4 hours at a concentration of 25 ~ M.

In vitro tests have shown that doxorubicin associated with poly (E-caprolactone) nanoparticles has an IC50 five times lower in the MCF-7 breast cancer line than that of free doxorubicin, with IC50 being 0.1 And 0.5 mM, respectively. Thus, doxorubicin bound to poly (Ecaprolactone) decreases its IC5o. The analysis of the cellular incorporation of doxorubicin also showed that the incorporation was 6.7 times higher in the case of the nanoparticle-associated doxorubicin (mean fluorescence of 1222 at 4 h) than when it was incubated with free doxorubicin (mean of fluorescence fluorescence 181 at 4h). These results suggest that the encapsulation of doxorubicin in the poly (E-caprolactone) nanoparticles increases the antitumor activity of the drug, so it could be used to decrease its toxicity (Figure 2). In addition, cell incorporation is superior when poly (E-caprolactone) is used compared to when other polymers such as poly (butylcyanoacrylate) are used.

Claims (33)

1.-A polymer nanoparticle with an average size of less than 200 nm, preferably less than 190 nm, comprising one or more polymers biodegradable, one or more active ingredients and at least one surfactant, where at least one biodegradable polymer is poly (E-caprolactone) and where at least An active substance is doxorubicin. 2. The polymeric nanoparticle according to the preceding claim, obtainable by the procedure comprising the following stages: i) dissolving poly (E-caprolactone) in an organic solvent, preferably dichloromethane; ii) the addition of the solution obtained in (i) on a mixture comprising an anti-solvent and a surfactant; iii) the isolation of the obtained particle; where the active substance doxorubicin is incorporated by association surface on the nanoparticle obtained after step (iii) or by association within the polymer matrix during step (i). 3.-The polymeric nanoparticle according to any of the claims above, characterized in that the nanoparticle has a particle size medium between 65 and 150 nm. 4. The polymeric nanoparticle according to the preceding claim, characterized because the nanoparticle has an average particle size of between 75 and 125 nm, preferably between 85 and 115 nm, and more preferably of approximately 95 nm. 5.-The polymeric nanoparticle according to any of the claims above, where the surfactant and poly (E-caprolactone) are not bound covalently 6. The polymeric nanoparticle according to any of the preceding claims, wherein the biodegradable polymer of poly (E-caprolactone) has a molecular weight of less than 100,000 Daltons, preferably between 5,000 and 45,000 Daltons, and even more preferably between 7 and 20,000 Daltons, and even more preferably is about 10,000 Dantons. 7. The polymeric nanoparticle according to any of the preceding claims, which comprises a biodegradable polymer consisting of poly (Ecaprolactone). 8. The polymeric nanoparticle according to any of the preceding claims, wherein the poly (E-caprolactone) and doxorubicin are in a sufficient ratio to remain associated. 9. The polymeric nanoparticle according to the preceding claim, wherein the ratio sufficient to remain associated with poly (E-caprolactone) and doxorubicin is a ratio between 1000: 1 and 1:50 p / p respectively, preferably it is a ratio of between 1: 5 and 1:15. 10. The polymeric nanoparticle according to the preceding claim, which comprises a biodegradable polymer consisting of poly (E-caprolactone). 11. The polymeric nanoparticle according to any of the preceding claims, comprising only doxorubicin as active ingredient. 12. The polymeric nanoparticle according to any of the preceding claims, comprising only a biodegradable polymer consisting of poly (E-caprolactone). 13. The polymeric nanoparticle according to any of the preceding claims, which comprises between 0.1% and 25% w / w of doxorubicin, preferably between 15% and 20% w / w. 14. The polymeric nanoparticle according to any of the preceding claims, comprising between 70% and 99.8% w / w poly (Ecaprolactone), preferably between 79% Yun 84.5% w / w. 15. The polymeric nanoparticle according to any of the preceding claims, which comprises between 0.1% and 2% w / w surfactant, preferably between 0.5% and 1% w / w. 16. The polymeric nanoparticle according to any of the preceding claims, wherein the surfactant is selected from polysorbate 20, polysorbate 60, polysorbate 80, polyvinyl alcohol of high or low molecular weight, polyvinyl pyrrolidone, phosphatidyl choline, lecithins, poloxamers of different types, solutol HS15, sodium glycocholate, sodium taurocholate, sodium tauroglycocholate, sodium taurodeoxycholate, cholesteryl hemisuccinate and tyloxapol, preferably polysorbate 80. 17. The polymeric nanoparticle according to any of the preceding claims, wherein the surfactant is a poloxamer, preferably a copolymer of poly (propylene oxide) and poly (ethylene oxide). 18. The polymeric nanoparticle according to claim 2, wherein the process further comprises an evaporation step of the organic solvent, preferably under reduced pressure. 19. The polymeric nanoparticle according to any of claims 2 or above, wherein step (iii) is carried out by centrifugation, preferably at less than 30,000 rpm, more preferably from 5,000 and 15,000 r.p.m. 20. The polymeric nanoparticle according to any of claims 2, 18 or 19, wherein the method further comprises a step of redispersion of the polymeric nanoparticle until the conductivity of the supernatant is less than or equal to 10¡JS / cm. 21. The polymeric nanoparticle according to any of claims 2 and 18-20, wherein step (ii) of the process is carried out sequentially. 22. The polymeric nanoparticle according to any of claims 2 and 18-21, wherein the antisolvent is water or mixtures of water and an organic solvent, preferably a C1-C4 alcohol. 23. The polymeric nanoparticle according to any of claims 2 and 18-22, wherein the antisolvent is water. 24. The polymeric nanoparticle according to any of claims 2 and 18-23, wherein the incorporation of doxorubicin by association inside the polymeric matrix during step (i) is carried out at a temperature below 60 ° C, preferably lower at 35 ° C, and even more preferably at about 25 ° C. 25.-A process for the synthesis of a polymeric nanoparticle with an average size of less than 200 nm, preferably less than 190 nm, comprising one or more biodegradable polymers, one or more active ingredients and at least one surfactant, where at least one Biodegradable polymer is poly (E-caprolactone) and where at least one active substance is doxorubicin: i) the dissolution of poly (E-caprolactone) in an organic solvent, preferably dichloromethane; ii) the addition of the solution obtained in (i) on a mixture comprising an anti-solvent and a surfactant; iii) the isolation of the obtained particle; where the active substance doxorubicin is incorporated by association surface on the nanoparticle obtained after step (iii) or by association within the polymer matrix during step (i). 26.-A pharmaceutical composition comprising: - a nanoparticle according to any one of claims 1 to 24; Y -a pharmaceutically acceptable excipient. 27. The pharmaceutical composition according to the preceding claim, characterized in that it comprises a diluent. 28. The pharmaceutical composition according to any of the two claims previous, characterized in that it includes water. 30. The pharmaceutical composition according to any of the two claims above, characterized in that it is fit for parental use, preferably for intravenous, intraarterial, intratumoral use, intramuscular or subcutaneous. 30. The pharmaceutical composition according to any of claims 26 or 27, characterized in that it comprises microcrystalline cellulose, lactose or Any of your combinations. 31. The pharmaceutical composition according to any of claims 26, 27 or earlier, characterized in that it is in oral solid form, preferably in tablets or capsules. 32. Use of a polymeric nanoparticle according to any of claims 1 to 24 or of a pharmaceutical composition according to any of claims 26 to 31 for the preparation of a medicament. 33. Use of a polymeric nanoparticle according to any of claims 1 to 24 or of a pharmaceutical composition according to any of claims 26 to 31 for the preparation of a medicament for the treatment of cancer, preferably breast cancer.