Polymer nanoparticle aggregate, method for producing same and use thereof for the treatment of venous thrombosis

Abstract

A first aspect of the present invention relates to a nanoparticle aggregate for the treatment of venous thrombosis characterised in that it comprises a combination of negatively charged nanoparticles comprising rtPA and positively charged nanoparticles comprising DNase per nanoparticle. A second aspect of the present invention relates to a method for producing the nanoparticle aggregate. A third aspect of the invention relates to a pharmaceutical composition comprising the nanoparticle aggregate according to the first aspect of the invention. Lastly, the present invention discloses the nanoparticle aggregate for use as a drug and for the treatment of thromboembolic diseases.

Description

[0001] DESCRIPTION

[0002] AGGREGATE OF POLYMERIC NANOPARTICLES, ITS OBTAINING PROCEDURE AND ITS USE IN THE TREATMENT OF VENOUS THROMBOEMBOLISM

[0003] TECHNICAL FIELD

[0004] The present invention relates to the pharmaceutical industry and nanotechnology and in particular refers to a new intravenous administration system of therapeutic enzymes for the treatment of venous thromboembolic diseases (VTEs).

[0005] BACKGROUND OF THE INVENTION

[0006] Venous thromboembolic disease (VTE) includes deep vein thrombosis (DVT) and pulmonary embolism (PE), which share risk factors, etiopathogenic mechanisms, and treatment. It occurs when a venous thrombus, usually in the lower limbs, fragments and impacts the pulmonary arteries, a potentially fatal situation. VTE has an incidence of 1 per 1000 people / year and is the third most common cardiovascular disease, after acute coronary syndrome and stroke (1). The total estimated annual cost, including direct and indirect costs, of VTE in Spain is €75.5 million (2).

[0007] Reperfusion therapy, usually with thrombolytics, is used in hemodynamically unstable PE patients and in patients with DVT who present with phlegmasia or venous gangrene (4). Its use is also being explored in normotensive PE patients with a likelihood of unfavorable outcome. Currently, fibrinolytic dose reductions, applied locally via a catheter, together with mechanical fragmentation using ultrasound, are in use, although not widely validated (5-7). The interest raised in this topic is reflected in currently planned clinical trials, such as the PEITHO III study (NCT04430569), which tests half-dose systemic fibrinolytic therapy versus standard treatment in normotensive PE patients with intermediate-high prognostic risk.

[0008] Extracellular neutrophilic tracts (NETs) are one of the components of thromboses and contribute to the resistance of thrombolysis mediated by t-PA. In this way, the document Ducroux C, Di Meglio L, Loyau S, Delbosc S, Boisseau 14 / , Deschildre C, Ben Maacha M, Blanc R, Redjem H, Ciccio G, Smajda S, Fahed R, Michel JB, Piotin M, Salomon L, Mazighi M, Ho-Tin-Noe B, Desilles JP. Thrombus Neutrophil Extracellular Traps Content Impair tPA-induced Thrombolysis in Acute Ischemic Stroke. Strokes. 2018 Mar; 49 (3): 754-757. doi: 10. 1161 / STROKEAHA. 117.019896. Epub 2018 Feb 8. PMID: 29438080 describe that the administration of DNasa (specifically DNasa I) together with t-PA significantly improves thrombolysis ex vivo for the resolution of thrombos in acute cerebrovascular ischemic accident.Another document that highlights the need to act against NETs so that t-PA can perform its function is Acute ischemic stroke thrombi have an outer Shekk that impairs fibrinolysis (Di Meglio et al., 2019), which reports a study on the outer layer of thrombi in acute ischemic stroke.

[0009] Polymeric nanoparticles as prolonged drug release systems.

[0010] In the cardiovascular field, there have also been major advances using nanomedicine, for example, BLAST assays, NANOM, and NanoAthero.

[0011] Among the new drug delivery systems, the use of polymeric nanoparticles stands out. These nanoparticles can remain in the body for an extended period, ensuring proper drug release. With the proper polymer selection and design, these systems can: reduce adverse effects; control drug release; prolong its therapeutic effect; reduce the number of daily doses; reduce treatment costs; reduce drug toxicity and side effects; and offer greater specificity for target tissues.

[0012] Targeting Strategies

[0013] To direct these systems towards the therapeutic target and achieve maximum efficacy and minimize side effects, there are several strategies: (i) active targeting and (i) passive targeting.

[0014] The former involve a surface modification of the nanoparticle with a biomolecule, thereby selectively interacting with a receptor and / or another biomolecule overexpressed in the target. The latter involve the system's response to some type of stimulus, endogenous or exogenous, seeking either a cumulative effect in the area of ​​interest (i.e., increased permeability effect in tumors) or the release of the drug in response to a specific stimulus (i.e., pH change, glutathione production, mechanical stimuli, etc.). In the context of cardiovascular pathologies, shear forces or shear stress play a fundamental role. Shear stress is defined as the force, per unit area, that blood flow exerts on the vascular wall and depends on blood viscosity and the blood flow velocity profile. Physiological values ​​in the venous system range between 1 and 6 dyne / cm. 2, while in the arterial system they are > 10-15 dyne / cm 2 Shear stress induces platelet aggregation, triggering endothelial cells to secrete prostacyclin (PGI2), which has vasodilatory action and inhibits platelet aggregation. It also secretes nitric oxide (NO), a potent vasodilator and inhibitor of platelet adhesion and aggregation. In response to vascular damage, platelets, which normally circulate as single cells, encounter the thrombogenic environment of the subendothelial matrix. This initiates interactions between adhesive proteins of the platelet wall, such as von Willebrand factor, and the corresponding receptors on the platelet membrane. This adhesive phase, in turn, facilitates the interaction of platelets with collagen and the initiation of the activation phase and thrombus formation.

[0015] In this situation, a change in the flow regime occurs from laminar to turbulent, increasing the shear stress.

[0016] Below are the most relevant documents to date for the treatment of thrombosis using nanoparticles:

[0017] The document Enhanced thrombolysis using tissue plasminogen activator (tPA)-loaded PEGylated PLGA nanoparticles for ischemic stroke (M. Zamanlu, M. Eskandani, J. Barar, et al. Journal of Drug Delivery Science and Technology, 53, 101165, 2019, https: / / doi.Org / 10.1016 / j.jddst.2019.101165) refers to the use of PEGylated PLGA nanoparticles loaded with t-PA for the treatment of ischemic stroke. The nanoparticles had a spherical shape, a size between 250-280 nm and a zeta potential between -7 mV and -12 mV, for PGLA and PGLA-PEG nanoparticles, respectively (see page 6, right column, last paragraph). Therefore, this document describes pegylated PGLA nanoparticles comprising t-PA.

[0018] The paper Chen HA, Ma YH, Hsu TY, Chen JP. Preparation of Peptide and Recombinant Tissue Plasminogen Activator Conjugated Poly(Lactic-Co-Glycolic Acid) (PLGA) Magnetic Nanoparticles for Dual Targeted Thrombolytic Therapy. Int J Mol Sci. 2020 Apr 13;21(8):2690. doi: 10.3390 / ijms21082690. PMID: 32294917; PMCID: PMC7215398 relates to a thrombolytic therapy for the delivery of an rt-PA to minimize the required dose of said activator. For this purpose, rt-PA and a fibrin-avid peptide were co-immobilized on PLGA magnetic nanoparticles (see abstract). Therefore, this paper describes PLGA magnetic nanoparticles that deliver rt-PA.

[0019] The document Uesugi Y, Kawata H, Jo J, Saito Y, Tabata Y. An ultrasound-responsive nano delivery system of tissue-type plasminogen activator for thrombolytic therapy. J Control Release. 2010 Oct 15; 147(2): 269-77. doi: 10.1016 / j.jconrel.2010.07.127. Epub 2010 Aug 6. PMID: 20696194 refers to a nanosystem of gelatin derivatives for releasing t-PA for thrombolytic therapy, in which t-PA has suppressed thrombolytic activity, which is recovered when exposed to an ultrasound source. For the synthesis of said system, different amounts of ethylenediamine are chemically introduced into gelatin such that the gelatin is cationized to form a complex with t-PA. The surfaces of these complexes were modified with polyethylene glycol (PEG). This paper demonstrates nanosystems capable of suppressing t-PA activity and reactivating it once they reach their target.

[0020] US patent application US 2013 / 0295.012 describes a system of nanoparticle aggregates that disaggregate under shear forces, with application for the treatment and / or diagnosis of pathologies involving restricted blood flow and increased shear forces. The invention describes PLGA-PEG nanoparticles coated with rtPA that form aggregates by spray-drying.

[0021] However, none of these documents cited here describes or suggests an aggregate formed by two types of nanoparticles with opposite surface electrical charges capable of disintegrating under the action of shear forces inside blood vessels for the co-administration of two therapeutic enzymes with a synergistic and / or additive fibrinolytic effect, with the release of said therapeutic enzymes being sustained.

[0022] Therefore, there is a clear need to develop new pharmaceutical formulations as a prolonged-release system for use in the treatment of thromboembolic diseases. BRIEF DESCRIPTION OF THE INVENTION

[0023] In a first aspect, the present invention relates to an aggregate of nanoparticles characterized in that it comprises negatively charged nanoparticles and positively charged nanoparticles, in which:

[0024] • said negatively charged nanoparticles comprise rtPA, and

[0025] • said positively charged nanoparticles comprise DNase.

[0026] In a second aspect of the present invention, to a method of obtaining the aggregate of nanoparticles described herein.

[0027] In a third aspect, the present invention relates to a pharmacological composition comprising the aggregate of nanoparticles according to the first aspect of the invention.

[0028] In a fourth aspect, the present invention relates to the pharmacological composition for use as a medicine.

[0029] And in a final aspect, the present invention relates to the pharmacological composition for use in the treatment of thromboembolic diseases.

[0030] BRIEF DESCRIPTION OF THE FIGURES

[0031] The following figures are described below, they illustrate the embodiments by way of example and do not limit their scope.

[0032] Figure 1. (1.a) Scheme of production of microaggregates of Nanoparticles containing rtPA or DNase and 1.(b) size distribution of the aggregate obtained.

[0033] Figure 2. (2.a) SEM image of a microaggregate; (2.b) of a cross-section of a microparticle aggregate showing its interior and where individual nanoparticles can be differentiated; (2.c) SEM image of a set of microaggregates showing a homogeneous size, sphericity and hollow structure; (2.d) Confocal microscopy image showing that the aggregate composition is similar in both types of nanoparticles and that they are hollow. Figure 3. (3.a) Image of a 12-well fibrin plate after incubation with different doses of free and encapsulated rtPA after incubation at 37°C for 30 min. (3.b) Percentage of lysed area versus time for 5 and 10 μg free rtPA doses (5 μg dark solid line; 10 μg light solid line) vs. Encapsulated (5 pg dark dashed line and 10 pg light dashed line).

[0034] Figure 4. Amount of DNase released over time evaluated according to results on agar plate at 37°C.

[0035] Figure 5. Microchip used to simulate different degrees of vascular obstruction in vitro. The figure shows a chip with a narrowed central region that simulates the stenosed region of a blood vessel. The stenosed region has dimensions of width “w” of 400 pm and height “a” of 160 pm. Where, Q = flow; Yw = wall shear; a: channel thickness (narrowing or stenosis); w: channel width (narrowing or stenosis).

[0036] Figure 6. (6.a) Particle size distribution and (6. b) appearance of the sample before and (6.c) after being subjected to shear forces simulating the stenosis of a blood vessel and (6.d) appearance of the sample after.

[0037] Figure 7. Results obtained for the Hemolysis test for both individual particles (positive and negative) (7.a) and microaggregates (7.b) at the indicated concentrations.

[0038] Figure 8. Results obtained for the platelet aggregation assay for both individual particles (positive and negative) (8.a) and microaggregates (8.b) at the indicated concentrations.

[0039] Figure 9. Results for the lymphocyte proliferation assay for both individual particles (positive and negative) (9.a) and microaggregates (9.b) at the indicated concentrations.

[0040] Figure 10. Results for the complement activation assay for both individual particles (positive and negative) (9.a) and microaggregates (9.b) at the indicated concentrations. Figure 11. Percentage reduction in clot weight after incubating rtPA and DNase at two different concentrations, at three different times (30, 45 and 60 min.) and combinations of the two drugs.

[0041] Figure 12. (12.a) Image and (12. b) Schematic of thrombus generated by the mouse vena cava thrombosis model: (1) inferior vena cava; (2) left renal vein; (3) IVC ligation; (4) lateral vein ligation; (5) developing thrombus; (6) iliac bifurcation.

[0042] Figure 13. Percentage of thrombus reduction “ex vivo” testing different therapeutic groups: (i) rtPA (25pg); (ii) DNase (2mg) and (iii) rtPA (25ug) and DNase (2mg); (iv) control group. Thrombi were weighed at 2, 3 and 4 hours of incubation with the drug (n=8).

[0043] DESCRIPTION OF THE INVENTION

[0044] Researchers have developed the present invention, which consists of size-controlled, hollow, spherical nanoparticle aggregates that are sensitive to endogenous mechanical changes, such as shear stress changes in the stenosed area of ​​the blood vessel. In this way, the aggregates disintegrate precisely at the site of the injury, allowing for the sustained co-release of two therapeutic enzymes with a synergistic fibrinolytic effect.

[0045] In a first aspect, the present invention relates to an aggregate of nanoparticles characterized in that it comprises negatively charged nanoparticles and positively charged nanoparticles, in which:

[0046] • said negatively charged nanoparticles comprise rtPA, and

[0047] • said positively charged nanoparticles comprise DNase.

[0048] In embodiments of the present invention, the nanoparticles are associated, meaning that they are held together by electrostatic forces of attraction due to their difference in electrical charge, and such binding can be reversed if these electrostatic forces of attraction are smaller than the shear forces within a blood vessel. In a preferred embodiment, the amount of rtPA in the negatively charged nanoparticles is between 3 and 6% by weight relative to the total number of negatively charged nanoparticles. In a more preferred embodiment, the amount of rtPA in the negatively charged nanoparticles is 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, or 6% by weight relative to the total number of negatively charged nanoparticles.

[0049] In a preferred embodiment, the amount of DNase in the positively charged nanoparticles is between 2.5 and 7% by weight relative to the total number of positively charged nanoparticles. In a more preferred embodiment, the amount of DNase in the positively charged nanoparticles is 2.5%, 3%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, or 7% by weight relative to the total number of positively charged nanoparticles.

[0050] In a preferred embodiment of the invention, the aggregate is spherical and hollow, which, in the context of the present invention, means that the aggregate of nanoparticles has the shape of a geometric body limited by a curved surface whose points are all at the same distance from an interior called the center and where said body is empty inside.

[0051] In this text, negatively charged nanoparticles are defined as type A nanoparticles, and positively charged nanoparticles as type B nanoparticles.

[0052] In embodiments of the present invention, the nanoparticle aggregate comprises the positively charged nanoparticles and negatively charged nanoparticles in a ratio of between 1:1 and 1:5. In preferred embodiments, the negatively charged nanoparticles to the positively charged nanoparticles are in a ratio of 1:1 and 1:5, preferably in a ratio of 1:1, 1:2, 1:3, 1:4 or 1:5.

[0053] In this way, a micrometric-sized aggregate is obtained that combines two types of nanoparticles, where the negatively charged nanoparticles, type A, comprise an active agent that is rtPA and the positively charged nanoparticles, type B, comprise an active agent that is DNase.

[0054] In the context of the present invention, rtPA is a tissue plasminogen activator that acts as a fibrinolytic.

[0055] In the context of the present invention, DNase is deoxyribonuclease I, which acts by degrading extracellular DNA. In a preferred embodiment, the nanoparticles form spherical, hollow aggregates of controlled size. In a preferred embodiment of the present invention, the nanoparticle aggregate has a diameter of 5 to 7 pm, and preferably has a size of 5, 6, or 7 pm.

[0056] In one embodiment of the present invention, the nanoparticle aggregate has a rough surface. In the context of the present invention, a rough surface refers to a surface that is not uniform and has folds or creases.

[0057] In the context of the present invention, the nanoparticles that constitute the nanoparticle aggregate, which are polymeric in nature, are attracted to each other by opposite surface electrical charges, thus forming nanoparticle aggregates, according to the first aspect of the present invention.

[0058] In one embodiment of the present invention, the nanoparticles comprising the aggregate according to the first aspect of the invention are polymeric nanoparticles. The negatively charged nanoparticles, of type A, are composed of the copolymer of lactic and glycolic acid (PLGA), and therefore have a negative surface electrical charge. On the other hand, the positively charged nanoparticles, of type B, are composed of the copolymer of lactic and glycolic acid (PLGA), and the copolymer of lactic and glycolic acid (PLGA) with polyethylene glycol PLGA-PEG, and preferably, an additional amino-type compound, which is stilamine, and therefore have a positive electrical charge.These type B nanoparticles are composed mainly of the biodegradable and biocompatible copolymer of lactic and glycolic acid (PLGA) found in an amount between 75-90% w / w and lactic and glycolic acid (PLGA) with polyethylene glycol (PLGA-PEG) found in an amount between 10-25% w / w covalently bonded.

[0059] Therefore, thanks to the combination of these two types of nanoparticles, a nanoparticle aggregate is obtained that offers a controlled and sustained release of the two active ingredients mentioned in this document, in such a way that the nanoparticle aggregate, according to the first aspect, releases both active ingredients at the same and constant speed for a period of at least 72 hours, preferably between 72 and 120 hours, and even more preferably for a period of 72 to 96 hours.In a preferred embodiment, the aggregate sustainably releases rtPA at a rate of 10 to 15%, preferably 15%, by weight relative to the total drug content comprising the particle each hour and releases 80% of DNase in the first 8 hours and then releases DNase at a rate of 5 to 80% by weight relative to the total drug content comprising the particle each hour for at least 72 hours when the aggregate disintegrates by at least 30% of its total volume.

[0060] It is worth noting that polyester nanoparticles such as PLGA exhibit high biocompatibility and are a very safe polymer, as their degradation products are absorbed. Therefore, nanoparticle aggregates act as hemocompatible and non-immunogenic systems. Furthermore, the presence of PEG chains confers greater stability, prevents protein absorption, and prevents opsonization by macrophages, increasing systemic circulation time.

[0061] Based on the above, the inventors have determined that the degree of pegylation of the positively charged nanoparticles has been key to the correct response of the aggregates to changes in shear stresses. In the context of the present invention, shear stresses are the force, per unit area, that blood flow exerts on the vascular wall, and depend on blood viscosity and the blood flow velocity profile. Thus, if the degree of pegylation is 0%, the aggregates do not rupture under physiological conditions of maximum occlusion (maximum shear stress). If the degree of pegylation is greater than 80%, the aggregates are not stable under standard handling conditions prior to administration.Therefore, in preferred embodiments of the present invention, the degree of pegylation of the positively charged nanoparticles is between 10%-60%, more preferably between 20%-50%, and even more preferably between 20%-40%. In preferred embodiments of the invention, the degree of pegylation of the positively charged nanoparticles is between 10%-25%. Therefore, in preferred embodiments of the present invention, the degree of pegylation of the positively charged nanoparticles is 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%. The inventors have been able to verify that by controlling this degree of pegylation of the positively charged nanoparticles, the forces of attraction between the positive and negative particles can be controlled, thereby indirectly controlling the disintegration of the nanoparticle aggregate when subjected to shear forces.

[0062] In this way, and with the proportion of both types of nanoparticles described in this document, aggregates of micrometric sized particles sensitive to pathological shear force changes are obtained, i.e. for wall shear forces greater than 100 dynes / cm. 2 which implies values ​​of shear rate or velocity gradient (rate at which adjacent layers of a fluid move relative to each other) greater than 40,000 s' 1 .

[0063] In the context of the present invention, the encapsulated therapeutic enzyme refers to the active agent, in the present invention being rtPA for negatively charged nanoparticles and DNase for positively charged nanoparticles.

[0064] In one embodiment of the present invention, the nanoparticles have an opposite surface electrical charge, referred to herein as Zeta Potential (ZP), which is less than 30 mV in absolute value. In preferred embodiments of the present invention, the nanoparticles have a ZP of 1-25 mV, with 1-20 mV being more preferred; and 1-15 mV being even more preferred.

[0065] In other embodiments of the present invention, the nanoparticles having a positive zeta potential comprise an amino compound, preferably stearylamine, in a proportion of 6.28-12.5% ​​w / w. In a preferred embodiment, the positively charged nanoparticles have a quantity of stearylamine of 2.5-5 mg.

[0066] In a preferred embodiment of the present invention, the aggregates according to the first aspect disaggregate by at least 30% of their volume when subjected to wall shear forces of 25,000 s -1 or higher. In preferred embodiments, the aggregates of the present invention are disaggregated by at least 30%, 40%, 50%, 60%, 50%, 60%, 70%, 80%, 90% or 100%.

[0067] In a second aspect, the present invention relates to a method for obtaining the aggregate of nanoparticles comprising the following steps: a) adding a suspension of negatively charged nanoparticles with rtPA according to the first aspect of the invention to a suspension of positively charged nanoparticles with DNase according to the first aspect of the invention in a ratio between 1:5 and 1:10, wherein said suspension of positive nanoparticles has a concentration comprised between 0.1-0.5% w / v, preferably 0.3% w / v and said suspension of positive nanoparticles has a concentration comprised between 0.1-0.5% w / v, preferably 0.3% w / v, b) mixing the suspension obtained in the previous step at a speed of 15,000-20,000 rpm, preferably at a speed of 17,000-18.000 rpm, for a time between 40 and 60 seconds, preferably 45 seconds, and a temperature between 15-20°C, preferably 18°C; c) centrifuging the solution obtained in the previous step at a speed between 1500-2500 rpm, preferably 2000 rpm, for a period of 3 to 8 minutes, preferably between 4 and 5 minutes to obtain the aggregates.

[0068] In a preferred embodiment, the suspension of negatively charged nanoparticles with rtPA has a concentration between 0.1-0.5% w / v. In particular embodiments of the present invention, the suspension of negatively charged nanoparticles with rtPA has a concentration of 0.1%, 0.2%, 0.3%, 0.4% or 0.5%. In an even more preferred embodiment, the suspension of negatively charged nanoparticles with rtPA has a concentration of 0.27% w / v. In another embodiment of the present invention, the suspension of negatively charged nanoparticles with rtPA has a concentration between 1-5 mg / mL, preferably being a concentration between 1.5-4 mg / mL, and even more preferred between 2-4 mg / mL. In particular embodiments of the present invention, the suspension of negatively charged nanoparticles with rtPA has a concentration of 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL or 5 mg / mL.

[0069] In a preferred embodiment, the suspension of positively charged nanoparticles with DNase has a concentration between 0.1-0.5% w / v. In particular embodiments of the present invention, the suspension of positively charged nanoparticles with DNase has a concentration of 0.1%, 0.2%, 0.3%, 0.4% or 0.5%. In one embodiment of the present invention, the suspension of positively charged nanoparticles with DNase has a concentration between 1-5 mg / mL, preferably being a concentration between 1.5-4 mg / mL, and even more preferred between 2-4 mg / mL. In particular embodiments of the present invention, the suspension of positively charged nanoparticles with DNase has a concentration of 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL or 5 mg / mL.

[0070] In a preferred embodiment, the method comprises an additional step prior to step a) where an antifoam is added at a concentration of 0.1-1% to a suspension of negative nanoparticles, preferably between 0.4-1%. In preferred embodiments of the present invention, the amount of antifoam is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, 0.9% or 1%. Furthermore, in the present invention, the antifoam is selected from the group consisting of a silicone oil, also called liquid polymerized siloxane or polydimethylsiloxane (PDMS). In an even more preferred embodiment, in step a) 50-150 pL 0.1-1% of antifoam is added in a volume between 1-3 mL of a suspension of negative nanoparticles at a concentration of 1-5 mg / mL.

[0071] In another preferred embodiment, in step b) the suspension from the previous step is mixed with 10-15 mL of a suspension of positive Nanoparticles at a concentration of 0.28% w / v.

[0072] Preferably, the suspension of positive nanoparticles comprises silicone oil as an antifoaming agent in an amount between 25-1 OOpL, preferably between 40 and 80 pL, and even more preferably 50 pL.

[0073] The inventors have been able to verify that these parameters are key to the optimization of aggregates in several aspects:

[0074] • quantity of individual nanoparticles that form aggregates;

[0075] • aggregate composition, which is the proportion of each type of nanoparticle; and

[0076] • aggregate size and particle size distribution (in injectable / in aerosols).

[0077] In this way, the aggregate of nanoparticles object of the invention is obtained, which exhibits an integrity of the individual nanoparticles once they form part of the aggregate, which gives it the ability to remain stable within the bloodstream and disintegrate only under the necessary conditions. As described herein, said aggregate disintegrates by at least 30% starting from wall shear forces of 100 dyna / cm2, and said aggregate also exhibits surprising sensitivity to changes in shear force. In a preferred embodiment, the aggregate disintegrates by at least 30% of its total volume when the shear forces on the wall of a blood vessel are at least 100 dyna / cm2.

[0078] In one embodiment of the present invention, the method object of the invention further comprises two subprocesses prior to the process of obtaining the aggregate of nanoparticles according to the first aspect.

[0079] Based on this, in a particular embodiment of the present invention, there is a previous method for obtaining negative nanoparticles comprising rtPA according to the first aspect of the invention, where said method comprises the following steps: a') preparing an ethyl acetate solution of the PLGA polymer in a concentration between 1.5 and 2% w / v; preferably 2% w / v, b') preparing an aqueous solution of the PVA surfactant at 0.1-2% w / v and water, and adding rtPA in an amount between 8%-12%, preferably 10% w / v; c') adding the solution from step b') to the oily solution from step a') in a 1:20 ratio under sonication for 1 to 10 min at a temperature of 20°C - 25°C at 45-55 W and a frequency of between 50 and 60 Hz to obtain an emulsion; d') adding the emulsion from step c') to an aqueous phase of PVA at 0.1-2% w / v in a ratio of 1:5 under stirring at 18,000-24,000 rpm, preferably between 20,000-22,000 rpm.000 rpm and for a period between 30-180 seconds, preferably between 45-100 seconds; e') evaporating the acetate at room temperature under gentle magnetic stirring, preferably at a speed between 300-900 rpm, more preferably 450-600 rpm; f') collecting the negatively charged nanoparticles with rtPA by ultracentrifugation at 2-12°C, at a speed of 5,000-12,000 rpm, for 35-60 min; g') washing the obtained negatively charged nanoparticles with rtPA, preferably two consecutive washes.

[0080] In this way, negatively charged nanoparticles containing rtPA are obtained, which are sized 150-220 nm. In a preferred embodiment of the present invention, the negatively charged nanoparticles have a PZ of between -10 and 30 mV. In a preferred embodiment of the present invention, the negatively charged nanoparticles have an rtPA encapsulation efficiency of between 45 and 65%. In a preferred embodiment, the negatively charged nanoparticles have an rtPA content of 3-6% w / w relative to the weight of the nanoparticle.

[0081] In the context of the present invention, encapsulation efficiency (EE) refers to the amount of encapsulated enzyme relative to the amount initially bound; that is, this variable defines how effective the entrapment of the active agent within the nanoparticle is. In the context of the present invention, enzyme content or loading is understood as the amount of active agent within the nanoparticle relative to the weight of the polymer.

[0082] In a preferred embodiment of the subprocess for preparing the negatively charged nanoparticles, in step a') prepare a solution in ethyl acetate of the PLGA polymer at a concentration of 4% w / v, being even more preferred at a concentration of 80 mg / mL.

[0083] Likewise, in another particular embodiment of the present invention, there is a prior subprocess for obtaining DNase-positive nanoparticles, where said method comprises the following steps: a") preparing a solution in ethyl acetate of a mixture of PLGA polymers in an amount between 75-90% w / w and PLGA-PEG polymer in an amount between 10-25% w / w, the total polymer concentration being 0.5-1% w / v, preferably 1% w / v, and optionally an amino component in an amount between 6.28-12.5% ​​w / w, b") preparing an aqueous solution of PVA surfactant at 0.1-1% w / v and water, and adding DNase in an amount between 1% - 2% w / v, preferably 1.3% w / v;c") adding the aqueous solution from step b") to the oily solution from step a") in a volume ratio of 1:13 under sonication for 1 to 10 min at a temperature of 20°C - 25°C at 45-55 W and a frequency of between 50 and 60 Hz to obtain an emulsion; d") adding the emulsion obtained in step c) to an aqueous phase of PVA at 0.1-1% w / v in a ratio of 4:10 under stirring at a speed of between 18,000-24,000 rpm, preferably between 20,000-22,000 rpm and for a period of time of between 30-180 seconds, preferably between 45-100 seconds; e") evaporate the ethyl acetate at room temperature under gentle magnetic stirring, preferably at a speed between 300-900 rpm, preferably 450-600 rpm; f") collect the positively charged nanoparticles with DNase by ultracentrifugation at 2-12°C, at a speed of 5,000-12,000 rpm, for 35-60 min;g”) wash the obtained positively charged nanoparticles with DNase, preferably two consecutive washes.;

[0084] In this way, positively charged nanoparticles containing DNase are obtained with a size of 190-310 nm, with a PZ of +10 to +30 mV and a DNase encapsulation efficiency of between 40-60%. In a preferred embodiment, the positively charged nanoparticles have a DNase content of 2.5-7% w / w relative to the nanoparticle weight. In a preferred embodiment, in step b"), 4 mg of DNase are added in 300 pL of the aqueous co-solution of the PVA surfactant at 0.1-1% w / v.

[0085] Finally, both positively charged and negatively charged nanoparticles can be preserved at -20°C by adding a 5% w / v trehalose solution that acts as a cryoprotectant.

[0086] A third aspect of the present invention is a novel pharmaceutical composition comprising the nanoparticle aggregate described herein and further comprising one or more pharmaceutically acceptable excipients. In other preferred embodiments of the present invention, the pharmaceutical composition comprises an additive selected from the group consisting of trehalose, sugar, low molecular weight alcohol, dimethylsulfoxide, or a combination thereof. In the present invention, trehalose is used as a cryoprotectant.

[0087] In a preferred embodiment, the pharmaceutical composition may be presented in the form of lyophilized powder.

[0088] In preferred embodiments, where the pharmacological composition is administered intravenously, the composition is a solution in water for injection, the concentration of aggregates being 1-10 mg / mL, and preferably at a concentration of 2.5 mg / mL. The administration would be single, or in other words, it is administered only once, with a corresponding dose of rtPA of 10 mg / kg and DNase of 40,000KUnit / kg, which corresponds to 46 mg / kg.

[0089] In the context of the present invention, water for injection is a sterile water that serves as a vehicle for the dilution and reconstitution of medications that are administered parenterally or intravenously.

[0090] In preferred embodiments, where the pharmacological composition is administered by inhalation, the composition is a saline solution with an aggregate concentration of 2.5-15 mg / mL, preferably at a concentration of 5 mg / mL. Administration is carried out using a vibrating membrane nebulizer. In a preferred embodiment, the saline solution comprises 0.9% w / v NaCl. The inventors have therefore been able to develop a truly advantageous system compared to what is known in the treatment of thromboembolic diseases, since, thanks to the nature of the nanoparticle aggregate according to the first aspect of the invention, this system is surprisingly sensitive to shear forces.

[0091] Thanks to this sensitivity that the aggregate object of the invention presents, the changes in shear force in the stenosed area of ​​a blood vessel cause the aggregates to disintegrate right in the stenosed area of ​​a blood vessel, allowing the sustained co-release of the active agents, also called in this document, the therapeutic enzymes with synergistic fibrinolytic effect, which are rtPA or DNase.

[0092] In the context of the present invention, a stenosed area of ​​a blood vessel is understood to be an area that presents a narrowing in its diameter.

[0093] In a fourth aspect, the present invention relates to the aggregate of nanoparticles for use as a medicament. Likewise, the present invention relates to the use of the aggregate of nanoparticles for the manufacture of a medicament for the treatment of thromboembolic diseases.

[0094] In a preferred embodiment, the present invention relates to the use of nanoparticles in thromboembolic diseases, preferably venous thromboembolic diseases (VTEs). In particular embodiments, these diseases are selected from the group consisting of stroke, ischemic heart disease, peripheral arterial embolism in the lower or upper limbs, arterial embolism in abdominal vessels, retinal arterial or venous thrombosis, venous thromboembolic disease, deep vein thrombosis (DVT) and superficial vein thrombosis in the lower and upper limbs, and pulmonary embolism (PE).

[0095] In the context of the present invention, venous thromboembolism is a condition that occurs when a blood clot forms in a vein.

[0096] In the context of the present invention, inflammatory and / or thrombotic material is a thrombus composed of fibrin networks and NETs that include aggregated platelets and coagulation factors. Another embodiment of the present invention is a pharmacological composition for use in thrombotic diseases, wherein said composition is administered intravenously or by inhalation.

[0097] From the technical characteristics of the invention described in this document, the following technical advantages are derived:

[0098] • allows co-administration of two agents with proven enhanced fibrinolytic effect.

[0099] • allows sustained fibrinolysis, especially useful in patients at intermediate-high risk of pulmonary embolism.

[0100] • It is sensitive to endogenous mechanical stimuli, ensuring the arrival and release of both active ingredients at the therapeutic target at the same time.

[0101] • Since it is based on passive targeting, it will not depend on the levels of the target molecule expressed and / or produced in the therapeutic target, thus avoiding possible competition for the target in the event that the two types of nanoparticles were administered simultaneously and individually.

[0102] PREFERRED EMBODIMENTS OF THE INVENTION

[0103] For illustrative purposes and in order to better understand the invention, the following examples are described below.

[0104] Example 1. Synthesis of nanoparticle aggregates

[0105] The nanoparticles were prepared by the modified solvent emulsion-evaporation method.

[0106] The negative nanoparticles are composed of a 50:50 PLGA (Resomer 504H®). The positive nanoparticles are composed of a mixture of PLGA and PLGA-PEG. The PLGA used is Resomer(R) 502, and the PLGA-PEG (Sigma) type used is composed of 50% glycolic acid and 50% lactic acid (Mn 11,500 Da), with covalently bonded PEG molecules of Mn 2,000 Da.

[0107] In a particular embodiment, stearylamine (5 mg) is used to modulate the electrical charge of the positive nanoparticles. The method comprises the following steps: (i) preparation of an ethyl acetate solution of a mixture of PLGA for the rtPA and in a mixture of PLGA and PLGA-PEG polymers for the DNase, (ii) preparation of an aqueous co-solution of the PVA surfactant at 0.5% w / v and the rtPA or DNase; (iii) addition of the aqueous phase to the oily phase under sonication; (iii) addition of the emulsion obtained to an aqueous phase of 0.5% PVA under high speed stirring (1 min, 24,000 rpm); (iv) evaporation of the acetate at room temperature under gentle magnetic stirring; (iv) collection of nanoparticles by ultracentrifugation (4°C, 10,000 rpm, 45 min) and washing with distilled water (2 cycles).

[0108] An organic phase consisting of a solution of the polymer in 2 ml of ethyl acetate (1.5% w / v) was prepared. An aqueous phase 1 consisting of a 0.5% w / v PVA solution in which 5 mg (6.25% w / w) of rtPA or 4 mg of DNase were dissolved was prepared.

[0109] This aqueous phase 1 is then emulsified onto the organic phase under sonication for 1 min. This emulsion is then re-emulsified under high-speed stirring (IKA, 20,000 rpm) in a second aqueous phase of 10 ml of PVA in an ice bath. The ethyl acetate was then evaporated by gentle stirring for 4 h at room temperature. After this process, the nanoparticles were washed with milliQ water and collected by ultracentrifugation (4°C, 60 min, 10,000 rpm, Eppendorf). Finally, the collected pellet was resuspended in 1 mL of a 5% w / v trehalose solution (VWR) and stored at -20°C.

[0110] To control the nanoparticle production process, empty particles without drug were prepared using the same methods described above.

[0111] The size and zeta potential of the nanoparticles were measured using dynamic light scattering (DLS) (Zetasizer Nano Z, Malvern, UK). A 100 pL aliquot of the nanoparticle suspension was added to a volume of 1 mL of milliQ water for DLS analysis.

[0112] The negative PLGA nanoparticles showed a particle size of 180 nm and a PZ of -20 mV. The positive PLGA-PEG nanoparticles showed a particle size of 270 nm and a PZ of +20 mV, and did not show significant differences with respect to the empty particles. In a particular embodiment, 3 mL of positive nanoparticles (2.82 mg / mL) are added to 10 mL of a suspension of negative nanoparticles (2.72 mg / mL) under stirring at 20,000 rpm for 60 seconds, controlling the temperature (Figure 1).

[0113] In a particular embodiment, a volume of silicone oil is added to the positive nanoparticles as an antifoaming agent (25-50-100 pL, preferably 50 pL).

[0114] In a particular embodiment, the aggregates are collected after a centrifuge spin at 2,000 rpm for 5 min. This results in aggregates of micron-sized particles (5-7 pm diameter), spherical and hollow, composed of 50% of each type of nanoparticle (Figure 2).

[0115] Example 2. Determination of the release profile of rtPA and Dnase from each type of nanoparticle

[0116] The fibrinolytic enzymatic activity of rtPA after the encapsulation process was evaluated in 12-well fibrin plates in which different doses of free and encapsulated drug were incubated at 37°C. After predetermined times, photographs of the plates were taken and the lysis halo produced was measured. Figure 3a, as an example, shows the lysis halo produced by doses of 5 and 10 pg of free and encapsulated rtPA. The image shows that, while the free drug has completely lysed the contents of the well, the encapsulated drug has not. This is because the active ingredient is released in a controlled manner from the nanoparticle, allowing for sustained lysis. The release profile was determined by analyzing the images of the fibrin plates and measuring the lysis halo using image analysis software (Image J). Figure 3b shows the release profile of the encapsulated drug versus free rtPA.

[0117] In the case of positive particles containing DNase, enzyme activity was determined on DNase agar plates with Toluidine Blue, used as a marker for deoxyribonuclease activity. To do this, the agar was prepared in Petri dishes according to the manufacturer's specifications. The sample to be tested was then incubated at 37°C. After predetermined times, the blue halo on the plate was evaluated. From these data, a graph can be constructed (Figure 4) to further evaluate the enzyme release profile from the nanoparticle.

[0118] In both cases, controlled release profiles were obtained for both drugs. Example 3. Determination of the mechanical properties of the aggregates

[0119] The obtained nanoparticle aggregates (1-5 pm) have been evaluated in vitro to check their sensitivity to changes in shear forces and to changes in hemodynamic flow using a 3D poly-dimethylsiloxane (PDMS) microfluidic system (Figure 5) where HUVEC (Human Vein Endothelial Cells) cells were cultured. Depending on the inlet speed of the formulation into the chip, and therefore the shear force, we can simulate 3 obstruction conditions: low, medium, and high.

[0120] To do this, the chip was coated with type I collagen (1 h, 37°C) at a rate of 50 pg / mL. Excess collagen was removed by washing with PBS using a low-speed syringe pump.

[0121] HUVEC cells were then seeded in suspension and left to rest for 4-24h. After 24h in static conditions, the cells were subjected to flow at a rate of 50 pL / h with culture medium. At this point, the cells modified their morphology from elongated to rounded in response to shear forces, simulating the physiological condition in vivo. To monitor the process, cell nuclei were labeled with Hoechst (1:1000 dilution). Next, a suspension of irrigated microorganisms consisting of positive nanoparticles labeled with Nile Red and negative particles labeled with FITC was introduced at a rate of 3.3 mL / min. This is equivalent to a shear force of 32,000 s-1. A Nikon Eclipse Ti-E fluorescence microscope was used for observation and analysis.

[0122] In addition, the particle size and size distribution (Partica Horiba) of the particle were evaluated after passing through the chip (Figure 6) and it was found that approximately 70% of the aggregates broke into individual nanoparticles.

[0123] Example 4. Hemocompatibility of aggregates

[0124] The resulting nanoparticle aggregates (1-5 μm) were evaluated in vitro to verify their hemocompatibility. Both hemolysis and platelet aggregation assays were performed.

[0125] 4.1. Hemolysis

[0126] The study was designed to assess the hemolytic capacity of different nanoparticle formulations. Briefly, blood collected from healthy volunteer donors was centrifuged for 5 minutes at 900g to remove the serum fraction. Once centrifuged, the serum was removed and the removed volume was replaced with 0.9% w / v NaCl for washing. After two washes, 2x108 red blood cells / mL were counted and the procedure was carried out incubating for 2 hours at 37 °C with the nanoparticle formulations at different concentrations (250, 100, 50, 25, 10, and 5 ug / ml) (1:1). PBS was used as a negative control (0% hemolysis), and a 1% w / v Triton X-100 solution was used as a positive control (100% hemolysis).

[0127] After incubation, centrifuge the samples (5 minutes, 900g) and read the absorbance of the supernatant in a plate reader at 545 nm.

[0128] The percentage of hemolysis is calculated as follows:

[0129] * 100

[0130] As can be seen in Figures 7a and 7b, none of the concentrations or formulations tested caused hemolysis of erythrocytes when incubated with them, compared to the positive control (Triton X-100 1% w / v). No significant differences were obtained in any type of nanoparticles or aggregates at any of the concentrations studied with respect to the negative control (PBS), suggesting that the formulations are perfectly compatible with the red series.

[0131] On the contrary, the positive control did present significant differences with the rest of the formulations (100% hemolysis).

[0132] 4.2. Platelet aggregation

[0133] For the platelet aggregation assay, platelet-rich plasma (PRP) must first be separated from platelet-poor plasma (PPP) by gentle centrifugation of whole blood. Once the PRP is obtained, the platelet count is adjusted to 200–250 x 109 platelets / liter. A PRP blank is set in each channel of the aggregometer, and 225 pL of PRP and 25 pL of each of the particle concentrations are added.

[0134] The calibration for light transmission through PPP (clear plasma) is set to 100%; the calibration for light transmission through PRP (platelet-rich plasma, turbid or opaque plasma) is set to 0%.

[0135] Once the plasma is positioned as described above, a platelet agonist (in our case, ADP) is added to the platelets, which causes them to aggregate. This results in an increase in light transmission (a decrease in plasma turbidity), which is detected and recorded as a function of time after the agonist is added. The greater the platelet aggregation, the greater the light transmission, and vice versa.

[0136] As observed in Figure 8a and 8b, for all types of Nanoparticles studied (white positive and negative, charged with positive and negative F and white and charged aggregates), no platelet aggregation percentages higher than 7% were obtained at any of the concentrations studied.

[0137] After the statistical study (one-way ANOVA for each particle type), no significant differences were observed between any of the concentrations studied in any case.

[0138] 5.1. Lymphocyte proliferation

[0139] The toxicity of the formulations on lymphocytes was carried out by studying their proliferation with the CelITiter 96® AQueous One Solution (MTS) reagent and using phytohemagglutinin (1 pg / mL) as a positive control.

[0140] To obtain lymphocytes, human blood from healthy volunteers was used using a Ficoll gradient following the manufacturer's instructions.

[0141] Once the lymphocytes were obtained, they were seeded at 5 * 105 PBMC in 120 pL of RPMI-1640 medium in each well of 96-well plates.

[0142] The samples and controls tested were 80 pL of:

[0143] Negative control: RPMI-1640

[0144] Positive control: phytohemagglutinin (10 pg / mL).

[0145] White: RPMI without cells

[0146] Samples: Nanoparticles filtered by 0.22 um and resuspended with RPMI-1640 at 10-50 and 250 pg / mL.

[0147] The percentage of cell proliferation was calculated as the following formula:

[0148] % cell proliferation = ((Sample absorbance - Negative control absorbance) / (positive control absorbance - negative control abs)) x 100%.

[0149] Statistically comparing each of the formulations and concentrations with the culture medium (normal cell proliferation), no significant differences were found in any case, unlike the positive control. Therefore, neither the nanoparticle formulations nor the aggregate formulations at any concentration induced an exacerbated lymphocyte response or caused cell death (Figure 9).

[0150] 5.2. Activating the plugin

[0151] This assay monitors the cleavage of complement component C3 after activation by measuring the formation of its activation peptide, the C3a chain (C3a desArg), using a commercially available C3a enzyme immunoassay kit. The assay was performed according to the manufacturer's instructions.

[0152] The results obtained (Figure 10) showed that the immunogenicity of the Nanoparticles (all types) is dose-dependent.

[0153] At low concentrations (5 and 10 ug / mL), no significant differences were observed between the different formulations (negative and positive charged and negative and positive white).

[0154] Regarding the aggregates (charged and blank), similar immunogenicity values ​​were obtained for all concentrations studied. No significant differences were found with respect to the negative control (SSF). This may be due to the larger size of the aggregates, since studies show that the larger the particle size, the lower the complement activation (reference: https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC37592).

[0155] Example 6. Additive fibrinolytic effect of both drugs

[0156] The additive thrombolytic capacity of both drugs has been demonstrated in vitro and ex vivo. Initially, clot generation was achieved in vitro using blood from volunteer donors. Blood was collected in tubes with EDTA (for neutrophil activation) and in tubes with coagulation activator (for clot formation). Neutrophils were activated with 100 nM PMA for 3 hours in a thermoblock at 37°C. They were then incubated with 250 pL of blood from the tube with activator for 30 minutes. The presence of neutrophils and extracellular networks of DNA and histones in histological sections of the incubated clots were observed by immunofluorescence with the anti-citrullulated Histone H3 antibody. The formed clot was incubated with 1 mL of serum from the same donor, together with the free drugs (12.5 pg / mL of tPA and / or 4 mg / mL of DNase) individually and in combination for 30, 45 and 60 minutes.

[0157] Incubation was carried out at 37°C with shaking at 70 rpm. Clot weight was measured at the beginning and after each incubation period with the drugs. The main results are shown in Figure 11, which shows the reduction in clot weight in % after incubation with rtPA and DNase at two different concentrations, at three different times (30, 45, and 60 min), and in combinations of the two drugs. The combination of the two drugs had a potentiating effect at a concentration of rtPA of 12.5 mg / mL + DNase 4 mg / mL after 45 minutes, reducing clot weight by 90%.

[0158] Ex vivo thrombi were generated from a mouse vena cava thrombosis model (Figure 12). This model was generated using the stasis model, in which the lateral collateral veins of the vena cava were ligated, followed by cauterization of the posterior collaterals, and finally, the vena cava was ligated below the left renal vein. The thrombi obtained were incubated in 1 ml of RPMI 1640 medium together with 25 pg / ml of tPA and / or 2 mg / mL of DNase, individually and in combination, for 1, 2, 3, and 4 hours. The thrombi were weighed at the beginning and after the incubation times. The decrease in weight is graphically represented in Figure 13.

Claims

CLAIMS 1. A nanoparticle aggregate characterized in that it comprises negatively charged nanoparticles and positively charged nanoparticles, wherein: said negatively charged nanoparticles comprise rtPA, and said positively charged nanoparticles comprise DNase.

2. The aggregate according to claim 1, wherein said amount of rtPA in the negatively charged nanoparticles is between 3 and 6% by weight with respect to the total negatively charged nanoparticles.

3. The aggregate according to claim 1 or 2, wherein said amount of DNase in the positively charged nanoparticles is between 2.5 and 7% by weight with respect to the total number of positively charged nanoparticles.

4. The aggregate of nanoparticles according to any one of claims 1 to 3, wherein said aggregate is spherical and hollow.

5. The aggregate of nanoparticles according to any one of claims 1 to 4, wherein the positively charged nanoparticles and negatively charged nanoparticles are in a ratio of between 1:1 and 1:

5.

6. The aggregate of nanoparticles according to any one of claims 1 to 5, wherein the aggregate has a diameter size of 5 to 7 pm.

7. The nanoparticle aggregate according to any one of claims 1 to 6, wherein the negatively charged nanoparticles comprise a polymer of lactic and glycolic acid (PLGA).

8. The aggregate of nanoparticles according to any one of claims 1 to 7, wherein the positively charged nanoparticles comprise a copolymer of lactic and glycolic acid (PLGA), and lactic and glycolic acid with polyethylene glycol (PLGA-PEG).

9. The nanoparticle aggregate according to claim 8, wherein the positively charged nanoparticles comprise an amino compound in an amount of between 6.28-12.5% w / w.

10. The aggregate of nanoparticles according to any one of claims 1 to 9, wherein the positively charged nanoparticles have a degree of pegylation between 10%-60%.

11. The aggregate of nanoparticles according to any one of claims 1 to 10, the negatively charged nanoparticles having a zeta potential between -10 and -30 mV.

12. The aggregate of nanoparticles according to any one of claims 1 to 11, wherein the positively charged nanoparticles have a zeta potential between +10 and +30 mV.

13. A method for obtaining negatively charged nanoparticles with rtPA from a nanoparticle aggregate according to claim 1, comprising the following steps: a') preparing an ethyl acetate solution of the PLGA polymer in a concentration between 1.5 and 2% w / v; b') preparing an aqueous solution of the PVA surfactant at 0.1-2% w / v and water, and adding rtPA in an amount between 8%-12%; c') adding the solution from step b') to the oily solution from step a') in a 1:20 ratio under sonication for 1 to 10 min at a temperature of 20°C - 25°C at 45-55 W and a frequency of between 50 and 60 Hz to obtain an emulsion; d') add the emulsion from step c') to an aqueous phase of PVA at 0.1-2% w / v in a 1:5 ratio under stirring at 18,000-24.OOOrpm and for 30-180 seconds; e') evaporating the ethyl acetate at room temperature under stirring at a speed between 300-900 rpm, being more preferred 450-600 rpm; f') collecting the negatively charged nanoparticles with rtPA by ultracentrifugation at 2-12°C temperature, at a speed of 5,000-12,000 rpm, for 35-60 min; g') washing the obtained negatively charged nanoparticles with rtPA.

14. A method for obtaining positively charged nanoparticles with DNase from a nanoparticle aggregate according to claim 1, comprising the following steps: a”) preparing a solution in ethyl acetate of a mixture of PLGA polymers in an amount between 75-90% w / w and PLGA-PEG polymer in an amount between 10-25% w / w, the total polymer concentration being 0.5-1% w / v, preferably 1% optionally amino component in an amount between 6.28-12.5% w / w, b”) preparing an aqueous solution of the PVA surfactant at 0.1-1% w / v and adding DNase in an amount between 1% - 2% w / v; c") add the solution from step b") to the oily solution from step a") in a 1:13 ratio under sonication for 1 to 10 min at a temperature of 20°C - 25°C at 45-55 W and a frequency of between 50 and 60 Hz to obtain an emulsion; d") add the emulsion obtained in step c") to an aqueous phase of PVA at 0.1-1 % w / v in a 4:10 ratio under stirring at 18,000-24.OOOrpm and for a period of time between 30-180 seconds; e") evaporate the acetate at room temperature under stirring at a speed between 300-900 rpm; f") collect the positively charged nanoparticles with DNase by ultracentrifugation at 2-12 ° C of temperature, at a speed of 5,000-12,000 rpm, for 35-60 min; g") wash the obtained positively charged nanoparticles with DNase.

15. A method for obtaining a nanoparticle aggregate according to any one of claims 1 to 12, comprising the following steps: a) adding a suspension of negatively charged nanoparticles with rtPA obtained according to claim 13 to a suspension of positively charged nanoparticles with DNase obtained according to claim 14 in a ratio between 1:5 and 1:10, wherein said suspension of positive nanoparticles has a concentration comprised between 0.1-0.5% w / v and said suspension of positive nanoparticles has a concentration comprised between 0.1-0.5% w / v, b) mixing the suspension obtained in the previous step at a speed of 15,000-20,000 rpm for a time comprised between 40 to 60 seconds, and a temperature comprised between 15-20°C, c) centrifuge the solution obtained in the previous step at a speed between 1500-2500 rpm, for a period of 3 to 8 minutes, to obtain the aggregates.

16. A pharmaceutical composition characterized in that it comprises a plurality of nanoparticle aggregates according to any one of claims 1 to 12, and one or more pharmaceutically acceptable excipients.

17. The pharmaceutical composition according to claim 16, comprising an additive selected from the group consisting of trehalose, sugar, low molecular weight alcohol and dimethyl sulfoxy, or a combination thereof.

18. The pharmaceutical composition according to claim 16 or 17, wherein said composition is a solution in water for injectable preparation in a concentration of 1-10 mg / ml.

19. The pharmaceutical composition according to claim 18, wherein said composition is administered intravenously.

20. The pharmaceutical composition according to claim 16 or 17, wherein said composition is a saline solution in a concentration of 2.5-15 mg / ml.

21. The pharmaceutical composition according to claim 20, wherein said composition is administered by inhalation.

22. The pharmaceutical composition according to claim 16 or 17, wherein the composition is in the form of lyophilized powder.

23. The aggregate of nanoparticles according to any one of claims 1 to 12, for use as a medicine.

24. The aggregate of nanoparticles according to claim 23, for use in the treatment of a thromboembolic disease selected from the group consisting of stroke, ischemic heart disease, peripheral arterial embolism in lower or upper limbs, arterial embolism of abdominal vessels, retinal arterial or venous thrombosis, venous thromboembolic disease, deep vein thrombosis (DVT) and superficial vein thrombosis of lower and upper limbs, and pulmonary embolism (PE).

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