ES2577056B2 - Coverage of carbon nanotubes for use as an anchoring system for nano and micrometric devices with therapeutic activity - Google Patents

Abstract

Coverage of carbon nanotubes for use as an anchoring system of nano and micrometric devices with therapeutic activity. # The present invention relates to a coverage of carbon nanotubes for use as an anchoring system to the cell membrane and transport to the Cytosol of micro-nanometric devices with therapeutic activity. Also contemplated is a composition comprising a) a micro-nanometric device with therapeutic activity, and b) a coverage of carbon nanotubes covering the micro-nanometric device. Finally, said composition is contemplated for use in antineoplastic therapies and therapies aimed at alterations of the nervous system.

Description

DESCRIPTION

Coverage of carbon nanotubes for use as an anchoring system for nano and micrometric devices with therapeutic activity.

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Object of the invention

The present invention has its field of application within the health sector, in particular, it refers to the use of carbon nanotube covers as anchoring systems to the cell membrane and transporting to the cytosol of nano and 10 micrometric devices with therapeutic activity. In particular, its application is contemplated in neoplastic therapies and in therapies aimed at alterations of the nervous system.

Background of the invention

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Cancer is a highly heterogeneous disease where some rapidly dividing cells escape all genetic control mechanisms, resulting in innumerable random mutations (Navin et al. "Tumor evolution inferred by single cell sequencing", Nature 2011, 472, 90- 94). Some of these mutations are incompatible with cell survival, but others provide substantial genetic advantages that inevitably lead to resistance to different drugs. As a result, therapies targeting genes or proteins are only transiently effective until new adaptations or cell mutations emerge that elude the effect of the drug. For this reason, genetic resistance to chemotherapy is one of the problems that pharmacology faces in the medium term. 25

As a result of resistance, more and more doses of drugs must be used to ensure the removal of the most resistant cells from the tumor. These high doses are often unfeasible due to their toxicity.

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For example, paclitaxel (taxol®) favors the stabilization of microtubules and causes the death of actively dividing cells such as cancer cells. But, despite being one of the most used antitumor agents in recent decades. Paclitaxel has numerous disadvantages, such as its insolubility in saline solvents. At the systemic level, all these drugs also have innumerable disadvantages, since they are inhibitors of the proliferation of all body cells, which has important side effects in the patient being treated.

In the targeted application of therapies, the use of targeted nano-dispensers would allow, for example, drugs that are not soluble in physiological solvents due to their hydrophobicity, to reach their area of application intact. In this sense, nanotechnology provides completely new alternatives.

In other therapeutic areas, such as neurodegenerative diseases, the clinical failure of many pharmacological candidates is often attributable to delivery methods that do not cross, for example, the blood brain barrier (BHE). In the case of the brain, passive diffusion of many drugs is restricted, which constitutes a major obstacle in the pharmacological treatment of the central nervous system (CNS). Therefore, methods that can improve the administration of drugs to the brain and nervous system are of great pharmaceutical interest. fifty

There are several therapy systems that use non-invasive methods, including drug manipulation that encompasses the transformation into lipophilic analogues, pro-drugs, chemical administration of drugs, administration of drugs mediated by vector-receptor or drug-mediated delivery and intranasal drug administration, which exploits the olfactory and trigeminal neuronal pathways to administer drugs to the brain.

On the other hand, invasive methods are also used such as the administration of direct intracranial drugs by intra-cerebral-ventricular, intracerebral or intrathecal administration after creating reversible openings in the head. All these methods 10 carry a significant biological risk so that, currently, other types of treatments are contemplated, such as targeted therapies, to ensure that these therapies can access the brain, or peripheral nervous system cells at the transcranial level. Some of these mechanisms are based on receptor-mediated endocytosis. fifteen

Some pathogens, such as viruses and certain bacteria, enter the central nervous system achieving direct physical passage through endothelial or neuronal cells to infect the brain using receptors. There are also toxins that preferentially interact with specific cell types to exert a wide range of biological effects on peripheral and central neurons. The mechanisms of interaction of these microorganisms or toxins with the cells of the nervous system can be used to design therapies that can enter the nervous system as ligands, generating innumerable designs of nanocarriers.

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Nanofilaments in general, and carbon nanotubes (NTCs) in particular, are very chemically stable. Like all nanomaterials, they have an extraordinary surface / weight ratio (approximately 1000-2000 m2 per gram of material).

NTCs represent a class of highly versatile materials that show 30 very interesting mechanical, thermal and electronic properties. NTCs are able to pass through tissues and penetrate inside cells by different mechanisms, mainly by endocytosis (Shi et al. "Cell entry of one dimensional nanomaterials occurs by tip recognition and rotation", Nature Nanotechnology 2011, 6, 714- 719; Lacerda et al. "Translocation mechanisms of chemically functionalized carbon 35 nanotubes across plasma membranes", Biomaterials 2012, 33, 3334-3343). The entry of NTCs into the cell depends on the molecules that are attached to its surface (functionalization).

The NTCs when they come into contact with biological systems are covered (that is, functionalized) with their components, constituting the so-called "biocorona" (Saptarshi et al. "Lnteraction of nanoparticles with proteins: relation to bio-reactivity of the nanoparticle ", Journal of Nanobiotechnology 2013, 11, 26). This bio-crown provides NTCs with biomimetic properties and causes cells to actively capture them inside via membrane receptors (Kostarelos et al. "Cellular uptake of 45 functionalized carbon nanotubes is independent of functional group and cell type", Nature Nanotechnology 2007, 2 108-113). There are many works that show how the protein coat can be modified to direct the NTCs to specific targets and thus be able to redirect these nanomaterials to target cells, for example that exhibit ligands that interact with cancer cells. This invention takes advantage of 50

These properties of the NTCs for the design of new nano-devices for targeted therapy and intracellular action.

The functionalization of nanofilaments with proteins allows to modify the physical properties of the surface of these nanomaterials, such as solubility and dispersibility, essential for them to exert their biological properties, allowing them to have a better interaction with the receptors (biological molecules) of the membranes of cancer cells (ES2478793B2).

In view of the need for alternative targeted therapies, the authors of the present invention, after an important work of experimentation, have developed a new composition of NTCs as a coating: a crown-shaped NTC coverage to facilitate the entry of micro and nanodevices inside the cells.

Within this application, in the state of the art there is also great interest in the 15 drug delivery systems encapsulated in nanoparticles that protect the drugs from degradation and which, combined with physical methods, significantly increase the penetration of the drug into target cells (for example, in the tumor), acting as vehicles thereof (Stephania Fleury Taveira and Renata Fonseca Vianna Lopez (2011). Topical Administration of Anticancer. Drugs for 20 Skin Cancer Treatment. Skin Cancers - Risk Factors, Prevention and Therapy, Prof. Caterina La Porta (Ed.), ISBN: 978-953-307-722-2, lnTech, Available from: http: llwwwintechopen.com/books/skincancers-risk- factors-prevention-and-therapy / topical -administration-of-anticancer-drugs-for-skin-cancertreatment).

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The composition of the present invention represents an important advance in the state of the art, since, unlike what has been disclosed so far, the NTCs act for their properties as anchoring micro and nanocapsules on the cell surface, subsequently favoring the outflow of these devices from the endo-lysosome and / or phago-lysosome into the cell interior, where progressive release of different therapies such as drugs or nucleic acids, for example interference RNA, would occur.

The composition of the invention therefore has important clinical applications, being able to be used in the adjuvant or neo-adjuvant treatment of many types of cancers or tumors, through different routes of application, as well as in therapies directed to other pathologies, such as of the nervous system

Description of the figures

Figure 1.- Diagram of intracellular input and release mechanism of model devices 40 surrounded by NTC coverage.

Figure 2.- Image of the nano-device model covered with the coverage of NTCs with transmission electron microscopy (left) and scanning (right).

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Figure 3.- Scanning electron microscopy image showing the interaction of the coverage of NTCs with the cell surface.

Figure 4.- Image of the nano-device released at the cytosolic level. Left: Images of confocal microscopy taken in a Z plane of the cells showing how the 50

nanodevices are located in the cell cytoplasm at the height of the nucleus. Right: measurement of the diameter of the nanodevices intracellularly

Figure 5.- Image of the nano-device released at the cytosolic level outside the endolysosomal vesicles. a) localization of cell lysosomes by specific staining (orange acridine). b) location of the nanodevices in the cytoplasm.

Figure 6.- Image of HeLa cells treated with nanodevices that release the therapy (dye) after being subjected to the reducing conditions of the endo-lysosomes.

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Description of the invention

Based on the needs of the technique in relation to the search for alternative targeted therapies, the authors of the present invention have developed a composition formed by a micro-nanometric device with therapeutic activity, surrounded by a coverage of carbon nanotubes that cover.

In this composition, the coverage of carbon nanotubes acts as an anchoring system to the cell membrane and transport to the cytosol of the device with therapeutic activity. twenty

In this way, these therapy systems are directed by the NTCs that, functionalized with certain ligands, are capable of introducing the therapies into certain cells selectively, and thus achieve high concentrations of drugs, image contrast agents, biotherapies or other intracellularly, providing an improvement in therapeutic performance at the local level and reducing unwanted side effects.

The carbon nanotubes that form the cover can be both multipared and single wall, and can have a diameter from 1 to 100 nm and a length from 30 hundreds of nanometers to microns.

The functionalization of the NTCs can be carried out with ligands, in particular proteins, peptides, nucleic acids, oligosaccharides or small molecules, which bind to cellular receptors of any nature, allowing micro-35 nanodevices to be directed to the target cells. In preferred embodiments of the composition of the present invention, the NTCs are functionalized with proteins. In particular, the functionalization can be carried out with serum, which allows modifying the physical properties of the surface of these nanomaterials, such as solubility and dispersibility, which are fundamental so that they can exert their biological properties, allowing them to have a better interaction with the receptors (biological molecules) of cancer cell membranes.

These proteins make the cells specifically recognize the nanotubes of the microstructure coverage as their own, actively endocitating them, so that, once in the cellular cytosol, they can act in numerous ways. If the nanotubes are free (not attached to the therapeutic device) they interfere with the dynamic processes of the microtubules forming beams. When this occurs, dividing cells that require a very active microtubular dynamics remain metaphase locked and eventually die. fifty

Likewise, it is possible to functionalize the NTCs by coating them with other types of proteins or peptides, for example microbiological interaction proteins that employ some pathogens, such as viruses and certain bacteria, which facilitate entry into the central nervous system, or certain toxins that preferably they interact with specific types of cells to exert a wide range of biological effects on peripheral and central neurons. The mechanisms of interaction of these microorganisms or toxins with the cells of the nervous system can be used to design therapies that can enter the nervous system as ligands, allowing the design of nano-vehicles "on demand".

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The devices with therapeutic activity used in the composition of the invention have dimensions, ranging from the range of nanometers to even microns, being useful in the administration of compositions for the treatment of tumors and other pathologies.

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The micro and nanodevices can be composed of hollow or solid micro-nanoparticles completely or partially coated by the carbon nanotubes. The holes may include different therapies, such as drugs or DNA-like nucleic acids. or RNA, for later release. The solids could be of various natures with therapeutic potential, for example nanoparticles that are heated by 20 infrared radiation effects, magnetic fields, nanoparticles with biocidal effects, etc.

Carbon nanotube coverage (NTCs) can be attached to the micro-nanometer device by:

i) covalent bonds.

ii) biodegradable polymers.

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iii) sensitive links to lysosome reduction processes, or

iv) links sensitive to the action of lysosome enzymes, the so-called acid hydrolases. These enzymes are only activated in the lysosome and in the membranous compartments of the intracellular degradative pathways. 35

In those embodiments in which the nanotube coverage is attached to the micro-nanometer device by means of covalent bonds (i), the nanotubes can in turn carry drugs and / or ligands anchored to their walls by means of sensitive links to reduction processes ( SS, disulfide bridges) or bonds whose rupture is catalyzed by 40 lysosome enzymes (amide bonds or peptide bonds). Breaking of the sensitive links to the reduction processes (S-S) occurs in the cell's entry pathways (endosomes-endolysomes).

Based on the coverage of nanotubes, and their use as anchoring of the micro-nanometer device to the cell membrane, the authors of the invention have developed compositions with different applications:

NTC coverage for the anchoring and entry of micro-nanometer devices with therapeutic activity in target cells. fifty

In this particular embodiment of the composition of the invention, the nanotube coverage is stably fixed to the surface of the micro or nanometric device by covalent bonds. Said covalent bonds are carried out through an amination (for example by the use of aminopropyl trimethoxysilane, APS) of the surface of the micro-nanometric devices combined with a reaction of 5 Steglich or carbodiimide method. In this way, the union is carried out by means of the link between the NH2 group of the nanodispenser and the COOH group of the NTCs.

Alternatively, click chemistry methods can also be used for anchoring NTCs to micro-nanometric devices (in particular copper catalyzed cycloaddition 10 between azides and alkynes, CuAAC). Click chemistry is defined as a chemical tool to generate substances quickly and reliably by joining small units together, and that inspired by nature does not respond to a specific reaction.

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The nature of these covalent bonds allows NTCs not to detach upon entry into the cellular cytosol.

In this embodiment of the composition of the invention, the coverage of NTCs completely surrounds the micro-nanometer device thus acting as an anchoring system to the cell membrane, and in turn facilitating the release of the drug in the cytosol of the cells. Diana.

NTC coverage of the anchor, entry and intravesicular / cytoplasmic release of therapies in target cells based on reducing conditions. 25

In this particular embodiment of the composition of the invention, the NTCs of the cover, in addition to being fixed to the surface of the micro-nanometer devices by means of covalent bonds, are attached, by means of sensitive links to the processes of reduction in the lysosome or bonds whose rupture is catalyzed by lysosome enzymes, therapies (drugs and / or ligands) that will be released intracellularly.

Therapies and / or ligands are anchored to the walls of the NTCs through these sensitive links to intracellular reduction processes, such as disulfide bridges (SS), eg through the use of cystamine, thus creating this link on the coverage of NTCs, 35 although other strategies based on organic chemistry are also contemplated, or through bonds whose breakage is catalyzed by lysosome enzymes, facilitating their release.

Coverage of NTCs for anchoring, entry and intracellular release of NTCs for cytotoxic therapy.

In this particular embodiment of the composition of the invention, the micro-nanometer devices can be anchored to the NTCs by:

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a) biodegradable polymers, or

b) bonds that are sensitive to processes of reduction in the lysosome or links whose breakage is catalyzed by lysosome enzymes.

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In option a), the NTCs are fixed to the micro-nanometric device through the use of biodegradable polymers, such as biodegradable charged polyelectrolytes, which release the nanotubes in a directed and controlled manner upon degradation (for example, sodium dextran sulfate or hydrochloride of poly-L-arginine). In turn, the coverage of NTCs covers, totally or partially, the device as an anchoring system to the cell membrane, 5 also facilitating the release of the drug contained in the device in the cytosol.

In option b) the NTCs are fixed to the micro-nanometer device by means of bonds that are broken in a reducing medium, such as disulfide bridges (eg cystamine), or bonds whose rupture is catalyzed by lysosome enzymes, causing release. of the NTCs. Similarly, the coverage of NTCs serves as an anchoring system to the cell membrane and allows the release of therapy in the cytosol. In this case, the devices, which have the coverage of NTCs attached to their surface, by the action of enzymes and intracellular pH, will release their NTC coating between approximately 48-72 hours from the arrival at the intracellular level, so that the NTCs effect your 15 antiproliferative action.

In both cases, the therapeutic action would derive from the combination of the antiproliferative effect of the NTCs and the neoplastic agents contained in the nanodispensers, when both are released. twenty

The composition of the invention has important clinical applications, being able to be used in antineoplastic therapies, in the adjuvant or neo-adjuvant treatment of many types of cancers or tumors, through different routes of application, as well as in therapies directed to other pathologies, such as those of the nervous system, those of the respiratory system, those of the digestive system and, in general, any tissue or organ.

Examples

Example 1. Obtaining SiO2 particles @ dye @ NTCs: 30

Synthesis of nanometric devices: SiO2 particles

(i) Silica spheres: following the Stober method, a solution of TEOS: EtOH: H2O: NH4OH (1.7: 18.2: 3.1: 1.97 mL) was vigorously stirred for 2 h to obtain a white cloudy suspension. The monodisperse spheres (-500 nm) were washed with ethanol by 3 redispersion / centrifugation cycles (9000 rpm, 10 min), and finally dispersed again in 25 mL of water. The final concentration was 0.0174 g / mL.

(ii) Coloring with silica particles: silica particles were coated with APS by diluting 87 mg of SiO2 in 10 mL of EtOH and adding 0.5 mL of APS. After stirring 3 h, the excess was removed by 3 redispersion / centrifugation cycles (7000 rpm, 20 min). After this, the SiO2 particles functionalized with APS were diluted in 10 mL of EtOH and added to 10 mL of a solution of rhodamine B isothiocyanate (0.32 mg / mL). After stirring 3 h, the excess 45 was removed by 3 redispersion / centrifugation cycles (7000 rpm, 20 min). The final concentration was 8.7 mg / mL.

(iii) SiO2 particle coating: 10 mL of previously prepared SiO2 spheres (8.7 mg / mL), diluted with water to 20 mL and a solution of polydialyldimethylamine chloride (PDDA) was added to 60 mL of 50 ) (1 mg / mL containing NaCI

0.5 M) under weak sonication. The reaction proceeded for 1 h to allow adsorption of the PDDA and then its excess was removed with 3 redispersion / centrifugation cycles (8000 rpm, 20 min). Polystyrene sulfonate (PSS) (1 mg / mL containing 0.5 M NaCl) was deposited on the coated SiO2 spheres in a similar manner and using the same conditions. The final concentration was 8.7 mg / mL.

Coating silica spheres with NTCs

The NTCs were pre-treated with acetone and ethanol to remove organic materials, frozen with N2 and lyophilized. After this, the carbon nanotubes were oxidized by the following procedure: 100 mg of NTCs were ultrasounded in 100 mL of a mixture of H2SO4 / HNO3 (3: 1) with a tip sonicator for 15 min and with a bath of ultrasound for 4 h. Then, the sample was washed with an aqueous solution of NaOH diluted with 3 redispersion / centrifugation cycles 15 (9000 rpm, 10 min). When the pH stabilized at 10, the sample was subjected to ultrasound with the tip sonicator for 2 h. Finally, the NTCs were dispersed in water, obtaining a stable dispersion of oxidized NTCs, mainly presenting carboxylic and hydroxylic groups on the walls, which give them a negative surface charge. The final concentration was 0.8 mg / mL. twenty

10 mL of SiO2 spheres previously functionalized with PSS / PDDA (8.7 mg / mL) were diluted with 100 mL of milliQ water. Subsequently, 2 mL of a 0.5 M NaCl solution and 5 mL of a dispersion of NTCs (0.8 mg / mL) were added. After an adsorption period of 15 h the excess of NTCs was removed by 3 cycles of centrifugation / redispersion (7000 rpm, 20 min). The final concentration was 3.8 mg / mL. Figure 1 shows the coverage of NTCs around the particles chosen as nanodispenser models, as well as the path of entry of devices with therapeutic activity until their therapy is released.

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NTCs coverage functionalization

The functionalization was carried out with serum proteins diluted in a saline solution, such as the cell culture medium, at a concentration of 20-30 mg / mL by moderate sonication in a tip bath for 2 min. 35

Example 2. Coverage of NTCs for anchoring and entry of nanodispensers of therapies in target cells.

Nanodispensers with a therapeutic content were prepared based on any of the technical solutions that exist in patents or in the literature. They were coated with the NTCs as indicated in the previous example, joining the nanotubes either by electrostatic forces (using charged polyelectrolytes) or by covalent bonds (eg, between the carboxylic group of the oxidized nanotubes and the amino group with which functionalized the nanodispenser). They were functionalized with serum proteins 45 or with ligands destined for binding with molecular targets of the cancer cell surface (Scott et al. "Antibody therapy of cancer", Nature Reviews Cancer 2012, 12, 278-287).

The HeLa cell line was used as the object of the study and subjected to a solution of 50 5 μg / mL of the functionalized nanodispensers with whey proteins as

indicated in the previous example. The treatment lasted 96 h. During the treatment the cells were observed in vivo and also fixed with 4% paraformaldehyde, permeabilized with 1% triton X100 in PBS and stained with Hoechst 33342 for observation by high-resolution microscopy. The result of this study showed how the corona coating of NTCs in these structures favors internalization 5 (Figure 2 and 3) and cytosolic intracellular release of these nanostructures (Figure 4) in cellular models.

Example 3. Coverage of NTCs for the intravesicular / cytoplasmic anchorage, entry and release of target cell therapies based on reducing conditions. 10

The nanodispensers were prepared (in this case any nanometric structure where NTCs can be anchored) is based on any of the technical solutions that exist in patents or in the literature. They were coated and functionalized, as indicated in Example 1. In this case the NTCs were stably anchored, either by electrostatic forces (using charged polyelectrolytes) or by covalent bonds (eg, between the carboxylic group of the oxidized nanotubes and the amino group with which the nanodispenser was functionalized) to the nanometric structures. In turn, a therapy by a disulfide bond was coupled to the external surface of the NTCs, in this case a dye only detectable once released from the nanotube. Said therapy was released intracellularly due to the rupture of said link once the nanodispensers passed through the lysosomes. The nanodevices eventually escape from the lysosome to the cytosol (Figure 5). A proof of concept is shown in Figure 6 where cells are observed showing staining with a dye after their release from the carbon nanotube anchor. 25

Example 4. Coverage of NTCs for anchoring, entry and intracellular release of NTCs for cytotoxic therapy.

The nanodispensers were prepared (in this case any nanometric structure 30 where the NTCs can be anchored) is used based on any of the technical solutions that exist in patents or in the literature. These were coated, as indicated in Example 1, with biodegradable polyelectrolytes (sodium dextran sulfate as a positively charged polyelectrolyte and poly-L arginine hydrochloride as a negatively charged polyelectrolyte) and subsequently coated with the NTCs. The 35 NTCs that were used in the coating had a length of 500-5000 nm and after 48-72 h were released by the degradation of the polymers that act as an anchor to the nanostructure. Their release allowed them to interact with cell components biomimetically and produce a cytotoxic, antiproliferative effect.

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Claims (13)

1. Coverage of carbon nanotubes for use as an anchoring system to the cell membrane and transport to the cytosol of micro-nanometer devices with therapeutic activity. 5 2. Composition comprising: a) a micro-nanometric device with therapeutic activity, and  10 b) a coverage of carbon nanotubes covering the micro-nanometer device. 3. Composition according to claim 2, wherein the carbon nanotubes are functionalized with ligands. fifteen 4. Composition according to claim 3, wherein the carbon nanotubes are functionalized with proteins, peptides, nucleic acids or oligosaccharides. 5. Composition according to claim 4, wherein the proteins are microbiological interaction proteins of pathogens or toxins. 6. Composition according to any of claims 2-5 wherein the carbon nanotube cover is attached to the micro-nanometer device by means of:  25 i) covalent bonds, ií) biodegradable polymers, iii) sensitive links to lysosome reduction processes, or 30 iv) lysosome-sensitive enzymatic action bonds. 7. Composition according to claim 6, wherein the nanotube coverage is attached to the micro-nanometer device by means of covalent bonds (i). 35 8. Composition according to claim 7, wherein the nanotubes can in turn carry drugs and / or ligands anchored to their walls by means of sensitive links to processes of reduction in the lysosome or bonds whose breakage is catalyzed by lysosome enzymes. 40 9. Composition according to claim 6, wherein the biodegradable polymers are selected from sodium dextran sulfate and poly-L-arginine hydrochloride. 10. Composition according to claim 6 wherein the bonds sensitive to reduction processes used are disulfide bridges. 11. Composition according to claim 6 wherein the bonds whose breakage is catalyzed by lysosome enzymes are peptide bonds or amide bonds.  fifty 12. Composition, according to claims 2-11, for use in antineoplastic therapies. 13. Composition, according to claims 2-11, for use in therapies aimed at alterations of the nervous system. 5