WO2012101309A1

WO2012101309A1 - Peptide-functionalised nanoliposomes

Info

Publication number: WO2012101309A1
Application number: PCT/ES2012/070037
Authority: WO
Inventors: David POZO PÉREZ; Rebecca KLIPPSTEIN MARTIN; Ricardo GONZÁLEZ CAMPORA; Inmaculada TRIGO SÁNCHEZ; María Teresa VARGAS DE LOS MONTEROS
Original assignee: Fundación Progreso Y Salud; Universidad De Sevilla; Servicio Andaluz De Salud
Priority date: 2011-01-24
Filing date: 2012-01-24
Publication date: 2012-08-02

Abstract

The invention relates to nanoliposomes functionalised with bioactive peptides on the surface thereof, which allow the identification of target cells and/or tissues and the release of drugs in a selective manner. More specifically, the invention relates to nanoliposomes functionalised with the VIP peptide. In addition, the invention relates to pharmaceutical compositions comprising said nanoliposomes, to a preparation method thereof and to the medical uses of same.

Description

NANOLIPOSOMES FUNCTIONALIZED WITH PEPTIDES

FIELD OF THE INVENTION

The present invention is within the field of medicine, chemistry, biochemistry and immunology, and refers to the use of nanoliposomes functionalized with peptides on its surface that allow the identification of tissues and / or target cells and the release of drugs selectively, and in particular, to the functionalization of nanoliposomes with the VIP peptide. The present invention also relates to the compositions, the preparation process and the uses of said nanoliposomes.

STATE OF THE PREVIOUS TECHNIQUE

There is a growing interest in the biological effects of nanoparticles, their toxicity and their scope depending on the environment (M. Tsoli et al. 2005. Small (2005); 1: 841), for example, their potential use as a therapeutic tool in cancer treatments (El-Sayed et al., 2005. Nano Letters Vol. 5, 5, 829-834).

Thanks to the enormous interest of these nano-bioconjugates, a wide range of applications such as drug distribution, molecular markers, ultrasensitive biochemical analyzes, development of "lab-on-a-chip" devices, construction of electronic nanocomponents, nano engines have been developed -molecular ... etc [C. M. Niemeyer, C. A. Mirkin Eds. Nanobiotechnology, Wiley-VCH 2004].

Among the different types of nanoparticles, nanoliposomes are of special relevance because they are the best clinically established nanometric systems for the transport and shipment of drugs because they are not cytotoxic, they are biocompatible and biodegradable, and also their synthesis is relatively cheap and Easy scaling in industrial processes. Liposomes in general have been used in therapies to treat cancer for more than a decade since they have been shown to systematically reduce side effects, toxicity and facilitate drug elimination. (Torchlin, 2005. Nat Rev. Drug Discovery 4, 145).

Nanoliposomes can be used as carriers of various substances both outside and inside and for a variety of biomedical applications such as gene therapy or for drug delivery, so that acids nucleic or the drug are protected inside avoiding its enzymatic degradation and its direct contact with other healthy cells. On the other hand they allow the sending of biologically active molecules of lipophilic character and sizes greater than 500 Da, two of the most important problems that an active molecule can present to end up as an active ingredient of a medicine.

Nanoliposomes of various sizes, usually smaller than 400 nm, can quickly enter areas where tumors exist since the vascular endothelial wall is fenestrated. In contrast, nanoliposomes remain in the bloodstream of healthy tissue through the wall of the non-fenestrated vascular endothelium.

One of the limitations for the clinical use of peptides in general, mainly of neuropeptides, and VIP peptides in particular, is their short half-life in circulation, which would necessitate chronic administration of the same, increasing economic costs and making it difficult Your dosage to the patient. The binding of peptides to liposomes increases the half-life of molecules bound to them, since it hinders their proteolytic attack.

In addition, the functionalization of the nanoliposomes with peptides that would recognize with greater selectivity the therapeutic targets, would allow addressing specifically in those cases in which it is known that said targets overexpress the receptors that recognize said peptides.

There are currently functionalized liposomes with peptides on their surface. For example, liposomes functionalized with tuftsin have allowed increasing the efficacy of sodium stibogluconate (Agrawal & Gupta, 2000. Adv. Drug Deliv. Rev. 41: 135-146; Gupta & Haq, 2005. Met ods Enzymol. 391: 291- 304) and amphotericin B (Gupta & Haq, 2005. Methods Enzymol. 391: 291-304, Agrawal et al., 2002. J. Drug Target. 10: 41-45).

So far, two ways of preparing functionalized liposomes with peptides have been developed, in which peptide ligands bind to the terminal end of a PEG chain. The first method involves incorporating PEG-lipid conjugates with their functionalized end into the liposomes and then conjugate it with the peptide ligands (Zalipsky et al., Bioconjug. Chem., 1995, 6, 705-8). However, when the terminal group of the functionalized PEG is conjugated to the peptide ligands, non-homogeneous conjugation can occur if there is more than one reactive group in the ligand. The second method is to directly incorporate the peptide-PEG-lipid conjugate into the liposomal membranes (Zalipsky et al., Bioconjug. Chem., 1997, 8, 1 1 1-8). However, the synthesis of these conjugates is difficult, since the chemical properties of the side chains in the peptides are diverse, the molecular mass of the PEG is heterogeneous and the nature of the lipids is amphiphilic. These properties make it difficult to protect the side chains in the process of synthesis, purification, and reaction. In fact, very few peptide-PEG-lipid conjugates have been synthesized. Application US2007 / 0106064 describes the preparation of this type of conjugates through the use of a peptide resin in which the amino groups are initially protected.

On the other hand, the biological effects of the VIP neuropeptide have a growing interest in its modulating capacity in pathologies in which there is an inflammatory and / or autoimmune component [Grimm, M. C. et al. (2003) J. Immunol. 171, 4990-4994; Pozo, D. (2003) Trends Mol. Med. 9, 21 1-217; Ganea, D., and Delgado, M. (2002). Crit. Rev. Oral. Biol. Med. 13, 229-237; Delgado, M. et al. (2003). Trends Immunol. 24, 221-224; Pozo et al. (2009). J Immunol 183, 4346-4359].

In the specific case of the VIP peptide, the process of nanoliposome functionalization with this peptide faces the added problem of designing a procedure in which the carboxyl end of VIP is free, since it is at this end that it interacts with its specific membrane receptors. It is necessary, therefore, to develop a method of functionalization of nanoliposomes with peptides, leaving the carboxyl end of said peptide free to interact with its receptor.

BRIEF DESCRIPTION OF THE INVENTION

The authors of the present invention have developed nanoliposomes functionalized with peptides on their surface, stable, non-toxic, soluble in water, and compatible with biological systems, as well as a procedure for obtaining them, which are useful for vehiculating active ingredients and / or pharmaceutical compositions The procedure that leads to these nanoliposomes allows functionalization with the peptide so that the carboxyl-terminal end is free, thus leaving intact its ability to interact with its specific receptors, which allows formulating in vivo drug detection and selective release strategies. on cells that are interested in eliminating (as is the case with tumor cells), or expanding (through the administration of trophic factors) depending on the pathology that is intended to be addressed.

In particular, it has been shown that through the functionalization of the VIP peptide on the surface of the nanoliposome, its entry into areas with tumors is maximized due to the fenestrated vascular endothelium, while its specific targeting is allowed in those cases in which that cancer cells overexpress the receptors of the VIP family of type seven transmembrane domains coupled to G proteins, as is the case with prostate cancer. In addition to allowing VIP to act as a therapeutic agent on target cells or as a mode of drug release on cells that overexpress VIP receptors, it increases the half-life of the molecule bound to it, since it hinders proteolytic attack.

In addition, it has been shown that the functionalization of nanoliposomes with the VIP peptide increases the cytotoxic capacity of antitumor drugs encapsulated inside the nanoliposome, such as doxorubicin, improving the effectiveness of this active ingredient, which allows the incorporation of the drug into the liposomal composition at lower concentrations than those used in other state-of-the-art formulations, such as the case of non-functionalized liposomes or in solution.

Thus, a first aspect of the invention relates to a nanoliposome functionalized with a peptide on its surface, hereinafter, nanoliposome of the invention. In a preferred embodiment, the carboxyl-terminal end of the peptide is free, being able to interact with its receptor through its carboxyl-terminal end. In another preferred embodiment, the nanoliposome of the invention comprises:

a lipid-spacer-maleimide conjugate; Y

- a peptide modified with a sulfhydryl group;

so that the peptide is covalently bound to the nanoliposome through the reaction between the maleimide of the conjugate and the sulfhydryl group of the modified peptide.

In a particular embodiment, the nanoliposome further comprises an active ingredient. Said active ingredient can be hydrophilic, being incorporated into the aqueous core of the nanoliposome, or it can be hydrophobic, in which case it is incorporated into the lipid bilayer of the nanoliposome.

In a second aspect, the present invention relates to a procedure for the preparation of a nanoliposome as previously defined.

A third aspect of the invention is a functionalized nanoliposome obtainable according to the procedure described above.

In a further aspect, the invention relates to a pharmaceutical composition comprising a nanoliposome as previously defined and an active ingredient capable of diagnosing, curing, mitigating, treating or preventing a disease.

A final aspect of the invention relates to the use of the nanoliposome as previously defined for the preparation of a medicament for the treatment of diseases that occur with cell proliferation.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Structure of the vasoactive intestinal peptide neuropeptide (VIP).

Figure 2: Determination of VIP peptide on the surface of the nanoliposome loaded with a biologically relevant substance, in this case doxorubicin.

Figure 3: Characterization of the size of the naoliposomes by Dynamic Light Scattering (DLS).

Figure 4: Functional characterization of the interaction with VPAC receptors (cAMP production, second intracellular messenger of the VIP receptor / effector system) on tumor lines of prostate cancer.

Figure 5: Optical microscopy (20X) after 48 hours of treatment of DU-145 cells. (A) untreated cells; (C) and (E) show the cells after being treated with nanoliposomes and nanoliposomes functionalized with VIP respectively. In (B), (D) and (F) cells treated with soluble doxorubicin, doxorubicin encapsulated in the nanoliposome, and doxorubicin encapsulated in the nanoliposome functionalized with VIP are shown.

Figure 6: Histogram representing the percentage of lactate dehydrogenase (LDH) release in the DU-145 cell line after exposure to the drug doxorubicin (10 g / mL) for 24 hours.

DETAILED DESCRIPTION OF THE INVENTION The invention relates to a nanoliposome functionalized with a peptide on its surface, preferably where the carboxyl-terminal of the peptide is free, being able to interact with its receptor through its carboxyl-terminal end.

In the context of the present invention, nanoliposomes are understood as essentially spherical aqueous compartments, surrounded by at least one closed lipid double layer with an average diameter of less than 1000 nm. In the present invention, preferably less than 500 nm, more preferably less than 400 nm and even more preferably less than 200 nm.

"Medium diameter" means the average diameter of the population of nanoliposomes dispersed in an aqueous medium. The average diameter of these systems can be measured by standard procedures known to the person skilled in the art, and which are described, for example, in the experimental part below.

Preferably, the nanoliposomes have an average diameter between 75 and 125 nm, preferably between 85 and 1 nm, more preferably between 95-105 nm.

The nanoliposomes of the invention preferably comprise a lipid-spacer-maleimide conjugate. In the context of the invention, conjugate should be understood as the product resulting from the covalent bond between the three constituent components of the conjugate, that is, the lipid, the spacer and the maleimide.

Lipid

The term "lipid", as used herein, is a natural or synthetic amphipathic molecule that has a hydrophilic part and a hydrophobic part in the same molecule and that can spontaneously form bilayer vesicles in an aqueous medium or can be stably incorporated in lipid bilayers.

The term lipid comprises acylglycerides, cerids, phospholipids, lysophospholipids and glycolipids (cerebrosides and gangliosides). Examples of these lipids include stearylamine, dodecylamine, hexadecylamine, acetylpalmitate, glycerol ricinolate, hexadecyl myristate, isopropyl myristate, amphoteric acrylic polymer, fatty acid amides, cholesterol, cholesterol ester, diacylglycerolsuccinate, fatty acid glycerol and similar fatty acid.

Also, cationic lipids consisting of a positively charged terminal group, such as an amine, polyamine or polylysine, may be employed attached to a portion lipophilic neutral character, such as a sterol, a hydrocarbon chain or two hydrocarbon chains. Examples of these cationic lipids include 1,2-dioleyloxy-3- (trimethylamino) propane (DOTAP); N- [1- (2,3-ditetradecyloxy) propyl] -N, N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N- [1- (2,3-dioleyloxy) propyl] -N, N - dimethyl-N-hydroxyethylammonium (DORIE), N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA) and dimethylammonium bromide (DDAB).

In a preferred embodiment, the term lipid refers to a phospholipid. The term "phospholipid" comprises phosphoaccylglycerols, that is, compounds that are composed of a glycerol molecule, two of whose hydroxyl groups are esterified by fatty acids (saturated or partially unsaturated straight chain monocarboxylic acids with 8 to 28 atoms of carbon), the third hydroxyl group being esterified by a phosphate group that binds glycerol to another organic molecule through a phosphodiester bond, which usually contains nitrogen, such as choline, serine or ethanolamine, and which may have an electric charge. Examples of phosphoacylglycerols are phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, phosphatidylcholine and phosphatidylserine. Also included within the term phospholipids are those complex mixtures extracted from natural products that essentially comprise phosphoacylglycerols such as lecithin.

In a preferred embodiment of the present invention, the phospholipid is selected from 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine (DSPE), 1, 2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1, 2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE) and combinations thereof.

Spacer

As a spacer, a linear hydrophilic polymer with functional terminal groups capable of binding to the lipid and the amino group of the maleimide can be used. Suitable spacers in the present invention include, but are not limited to, polyglycine, polyethylene glycol, polypropylene glycol, polymethyl acrylamide, polydimethyl acrylamide, polyhydroxyethyl acrylate, polyhydroxypropyl methacrylate and polyoxyalkene. Preferably, the spacer is polyethylene glycol (PEG).

The conjugation of the lipid with the PEG requires that both the PEG and the lipid have a suitable functional terminal group. When the resulting binding between both components is an amino group (lipid-NH-PEG) the PEG can be functionalized with a halide or a sulphonate so that it is coupled with an amino terminal group of the lipid. When the resulting union between both components is a urethane (lipid-NHC (O) O-PEG) group, the PEG can be functionalized by an activated carbonate (-C (O) -imidazolyl, -OC (0) -para-nitrophenyl, - OC (0) -succinimide, -OC (0) -2,4,4-trichlorophenyl) such that it is coupled with an amino terminal group of the lipid. When the resulting binding between both components is an amido group (lipid-NHC (O) -PEG) the PEG can be functionalized by an activated carboxyl group (carboxyl group activated by DCC (dichlorohexylcarbodimide) / HOBt (N-hydroxybenzotriazole), DCC / DMAP (dimethylaminopyridine), DIPCDI (1,3-diisopropylcarbodiimide) / HOBt, EDC (1 - (3-dimethylaminopropyl) -3-ethyl-carbodiimide)) / NHS (N-hydroxysuccinimide)) so that it is coupled with an amino terminal group of the lipid.

In a preferred embodiment, the phospholipid binding with the PEG is carried out through an amide bond, resulting from the reaction between the carboxyl group with which the PEG is functionalized and the amino terminal group of the organic phospholipid molecule.

For its part, the conjugation of the PEG with the maleimide molecule also requires that the PEG at the other end have a suitable functional terminal group capable of reacting with the maleimide amino group. For this, any of the aforementioned functional groups can be used.

In a preferred embodiment, the lipid-spacer-maleimide conjugate has the following structure:

which corresponds to 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [amino (polyethylene glycol) -maleimide] or (DSPE-PEG-maleimide).

Said conjugate is commercially available.

Peptide

According to the present invention, the peptide is a synthetic or natural compound peptide by a chain of amino acids. In a particular embodiment, the peptide is selected from the group consisting of hormones, cytokines, toxins, quemotaxins and peptides of the extracellular matrix for cell adhesion.

Said peptide can bind to a receptor such as somatostatin receptors, vasoactive intestinal peptide receptors, integrin receptors or growth factor receptors, among others.

In a preferred embodiment, the peptide is selected from a list comprising: glucagon, gastric inhibitory polypeptide (GIP), secretin, growth hormone, somatoliberin, somatotropin, PHI peptide (peptide histidine isoleucine), PHM peptide (peptide histidine- methionine), PACAP (pituitary adenylate cylase-activating peptides), adrenomedulin, corticostatin and vasoactive intestinal peptide (VIP). Preferably, the peptide is a neuropeptide, and more preferably it is the vasoactive intestinal peptide (VIP) whose structure is represented in Figure 1.

As understood herein, a neuropeptide refers to small protein-like molecules of a peptide bond of two or more amino acids and which differ from proteins by their length, and because they originate from cerebral synaptic transduction. Its size can vary from two amino acids, such as carnosine, to more than forty such as CRH (corticotropin releasing hormone). They have both stimulating and inhibitory brain function, producing effects such as analgesia, appetite or sleep, among others.

In this report, vasoactive intestinal peptide (VIP) is understood as a polypeptide hormone formed by 28 amino acid residues and produced by many structures of the human body such as the digestive system, the pancreas and the suprachiasmatic nucleus of the hypothalamus in the brain. It is characterized by its vasodilator property and its activity in the peripheral nervous system (for example, VIP relaxes the lungs, trachea and gastric musculature). It inhibits the secretion of gastric enzymes and stimulates the secretion of glucagon, insulin and somatostatin, increases adenyl cyclase, as well as bile secretion in the liver.

The VIP is also referred to as PHM27 or MGC13587. It is encoded by a gene found on chromosome 6 (6q25). Its amino acid sequence is found in SEQ ID NO: 1. In the context of the present invention, VIP is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 1, and which would comprise various variants from: a) acid molecules nucleic encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1,

b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),

c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code,

d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 1, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the VIP peptide.

Three VIP receivers known as VPAC1, VPAC2 and PAC1 are known. The VPAC1 receptor is expressed in malignant epithelial neoplasms, lung cancer and other cancers such as stomach, colon, breast, prostate, liver and urinary bladder, while VPAC2 has only been found in a few tumors. PAC1, on the other hand, is expressed primarily in tumors originating in the neural and endocrine systems, such as glial tumors (glioblastomas, neuroblastomas, astrocinomas, etc.), or pituitary adenomas.

In order to functionalize the nanoliposome with the peptide, it is modified by incorporating a terminal sulfhydryl group by methods known in the state of the art. For example, the peptide is reacted with the Traut reagent (2-iminothiolane hydrochloride) according to the following scheme:

Peptide -NH., ··· AR— S

where AR-S is Traut's reagent.

The addition of EDTA to the reaction helps prevent oxidation of the sulfhydryl group by preventing the formation of disulfide bonds. The peptide binds to the nanoliposome by reaction of the sulfhydryl group with the maleimide molecule of the conjugate present in the nanoliposome and described above, according to the following scheme:

In a particular embodiment, the peptide is modified with a cysteine molecule, so that the free -SH group is also left to react with the conjugate maleimide.

The nanoliposomes may further comprise other phospholipids that are not conjugated but free, such as those mentioned above. In a particular embodiment, these phospholipids are selected from 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl- sn-glycerol-3-phosphocholine (DPPC), 1, 2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE) and combinations thereof. Preferably, these phospholipids are 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) represented in the formula (I), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) represented in the formula (II) or any of its salts, derivatives or analogs, or any of its combinations of the compounds of formula (I) and (II).

(I)

(II)

These phospholipids can also be conjugated to a PEG molecule, called pegylated phospholipids. In a preferred embodiment, the nanoliposomes comprise 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [amino (polyethylene glycol)] (DSPE-PEG).

Preferably, the nanoliposomes of the invention comprise, in addition to the lipid-spacer-maleimide conjugate and the modified peptide, 1,2-distearoyl-sn-glycer-3-phosphoethanolamine-N- [amino (polyethylene glycol)] (DSPE-PEG), 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) and 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC).

Even more preferably, the nanoliposomes of the invention comprise:

1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [amino (polyethylene glycol) -maleimide] or (DSPE-PEG-maleimide);

1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [amino (polyethylene glycol)] (DSPE-PEG);

1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC),

1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), and

a peptide modified with a sulfhydryl group;

Preferably, the peptide is the VIP peptide modified with a cysteine molecule.

Preferably, the DPPC and the DSPC are in a mass ratio of approximately 6: 4. Preferably, the DSPE-PEG and the DSPE-PEG-Maleimide conjugate are at a final concentration of about 5 and 10%, respectively. The lipid bilayer of the nanoliposomes may also contain one or more sterols such as cholesterol, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol, phytosterol, stigmasterol, sitosterol, campesterol and brasicasterol, sugars such as glycerol and sucrose, esters of glycerin fatty acids such as triolein and trioctanoin.

It may also contain antioxidant substances such as tocopherol, propyl gallate, ascorbyl palmitate and butylated hydroxytoluene, compounds that provide positive charge such as stearylamine and oxylamine, compounds that provide negative charge such as di-methyl phosphate, membrane proteins.

Procedure for obtaining nanoliposomes

In a second aspect, the invention relates to a process for the preparation of the nanoliposomes defined above, comprising:

to. forming a lipid film, for example by reverse phase evaporation, of a compound comprising phospholipids, preferably

DSPC and DPPC and at least one pegylated phospholipid,

b. rehydrate the film with a buffer solution at an essentially neutral pH and add an organic solvent to form a two phase system;

C. subject the two phase system to sonication to form dispersed liposomes in a single phase

d. remove the organic solvent until a gel phase is obtained, e. convert the gel into an aqueous suspension, where liposomes have formed

F. convert liposomes of (e) into nanoliposomes, for example by sonication,

g. add a peptide, described above, to the suspension of the nanoliposomes of step (f). Preferably, the lipid film of step (a) comprises a lipid-spacer-maelimide conjugate as previously defined; More preferably, the lipid-spacer-maleimide conjugate is in a proportion of about 10% by weight with respect to the total weight of the lipid film. In a preferred form, the lipid film further comprises at least one phospholipid and / or at least one phospholipid conjugated to a PEG molecule. Within this preferred embodiment, the lipid film further comprises 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1, 2- dipalmitoyl-sn-glycer-3-phosphocholine (DPPC) and 1, 2- distearoyl-sn-glycer-3-phosphoethanolamine-N- [amino (polyethylene glycol)] (DSPE-PEG). Preferably, the mass ratio of the DPPC: DSPC phospholipids is approximately 6: 4. Also preferably, the proportion of DSPE-PEG is around 5% by weight with respect to the total weight of the lipid film.

In another preferred embodiment, the buffer solution is a solution of HEPES (4- (2-hydroxyethyl) -1 -piperazinethanesulfonic acid) of pH 7.4.

In another preferred embodiment, the organic solvent is a mixture of diethylether and chloroform in an approximate ratio of 1: 1 by volume. More preferably, the volume ratio of organic phase: aqueous phase is about 4: 1.

In particular, the sonicating of step c) is performed in a sonic bath until the liposomes form spontaneously and the sample appears dispersed in a single phase. Preferably this sonication stage is carried out for 3 to 5 minutes.

Preferably, the removal of the organic solvent is carried out by evaporation, for example with rotary evaporator, at a temperature between 35 and 55 ° C, more preferably at 40 ° C, and at a rotation speed between 100 and 150 rpm, more preferably at 120 rpm.

Preferably, the sonication of step (f) is performed in a sonic bath at a temperature higher than that of its transition phase, preferably at more than 60 ° C, to control the size of the nanoliposome. Preferably, the nanoliposome is adjusted to an average size between 75 and 125 nm, preferably between 85 and 1 nm, more preferably about 95-105 nm.

Preferably, the functionalization of the nanoliposome with the peptide (step (g)) is carried out by adding at least 100 μg of the modified peptide, more preferably at least 125 μg and even more preferably at least 150 μg, to the sample of Nanoliposomes kept under stirring and room temperature for more than 10 hours, and more preferably between 12 and 16 hours. Preferably, the peptide is dissolved in a buffer solution at a pH of around 6-6.5.

Said peptide is modified with a sulfhydryl group at one end thereof. The maleimide present in the lipid-spacer-maleimide conjugate reacts with the peptide through the sulfhydryl -SH group. Said reaction is preferably carried out under nitrogen bubbling for at least one hour to prevent sulfhydryl groups from being oxidized. The sample is incubated at room temperature and with stirring.

In order to protect the free groups from maleimide, excess mercaptoethanol or free cysteine can be added, subsequently dialyzing to remove the excess of unreacted β-mercaptoethanol and peptide.

Preferably, the peptide is a neuropeptide and even more preferably it is the VIP peptide.

In a particular embodiment, the nanoliposomes obtained after step (f) can be purified by, for example, a Sephadex column, preferably G-50.

Also, the functionalized nanoliposomes obtained after step (g) can be purified by, for example, dialysis against deionized water for at least 15 hours, preferably for at least 20 hours, and much more preferably for 24 hours.

The procedure that leads to these nanoliposomes allows functionalization with the peptide so that the carboxyl-terminal end is free, thus leaving intact its ability to interact with its specific receptors, which allows formulating in vivo drug detection and selective release strategies. on cells that are interested in eliminating (as is the case with tumor cells), or expanding (through the administration of trophic factors) depending on the pathology that is intended to be addressed. Therefore, the nanoliposomes of the invention can also incorporate an active ingredient.

As used herein, the term "active ingredient" means any component that potentially provides a pharmacological activity or other effect different in the diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body. of man or other animals. The term includes those components that promote a chemical change in processing of the drug and are present therein in a modified form intended to provide the specific activity or effect.

The active substance can be both hydrophilic and hydrophobic. In the first case it is incorporated into the aqueous nucleus of the liposome, while in the second case it is incorporated into the lipid bilayer of the nanoliposome.

In a preferred embodiment, the active ingredient has an antitumor activity. In another preferred embodiment of this aspect of the invention, the active ingredient has anti-inflammatory activity. More preferably, the active substance is doxorubicin.

The encapsulation of the active substance in the nanoliposome can be performed using the osmotic gradient technique as described in the experimental part.

In a further aspect, the invention relates to a pharmaceutical composition, hereinafter pharmaceutical composition of the invention, comprising a nanoliposome as previously defined and an active ingredient capable of diagnosing, curing, mitigating, treating or preventing a disease. Preferably, the composition further comprises a pharmaceutically acceptable carrier. More preferably, the pharmaceutical composition of the invention further comprises another active ingredient.

The pharmaceutical compositions of the present invention can be formulated for administration to an animal, and more preferably to a mammal, including man, in a variety of ways known in the state of the art. Thus, they can be, without limitation, in sterile aqueous solution or in biological fluids, such as serum. Aqueous solutions may be buffered or unbuffered and have additional active or inactive components. Additional components include salts to modulate ionic strength, preservatives including, but not limited to, antimicrobial agents, antioxidants, chelators, and the like, and nutrients including glucose, dextrose, vitamins and minerals. The compositions may be combined with various inert vehicles or excipients, including but not limited to; binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose; dispersing agents such as alginic acid or corn starch, etc.

Such compositions and / or their formulations can be administered to an animal, including a mammal and, therefore, to man, in a variety of ways, including, but not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intracecal, intraventricular, oral, enteral, parenteral, intranasal or dermal.

The dosage to obtain a therapeutically effective amount depends on a variety of factors, such as the age, weight, sex, tolerance, ... of the mammal. In the sense used in this description, the term "therapeutically effective amount" refers to the amount of active ingredient, or its salts, prodrugs, derivatives or analogs, or combinations thereof, that produce the desired effect and, in general, It will be determined, among other causes, by the characteristics of said prodrugs, derivatives or analogues and the therapeutic effect to be achieved. The "adjuvants" and "pharmaceutically acceptable carriers" that can be used in said compositions are the vehicles known to those skilled in the art.

The term "excipient" refers to a substance that aids the absorption, distribution or action of any of the active ingredients of the present invention, stabilizes said active substance or aids in the preparation of the medicament in the sense of giving it consistency or providing flavors. Make it more enjoyable. Thus, the excipients could have the function of keeping the ingredients together such as starches, sugars or cellulose, sweetening function, dye function, drug protection function such as to isolate it from air and / or moisture, function filling a tablet, capsule or any other form of presentation such as dibasic calcium phosphate, a disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph.

The term "pharmaceutically acceptable" excipient refers to the excipient being allowed and evaluated so as not to cause damage to the organisms to which it is administered.

In addition, the excipient must be pharmaceutically suitable, that is, an excipient that allows the activity of the active ingredient or of the active ingredients, that is, that is compatible with the active ingredient, in this case, the active ingredient is any of the compounds of the present invention.

A "pharmaceutically acceptable carrier" refers to those substances, or combination of substances, known in the pharmaceutical sector, used in the preparation of pharmaceutical forms of administration and includes, but are not limited to, solids, liquids, solvents or surfactants. The vehicle, like the excipient, is a substance that is used in the medicament to dilute any of the compounds of the present invention to a certain volume or weight. The pharmaceutically acceptable carrier is an inert substance or action analogous to the active ingredients of the present invention. The function of the vehicle is to facilitate the incorporation of other compounds, allow a better dosage and administration or give consistency and form to the pharmaceutical composition. When the form of presentation is liquid, the pharmaceutically acceptable carrier is the diluent.

Another aspect of the invention relates to the use of the nanoliposome of the invention, in the preparation of a medicament for the treatment of diseases that occur with cell proliferation. In a preferred embodiment, the disease that occurs with cell proliferation is selected from the list comprising: malignant epithelial neoplasms, lung cancer and other cancers such as stomach, colon, breast, prostate, liver and urinary bladder. In a more preferred embodiment, the disease that occurs with cell proliferation is prostate cancer.

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 features 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.

EXAMPLES

Example 1. Preparation of nanoliposomes functionalized with VIP peptide

As a first step, a lipid film was obtained by reverse phase evaporation of a mixture containing 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1, 2- dipalmitoyl-sn-glycerol-3-phosphocholine ( DPPC), 1,2-distearoyl-sn-glycerol-3-phosphoethanol amine-N- [amino (polyethylene glycol)] (DSPE-PEG) and 1,2-distearoyl-sn-glycerol-3- phosphoethanolamine-N- [amino (polyethylene glycol) -maleimide] or (DSPE-PEG-maleimide). The film formed was rehydrated with a HEPES buffer solution at pH 7.4 and a mixture of diethyl ether and chloroform was added in a 1: 1 volume ratio, such that the volume ratio of the organic phase: aqueous phase was 4 :one . Thus a two-phase system was formed. This system was subjected to a sonication stage for about 3-5 minutes until the sample appeared dispersed in a single phase. The organic solvent was removed by rotoevaporation at a temperature of 40 ° C and at a speed of 120 rpm, until a gel phase was reached. Then the gel became an aqueous suspension where the liposomes were dispersed. These liposomes were subjected to an additional stage of sonication to reduce their size and thus convert them into nanoliposomes. The sonication was performed in a sonic bath at a temperature above 60 ° C. Next, the nanoliposomes were purified using a Sephadez G-50 column.

The functionalization of the nanoliposomes with the VIP was carried out by adding 150 μg of said modified peptide with a cysteine molecule on the aqueous suspension of the nanoliposomes, while stirring at room temperature for 12 hours.

Subsequently, the nanoliposomes were purified by dialysis against deionized water for 24 hours. Once the nanoliposomes were functionalized, the surface VIP was quantified using a commercial system called LavaPep - peptide quantification kit ™ (Fluorotechnics) based on the fluorescence detection of the peptide / fluorophore complexes. The results related to this quantification are shown in Figure 2. Likewise, the functionalized nanoliposomes were characterized by their size using the Dynamic Light Scattering (DLS) technique, obtaining values of around 100 nm as they were shown in figure 3.

Example 2. Specificity and effectiveness of nanoliposomes functionalized with VIP in the specific recognition of cells expressing their receptors.

DU145 cells (n = 4) (human prostate cancer cell line) were stimulated for 30 minutes with the VI-functionalized nanoliposomes obtained according to Example 1 at an effective functionalized VIP concentration of 10 ^"7 M.

CAMP production was measured by a bioluminescence assay using the Promega cAMP-GLO ™ assay according to the manufacturer's instructions. This assay is based on the decrease in luciferase-coupled light production when increased concentrations of cAMP produce a protein activation. kinase A and a concomitant decrease in ATP available for the detection reaction indicated above.

Figure 4 shows the results obtained in cells and demonstrates a specifically increased cAMP production after stimulation of the cells with the functionalized nanoliposomes.

Example 3. Encapsulation of doxorubicin

Doxorubicin encapsulation is performed using the osmotic gradient technique, in which two different pH buffers are used. The first consists of 250 mM ammonium sulfate pH 5.5, which is the aqueous phase in the synthesis of reverse evaporation, so this buffer is inside and outside the nanonanoliposome. Subsequently, the nanonanoliposome sample is passed through a column of Sephadex G-50 previously washed with saline HEPES buffer pH 7.4 to collect the nanoliposomes dispersed in this same buffer. This difference in osmolarity inside and outside the nanoliposome creates an osmotic gradient across the membrane. Once this osmotic gradient is created, doxorubicin dissolved in water is added to the nanoliposome sample and allowed to incubate for 90 minutes at a temperature higher than the transition, in our case at 60 ° C, since the transition temperature of our nanoliposome It is 46 ° C. This temperature causes the nanoliposomes to become more fluid, gaps form in their membrane, and doxorubicin penetrates. The movement of water into the nanoliposomes is due to the osmotic gradient and the gaps in the membrane that facilitates the encapsulation of doxorubicin and allows to reach encapsulation efficiency values close to 90%.

The sample is subsequently purified by means of a Sephadex G-50 column to eliminate unincorporated doxorubicin and the encapsulation efficiency is measured by the fluorescence spectrophotometer, measuring the sample before and after being purified and also after adding 1% triton (In this way the nanoliposome is broken and the drug inside is released).

Using this equation the percentage (%) of drug encapsulation is calculated:

Encapsulation efficiency = (A-B / C) X 100

A = Intensity of the purified sample after adding 1% triton. B = Intensity of the purified sample without 1% triton.

C = Intensity of the sample before purification and after adding 1% triton.

Example 4. Cytotoxic capacity of doxorubicin encapsulated in a VIP functionalized nanoliposome

DU-145 cells (n = 4) (human prostate cancer cell line) were treated for 48 hours with soluble doxorubicin, with doxorubicin encapsulated in liposomes and with doxorubicin encapsulated in VIP-functionalized nanoliposomes. Figure 5 shows the optical microscopy photographs obtained in all cases and their comparison with a sample of untreated cells. The increased cytotoxic effect of doxorubicin is observed in the presence of nanoliposomes functionalized with the VIP peptide.

Likewise, as a reference method to study cytotoxicity in vitro, the release of LDH (lactate dehydrogenase) was used. For this, said cell line was exposed to the drug (10 g / mL) for 24 hours. Figure 6 shows the histrogram obtained for all the samples analyzed. The greater the number of dead cells, the greater release into the environment of the LDH enzyme. In this way, cell death is proportional to the quantification of LDH activity. The result of treating the cells with a detergent (1% Triton X100) is used as a reference for absolute cytotoxicity, which logically dissolves the cell membranes and kills the total exposed population.

A greater release of LDH (a larger number of dead cells) can be observed when doxorubicin is encapsulated in VIP-functionalized nanoliposomes.

This test shows that the functionalization of nanoliposomes with the VIP peptide increases the cytotoxic capacity of doxorubicin, improving the effectiveness of this active substance, which allows the incorporation of the drug into the liposomal composition at lower concentrations than those used in other conventional formulations of the prior art.

Claims

1 - A functionalized nanoliposome with a peptide on its surface.

2- The nanoliposome according to the preceding claim, wherein the carboxyl-terminal end of the peptide is free.

3- Nanoliposome according to any of claims 1-2, further comprising:

a lipid-spacer-maleimide conjugate; Y

a peptide modified with a sulfhydryl group;

4- Nanoliposome according to claim 3, wherein the lipid present in the conjugate is a phospholipid.

5- Nanoliposome according to claim 4, wherein the phospholipid is selected from 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycerol-3- phosphoethanolamine (DSPE), 1, 2 -dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1, 2- dipalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE) and combinations thereof.

6- Nanoliposome according to any of claims 3 to 5, wherein the spacer present in the conjugate is polyethylene glycol (PEG).

7- Nanoliposome according to any one of claims 3 to 6, wherein the lipid-spacer-maleimide conjugate is 1,2-distearoyl-sn-glycerol-3- phosphoethanolamine-N- [amino (polyethylene glycol) -maleimide] (DSPE-PEG- maleimide). 8- Nanoliposome according to any of the preceding claims, wherein the peptide is selected from the list comprising: glucagon, GIP, secretin, growth hormone, somatoliberine, somatotropin, PHI peptide, PHM peptide, PACAP, adrenomedulin, corticostatin and vasoactive intestinal peptide VIP

9- Nanoliposome according to any one of claims 1-8, wherein the peptide is a neuropeptide. 10. Nanoliposome according to claim 9, wherein the neuropeptide is the VIP vasoactive intestinal peptide.

1- 1- Nanoliposome according to any one of claims 1 to 10, wherein the peptide is modified with a cysteine molecule.

12- Nanoliposome according to any of claims 1-1 1 which further comprises one or more free phospholipids and / or one or more polyethylene glycol conjugated phospholipids.

13- Nanoliposome according to claim 12, wherein the free phospholipids are selected from 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine (DSPE), 1 , 2-dipalmitoyl-sn-glycerol-3-phosphocholine

(DPPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE) and combinations thereof.

14- Nanoliposome according to any of claims 12 and 13, wherein the phospholipid conjugated with polyethylene glycol is 1,2-distearoyl-sn-glycerol-3- phosphoethanolamine-N- [amino (polyethylene glycol)] (DSPE-PEG).

15- Nanoliposome according to any one of claims 1 to 1, which further comprises 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [amino (polyethylene glycol)] (DSPE-PEG), 1, 2-distearoyl -sn-glycerol-3-phosphocholine (DSPC) and 1, 2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC).

16- Nanoliposome according to any of claims 1 to 15 comprising:

1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC),

1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), and

a peptide modified with a sulfhydryl group;

so that the peptide is covalently bound to the nanoliposome through the reaction between the maleimide of the conjugate and the sulfhydryl group of the modified peptide. 17- Nanoliposome according to claim 16, wherein the DSPE-PEG-Maleimide conjugate is found in a proportion of about 10% by weight with respect to the total lipid weight.

18- Nanoliposome according to any of claims 16 and 17, wherein the DSPE-PEG are in a proportion of about 5% by weight with respect to the total lipid weight.

19- Nanoliposome according to any of claims 16 to 18, wherein the DPPC and the DSPC are in a mass ratio of approximately 6: 4.

20- Nanoliposome according to any of claims 16 to 19, wherein the peptide is the vasoactive intestinal peptide (VIP) modified with a cysteine molecule.

21- Nanoliposome according to any of claims 1-20, wherein its average diameter is between 75 and 125 nm.

22- A process for the preparation of nanoliposomes as defined in claims 1 to 21, comprising:

a) form a lipid film;

b) rehydrate the film with a buffer solution at an essentially neutral pH and add an organic solvent to form a two phase system; c) subjecting the two phase system to sonication to form dispersed liposomes in a single phase;

d) remove the organic solvent until a gel phase is obtained;

e) convert the gel into an aqueous suspension where the liposomes remain; f) convert liposomes to nanoliposomes;

g) add the modified peptide to the nanoliposome suspension to form functionalized nanoliposomes.

23. Method according to claim 22, wherein the lipid film comprises a lipid-spacer-maleimide conjugate as previously defined.

24. Method according to any of claims 22-23, wherein the lipid film further comprises at least one phospholipid and / or at least one phospholipid conjugated to a PEG molecule. 25. Method according to any of claims 22-24, wherein the lipid film comprises 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1, 2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC ) and at least one pegylated phospholipid.

26. The method according to claim 25 wherein the pegylated phospholipids is 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [amino (polyethylene glycol)] (DSPE-PEG) or DSPE-PEG-Maleimide.

27- Method according to claim 26 wherein the DSPE-PEG and the DSPE-PEG-Maleimide are at a final concentration of about 5 and 10%, respectively, with respect to the total lipid weight.

28. The method according to any of claims 22 to 27, wherein the DPPC and the DSPC are in a mass ratio of approximately 6: 4.

29. Method according to any of claims 22 to 28, wherein the organic solvent consists of a mixture of diethyl ether and chloroform in an approximate ratio of 1: 1 by volume.

30. Method according to any of claims 22 to 29, wherein the volume ratio organic phase: final aqueous phase after adding the organic solvent, is approximately 4: 1.

31. Method according to any of claims 22 to 30, wherein the conversion of liposomes into nanoliposomes is performed by subjecting the aqueous suspension to sonication.

32. The method according to claim 31, wherein the sonication in step (f) is carried out at a temperature greater than 60 ° C.

33- A functionalized nanoliposome with a peptide on its surface obtainable according to a method as defined in any of the claims

22 to 32.

34. A nanoliposome according to any of claims 1 to 21 and 33, which further comprises an active ingredient capable of diagnosing, curing, mitigating, treating or preventing a disease.

35- Nanoliposome according to claim 34, wherein the active ingredient has an antitumor activity. 36- Nanoliposome according to claim 35, wherein the active substance is doxorubicin.

37- Nanoliposome according to claim 34, wherein the active ingredient has an anti-inflammatory activity.

38. A pharmaceutical composition comprising a nanoliposome as defined in any one of claims 1 to 21 and 33 to 37.

39- Pharmaceutical composition according to claim 38, further comprising a pharmaceutically acceptable carrier.

40- Use of the nanoliposome according to any of claims 1-21 and 33-37, or of a pharmaceutical composition according to any of the rindications 38-39, in the preparation of a medicament for the treatment of diseases that occur with cell proliferation.

41- Use of the nanoliposome according to claim 40, wherein the disease that occurs with cell proliferation is selected from the list comprising malignant epithelial neoplasms, lung cancer and other cancers such as stomach, colon, breast, prostate, liver and urinary bladder cancer .

42. Use of the nanoliposome according to the preceding claim, wherein the disease that occurs with cell proliferation is prostate cancer.