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    • 专利摘要:
    New vehicles for the transfection of mirnas. The present invention relates to a nanoparticle comprising (I) between 60% and 99% by weight, based on the total weight of the nanoparticle, of a sorbitan ester; (II) a positively charged substance; and (III) a mirna; to its manufacturing methods, and its uses, especially in therapeutic uses, such as cancer treatment. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2636646A1
    申请号:ES201630417
    申请日:2016-04-05
    公开日:2017-10-06
    发明作者:Alejandro SÁNCHEZ BARREIRO；Inés FERNÁNDEZ PIÑEIRO；Iker BADIOLA；Joana MÁRQUEZ
    申请人:Euskal Herriko Unibertsitatea；Universidade de Santiago de Compostela；
    IPC主号:
    专利说明:

    FIELD OF THE INVENTION The present invention relates to new systems comprising miRNAs for application in the pharmaceutical, cosmetic or nutritional fields, among others. These systems allow the most efficient administration of different miRNAs, for example, in applications for cancer treatment.
    BACKGROUND OF THE INVENTION MicroRNAs, also commonly referred to as miRNAs, are short RNA sequences that have the ability to interfere with cellular processes. This capacity has aroused interest in its potential for medical, cosmetic or nutritional applications, among others. It has been possible to verify the potential of many of these miRNAs in biological tests, and to date approximately one thousand of these natural substances with potential in various applications have been identified. So far, the main problem for its use is its short life when administered. The miRNAs are especially sensitive to the attack of exonucleases and have a half-life of minutes in the biological environment. This has triggered interest in the search for means of administration that allow transporting the miRNAs efficiently, avoiding their rapid degradation in vivo. One of the first Strategies contemplated was the structural modification of the miRNAs. One of the most widespread variations has been the modification of the 2 '· OH group of ribose (Wu SY, Yang X, Gharpure KM, Hatakeyama H, Egli M, et al. (2014) 2'.OMe-phosphorodithioate-modified siRNAs show increased loading into the RISC complex and enhanced anti · tumour activity. Nat Commun 5: 3459.). Examples of these modifications are the substitution in this position by 2'-f1uoro, 2'-Q-methyl or 2'-O-methoxymethyl, just to mention a few examples. Another strategy followed has been the use of viral vectors, for example, lentiviruses or adenoviruses, as explained in Kasar S, Salemo E, Yuan Y. Underbayev C, Vol1enweider D, et al. (2012) Systemic in vivo lentiviral delivery of miR-15a / 16 reduces malignancy in the NZB de novo mouse model of chronic Iymphocytic leukemia. Genes Immun 13: 109-119; or in Brandt MR, Kirste AG, Pozzuto T, Schubert S, Kandolf R, et al. (2013) Adenovirus vector-mediated RNA interference for the inhibition of human parvovirus B 19 replication. Res virus
    176: 155-160. However, and although they have been genetically modified to eliminate their virulent burden, the safety of these vectors always arouses concern. The field of oncology is where miRNAs have possibly received more attention so far. This interest stems from the discovery that miRNAs are deregulated in cancerous tissues and surrounding tissues, and in the ability of miRNAs to regulate multiple genes (for example, siRNAs are specific and allow action on a single gene), since they can bind to multiple RNA messengers (mRNA) (GARZON, R., MARCUCCI, G. and CROCE, CM, 2010. Targeting microRNAs in cancer: rationale, strategies and challenges. Nature reviews.Drug discovery, 9 (10) , pp. 775-789). An example is the case of colorectal cancer and liver metastasis, one of its most frequent regions of metastasis. Sinusoidal endothelial liver cells (LSECs) play a key role in the development and regulation of liver metastases. It has been found that some miRNAs (miR-20a; miR-29 and miR-652) are deregulated in LSECs, and the recovery of their normal levels is presented as a promising therapeutic alternative. In view of the potential of miRNAs and their difficulties in administration, the use of non-viral vectors has been tested, for example, liposomes, peptides, antibodies and other ligands, such as chitosan. Thus, the improvement in the transfection of different miRNAs with the usual agents such as those of the lipofectamine® family (DOTAP, DOTMA or DOPE) or Smarticles® (amphoteric derivatives) has been sought. For example, miR34a is now in clinical phases in the form of Smarticles® liposomes for the treatment of liver cancer, and miR-16 in the form of EnGenelC nanoparticles (minicell; EP23B6640) for the treatment of malignant pleural mesothelioma. This situation contrasts with that of drugs based on siRNAs, another different family of RNAs that have double strands, which are not finding so many problems in the search for suitable vehicles for transfection, and for which there are already many clinical trials underway . Given the recent interest aroused by miRNAs as therapeutic agents, there is a need to find and develop new vehicles for more effective administration (LAM, JK, CHOW, MY, ZHANG, Y. and LEUNG, SW, 2015. siRNA Versus miRNA as Therapeutics for Gene Silencing. Molecular therapy.Nuc / eie ae ids, 4, pp. E252). However, efforts in the case of miRNAs are being especially difficult because they are hydrophilic molecules, high molecular weight and negative charge, which prevent their passage through the cell membrane. The use of non-viral vectors mentioned above reduces these problems, however, especially in the case of nanometric systems, it creates other difficulties. The reticuloendothelial system (RES) rapidly absorbs nanometric systems, and is the cause of the poor in vivo stability (and therefore, poor efficiency) of many of them (RINKENAUER, AC, PRESS, AT, RAASCH, M., PIETSCH , e., SCHWEIZER, S., SCHWORER, S., RUDOLPH, KL, MOSIG, A., BAUER, M., TRAEGER, A. and SCHUBERT, US, 2015 Comparison of the uptake of methacrylate-based nanoparticles in static and dynamic in vitro systems as well as in vivo. Joumalo (confrolled r elease. or fficial jau mal o (f he e onfrolled R elease Soc iefy, 216, pp. 158-168; SADAUSKAS, E., WALLlN, H., STOL TENBERG, M., VOGEL, U., DOERING, P., LARSEN, A. and DANSCHER, G., 2007. Kupffer cells are central in the removal of nanoparticles from the organism Pariicle and fiber toxicology, 4, pp. 10) The RES comprises phagocytic cells such as monocytes or macrophages, for example, the cells of
    5 Kupffer Although it is generally accepted that there is a correlation between the positive surface charge of the nanoparticle and a lower absorption by phagocyte cells, it is not known exactly what factors influence this absorption, which makes it difficult to predict the type of nanometric systems that will have adequate stability in vivo The development of vehicles with improved properties for the administration of miRNAs is therefore of great interest
    BRIEF DESCRIPTION OF THE INVENTION
    The present invention solves the aforementioned problems, improving the stability of
    miRNAs in viv o. Researchers have been able to verify that the systems of the present invention
    15 surprisingly improve the in vivo transport of miRNAs, and their efficiency is surprisingly increased. It has been found that prevents degradation by the RES, and in particular prevents phagocytization by Kupffer cells. Therefore, a first aspect of the invention is a nanoparticle comprising (i) between 60% and 99 % by weight, with respect to the total weight of the nanoparticle, of a sorbitan ester; (ii) a positively charged substance; and (iii) a miRNA. The stability of these systems and the improvement in the transfection of miRNAs that they provide make the nanoparticles of the invention suitable for, for example, applications in the field of pharmacy, cosmetics or nutrition. An additional aspect is the use of a nanoparticle of the invention for the preparation of a medicament.
    A nanoparticle of the invention for use as a medicament is also an additional aspect. An additional aspect is the use of a nanoparticle of the invention for the manufacture of a medicament for the treatment of an indication that is selected from the group consisting of cancer, diabetes and neurodegenerative diseases. A nanoparticle of the invention is also an aspect for use in the treatment of an indication that is selected from the group consisting of cancer, diabetes and neurodegenerative diseases. Another advantage of the invention is that the nanoparticles are easy to prepare, and the incorporation of the miRNAs can be performed simultaneously to the formation of the nanoparticle itself or at a later stage of incubation, depending on the nature of the components , thus providing flexibility to its preparation. Thus, a further aspect of the invention is a process for the preparation of a
    The nanoparticle of the invention comprising (i) the step of adding an organic solution comprising an organic solvent and a sorbitan ester over an aqueous solution; wherein said organic solution or said aqueous solution or both comprise a positively charged substance; and (ii) the stage of evaporating organic solvent and water; and (iii) the optional step of incubating the nanoparticle resulting from step (ii) in the presence of other substances; wherein a miRNA is incorporated (a) during stage (i) as part of the aqueous solution, (b) in the incubation stage (iii), or in both stages (i) and (ii) An additional aspect of the The invention is a pharmaceutical composition comprising the nanoparticle of the invention and a pharmaceutically acceptable excipient.
    BRIEF DESCRIPTION OF THE FIGURES
    45 Figure 1. Electrophoresis gel that confines the existence of an effective association between a miRNA and nanoparticles. A: band corresponding to free miR-20a (5 J.lg / ml) B: band in which free miR-20a is not observed, corresponding to SP80-0A-CS-miR-20a (50 J.lgfml miR-20a ).
    Figure 2. Demonstration of the therapeutic effect in vivo. Clinical regulation of liver metastasis through the use of nanoparticles according to the invention SP80-0A-CS-miR-20a. C26 murine colorectal cancer cell lines (200,000 cells / animal) were injected into mice. Animals were treated from day 3 after tumor inoculation, and subsequently every 3 days until day 21. On day 21 the animals were sacrificed and their livers were processed for histological analysis. The mice were separated into 5 treatment groups. Group 1 was treated with placebo (glucose); group 2 (according to the invention) with SP80-0A-CS-miRNA-20a; group 3
    55 with free miRNA-20a, without any specific vehicle; group 4 with an SP80-0A-CS nanoparticle associated with a miRControl (a miRNA that does not attack any target); Group 5 with SpaO-OA-CS nanoparticles without any miRNA. The macroscopic evaluation of the livers confined the therapeutic effect in terms of clinical regulation in animals treated with SP80-0A-CS-miRNA-20a (group 2). Figure 3. Demonstration of the therapeutic effect in vi vo. Histological Analysis Sections of the livers evaluated in Figure 2 were stained with hematoxylin-eosin, and the area occupied by the tumor was quantified under the microscope. The different groups were related to the control group (group 1 treated with placebo) using the T-test. The significant statistical differences (p <0.05) are marked with 'Group 2 was also compared (treated with SP80-0A-CS -miRNA-20a) with group 5 (treated with SP80-0A-CS without miRNA) and its significant statistical difference (p <0.05) indicated with - +
    Detailed description
    Definitions For the nomenclature of the nanoparticles of the invention, the formula [sorbitan ester Positively charged substance] is used. If the nanoparticle has other components, for example, a negatively charged substance, it is indicated below separated by a hyphen. In some cases the name of the miRNA used is indicated at the end. The following abbreviations' SPBO are also used: it is understood as Span-BO®. Span-BO® is a substance that results from esterifying sorbitan with cis-9-octadecenoic acid (commonly known as oleic acid): that is, a molecule with the following formula:
    OR
    CH2 O-C-CH2 (CH2) s CH2 CH = CHCH2 (CH2) sCH3
    "
    I
    HO · "C O H:) ~~.: TOH
    Span-BQ® OA-oleylamine, ie (Z} -octadec-9-enylamine CS: chondroitin sulfate. Its structure and properties are explained below in greater detail. HA 'hyaluronic acid. Its structure and properties are explained below with greater detail Thus, for example, a nanoparticle that is abbreviated SPBO-OA-HA-miR-20a, will be one that incorporates Span-BO®, oleylamine, hyaluronic acid and miR-20a, prepared according to the procedures described here. This document has been followed by the standard nomenclature for miRNAs. The prefix "miR-" followed by a hyphen and a number has been used. According to the standard nomenclature the prefix "miRO with capital" R "is reserved for mature miRNAs , while the prefix "mir-" with lowercase "r" is usually reserved for pre-miRNAs, and the prefix "MIR" for the gene that encodes them. For the purposes of the present invention, the prefix "miR-" it includes all, mature miRNAs, pre-miRNAs and the genes that encode them. the number some miRNAs incorporate a letter that allows to distinguish miRNAs with very similar sequences. "Alkyl" means a linear or branched hydrocarbon chain that does not contain any from 1 to 40 carbon bonds unless otherwise indicated, optionally substituted with one, two or three substituents selected from -ORb, -SRb, -NRaRb, -C (O) Rb, -C02Rb, -C (O) NRaRb, -NRaC (O) Rb, NRaC {O) ORb, -NRaC (O) NRaRb, -CF3, -OCF3; where Ra and Rb independently selected from hydrogen, Cl-CS alkyl, C: c-C6 alkenyl and C2-C6 alkynyl. "Alkenyl" means a linear or branched hydrocarbon chain containing at least one double bond, from 2 to 40 carbon bonds unless otherwise indicated, optionally substituted with one, two or three substituents selected from -ORb, -SRb, -NRaRb, -C (O) Rb, -C02Rb, -C (O) NRaRb, NRaC (O) Rb , -NRaC (O) ORb, -NRaC (O) NRaRb, -CF3, -OCF3, where Ra and Rb are independently selected from hydrogen, Cl-Cs alkyl, C2-CS alkenyl and C2-CS alkynyl. alkynyl "a hydrocarbon chain linear or branched ada containing at least one triple bond, from 2 to 40 carbon atoms unless otherwise indicated, optionally substituted with one, two or three substituents selected from -ORb, -SRb, -NRaRb, -C ( O) Rb, -C02Rb, -C (O) NRaRb, -NRaC (O) Rb, -NRaC (O) ORb, -NRaC (O) NRaRb, -CF3, -OCF3; where Ra and Rb are independently selected from hydrogen, Cl-Cs alkyl, C2-CS alkenyl and C2-CS alkynyl Unless otherwise indicated, the weight percentages indicated herein are calculated with reference to the sum of all components added to the mixture in the formation of the nanoparticle, with the exception of solvents. For example, for a SP80-0A-HA-miR-20a nanoparticle, the weight percentage of SPBO will be the grams of SP80 added for the preparation of the nanoparticle, multiplied by one hundred, and divided by the sum of grams of SPBO, OA , HA and miR-20a added for the preparation of the nanoparticle. When the particle "a" or "a" is used, it should be understood as "at least one" or "one or more" For example, "a miRNA" indicates that the number of miRNAs present is at least 1, but that there may be mixtures of 2 or more miRNAs.
    Components of the nanoparticles of the invention The nanoparticles of the invention comprise sorbitan esters. Sorbitan is made up of a mixture of cyclic anhydrides of sorbitol, such as 1,4-anhydrosorbitol, 1,5-anhydrosorbitol and 1,4,3,6-dianhydrosorbitol. Sorbitan esters are considered non-ionic surfactants because they contain two localized regions, one of a hydrophilic nature and another hydrophobic. The term 'sorbitan esters' means esterified derivatives of sorbitan where the ester groups have a substituent selected from alkyl, alkenyl and alkynyl.The sorbitan esters include derivatives in which one, two, three or four hydroxyl groups are esterified, and even include esterified derivatives in which one ester molecule is present for every two sorbitan molecules (in which case they are named with the prefix "sesqui-"). Thus, for example, sorbitan monooleate is the ester of sortitan resulting from the esterification of a hydroxyl group with oleic acid; somitanium iolealo is the somitan ester resulting from esterification of three hydroxyl groups of the somitan with oleic acid There are many different types of sorbitan esters according to n esterified hydroxyl number, the structure of the ester, the mixture of anhydrosomitol, and other factors. The person skilled in the art can choose between
    5 different types within the scope of the invention and is not limited to a specific type Commonly used sorbitan esters are, for example, those marketed under the name Span® (without polyoxyethylene blocks) or Tween® (with polyoxyethylene blocks). To mention a few examples, common sorbitan esters of the Span @ family may be Span-8Q® (sorbitan monooleate), Span-2Q® (somitan monolaurate), Span-40® (somitan monopalmitate), the Span-65 (somitan triestearato), or the
    10 Span-85® (sorbitan trioleate). Common Tween® sorbitan esters are Tween® 20 (Polyoxyethylene sorbitan monolaurate), Tween® 40 (Polyoxyethylene sorbitan monopalmitate), Tween® 60 (Polyoxyethylene sorbitan monostearate) or Tween® 80 (Polyoxyethylene sorbitan monooleate). The formation of the nanoparlicles of the invention may comprise mixtures of two or more different sorbitan esters. For example, a mixture of a sorbitan ester without polyoxyethylene blocks with another ester of
    15 sorbitan with polyoxyethylene blocks. In preparing the nanoparticles of the invention, the person skilled in the art can then choose various sorbitan esters to combine with the positively charged substance, and by way of example, it can be selected from the group comprising sorbitan monooleate, sorbitan dioleate, trioleate sorbitan, sorbititan sesqui-oleate, 5Orbitan monolaurate, sorbitan dilaurate, sorbitan trilaurate, sorbitan sesqui-Iaurate, sorbitan monostearate, sorbitan distearate, trytearate
    20 sorbitan, sorbitan sesqui-stearate, somitan monopalmitate, sorbitan dipalmitate, sorbitan tripalmitate, sorbitan sesqui-palmitate and combinations thereof Considering that said sorbitan esters optionally comprise polyoxyethylene blocks, the sorbitan ester used in the nanoparticles of the invention it can be a compound of formula I ov- ~: 5
    Formula I wherein each of RS, R6, R7 and Re is independently selected from the group consisting of ~ H, - (C = O) Cl-C40 alkyl, - (C = O) -C2-C40 alkenyl, - (C = O) -C-alkynyl; ¡-C40, with the proviso that at least one of RS, R6, R7Y RS is not -H; Y
    30 each of a, b, c and d is independently a number between O and 100. Those most common are those commercially available and usually correspond to a linear alkyl comprising between 2 and 20 carbon atoms, for example, one with 9, 11 , 13, 15 or 17 carbon atoms, or a linear alkenyl group comprising between 4 and 25 carbon atoms. Thus, an example may be an alkenyl group of the formula - (CH2) n-CH = CH- (CH2) m-CH3, where n is an integer between 1 and 10, Y
    35 m is an integer between 1 and 10. One usually used in which n is 7 and m is 7, corresponding to oleic acid. As can be seen, the polyoxyethylene blocks are optional, and each of a, b, c and d may be O. In the case of including said polyoxyethylene blocks, the sum of a, b, c and d may be, for example, of between 10 and 50 oo between 10 and 30 or between 15 and 30. For example, in the case of Tween® 80 (Polyoxyethylene sorbitan monooleate) a, b, c and d add 20.
    According to a preferred embodiment, R6, R7 and R6 are -H. That is, it is a sorbitan monoester, for example, one that is selected from the group consisting of sorbitan monooleate, sorbitan monolaurate, sorbitan monostearate, sorbitan monopalmitate, and combinations thereof. This group of sorbitan monoesters can be represented by formula 11
    or
    HO
    HO OH
    Formula 11 wherein R5 is selected from the group consisting of - (C = O) - Cl-C4 alkyl (J and - (C = O) - Cl -C40 alkenyl; and
    5 b is a number between O and 100, preferably O Again, one is preferred where R5 is an alkenyl of the formula - (CH2) n-CH = CH- {CH2) m-CH3, where n is a number integer between 1 and 10, and m is an integer between 1 and 10. The proportion of sorbitan esters in the nanoparticles is between 60% and 99% by weight with respect to the total weight of the nanoparticle. Typical proportions in which sorbilan esters are found are among
    10 80% and 98% by weight with respect to the tolal weight of the nanoparticle, for example, between 85% and 95%. The proportion of sorbitan esters is usually greater than 87% by weight with respect to the total weight of the nanoparticle. Another essential component of the invention is the inclusion of a positively charged substance. In the context of the present invention, "positively charged substance" is understood to be that charged molecule
    15 positive electric. In the field of the present invention, these substances are used to modulate the properties of the particles formed, and a person skilled in the art has at their disposal a wide variety of them. These substances are widely discussed in numerous reference books, for example, in "Dekker Encyclopedia of Nanoscience and Nanotechnology, Volume 4", James A. Schwarz, Cristian 1. Contescu, Karol Putyera, Editor: Marcel Dekker, 2004, or in " Encyclopedia of Polymer Science and Technology,
    20 Concise ", Herman F. Mark, third edition, Editor: Wiley, 2007. Non-limiting examples are ammonium salts, cationic polymers and fatty or lipophilic amines. The cationic polymer can be selected from the group consisting of protamine, polyglutamic , cationized dextran, cationized pululane, polyamino acids, cationized proteins, and their salts Polyamino acids are another family of positively charged substances that can be used in the nanoparticles of the invention.
    25 which may be selected from the group consisting of polylysine and polyarginine "Cationized proteins may be selected from the group consisting of gelatin, albumin, collagen, atelocollagen, and their cationized derivatives. Ammonium salts may be substances comprising an ammonium or attached amine group. to one, two or three residues that are independently selected from the group consisting of Cl-Cto alkyl, C2-C40 alkenyl,
    C2-C40 alkynyl, for example, selected from the group consisting of cetyl trimethyl ammonium bromide (CTAB) and benzalkonium chloride (BZC). The fatty amine can be oleylamine. Positively charged substances suitable for the present invention may be ammonium salts or fatty amines, for example CTAB, BZC, oleylamine or mixtures thereof. Thus, said positively charged substance may have the formula (RlO) p (R ") (R'2) NR9, wherein
    Each of R'o, Rl1 and R12 is independently selected from the group consisting of -H, ClC4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, and C7-CI 5 phenylalkyl; R9 is selected from the group consisting of C, -Cto alkyl, C2-CoIO alkenyl, C2-C40 alkynyl; pesOo1;
    yen where it also includes a counter-anion in the case where p is 1. What is the counter-anion is not critical and
    40 can be, for example, a halide (F ", CI-, Br or 1") The negatively charged substance can be added in proportions ranging from 1% to 39% by weight with respect to the total weight of the nanoparticles . Preferred amounts range between 2% and 20%, typically between 3% and 12% or between 12% and 9% or between 3% and 8%, by weight with respect to the total weight of the nanoparticle.
    An optional component of the nanoparticles of the invention is a negatively charged substance. These substances are known to those skilled in the art and in the case of the present invention, those capable of forming a coating layer in the nanoparticle are preferred. The family of negatively charged substances can be according to the present invention an anionic polymer, an anionic polymer being understood as a polymer with a negative charge
    50 Researchers have proven that the incorporation of one or more negatively charged substances provides surprising advantages. Contrary to what one would expect from a system with a negative surface charge, they support RES attacks (no phagocytization was observed by Kupffer cells) and nanoparticles improve the transfection capacity of the transported miRNA. This is contrary to the observations described in the literature in which a positive correlation is established between the negative charge and
    greater phagocytization by Kupffer cells.
    The anionic polymers are preferably polysaccharides. There is a great variety of these polysaccharides to
    available to the expert in the field, and which are frequently used in this field. A favorite variety
    5 are those that contain a carboxyl group (-COOH) or sulfate (.S03H) in the repeating monomer. Polysaccharides having at least one glucuronic acid in the repeating structure have been shown to be particularly suitable for the present invention. For example, said negatively charged substance may comprise a polysaccharide whose repetitive unit has the phonula [X-Y- (Z) n] where n is O or 1; X, Y YZ are each independently selected from the group consisting of monosaccharides, disaccharides and
    10 polysaccharides; with the proviso that at least one of X, Y Y Z comprises an acid sugar and wherein the groups X, Y and Z join together through -O-glycosidic bonds. According to an embodiment of the invention, said acid sugar is selected from the group consisting of aldonic acids, ulosonic acids, uronic acids, aldaric acids and mixtures thereof. For example. And it can comprise an acid sugar, for example, a uronic acid. A non-limiting example of said uronic acid may be one of the formula 111 O O O-
    He has R 'R'R2
    Formula 111
    wherein each of R1, R2 and R3 is independently selected from the group consisting of -H, -OH, -0-, OR4, N (H) -R4 and -0-S03, with the proviso that at least one of Rl, R2 and R3 be -0-,
    20 wherein -0 -fonnan the glucosidic bonds, and where R4 is selected from the group consisting of alkyl e, -C4, C2-C4 alkenyl, C2C4 alkynyl, - (C = O) -C-alkyl, -C! , - (C = O) -C2-C4 alkenyl, - (C = O) -C2-C4 alkynyl. Non-limiting examples of negatively charged substances having this formula are selected from the group consisting of hyaluronic acid, chondroitin sulfate and xanthan gum.
    The negatively charged substances that can be incorporated into the nanoparticles of the present invention may also be those that are selected from the group consisting of hyaluronic acid, colominic acid, polysalic acid, chondroitin, queratane, dextrans, heparin, carrageenans, furcelerans, alginates, agar agar, glucomannan. gellan gum. garrotine rubber. rubber guaro rubber tragacanto. gum arabic xanthan gum Karaya gum, pectins, celluloses, starches, their salts, fragments, derivatives and mixtures thereof
    The hyaluronan is a linear polymer that comprises the repetition of a disaccharide structure formed by the alternate addition of D-glucuronic acid and D-N-acetylglucosamine, linked alternating beta-1,4 and beta-1,3 glucosidic bonds. In the context of the present invention, hyaluronic acid with a wide range of molecular weights can be used. High molecular weight hyaluronic acid is commercially available, while lower molecular weight can be obtained by fragmenting hyaluronic acid from
    High molecular weight, using, for example, a hyaluronidase enzyme. The term "hyaluronic acid, hyaluronic acid, hyaluronan · as used herein includes either hyaluronic acid or a conjugate base thereof (hyaluronate). This conjugate base may be an alkaline salt of hyaluronic acid including inorganic salts such as, for example, sodium, potassium, calcium, ammonium, magnesium, aluminum and lithium salts, organic salts such as basic amino acid salts at neutral pH, preferably
    Said salts are pharmaceutically acceptable. In a preferred embodiment of the invention, the alkaline salt is the sodium salt of hyaluronic acid. The family of polysalic acids, tenin that includes colominic acid, is composed of linear polymers consisting of residues of N-acetylneuraminic acid (NeuSAc; also known as sialic acid), a natural constituent of cells and tissues, linked by glycosidic bonds a- {2 - + 8). Every
    The N-acetylneuraminic acid residue has a carboxyl group, responsible for the negative charge of colominic acid. It is a biocompatible and biodegradable, non-immunogenic material, whose degradation products are not toxic (Gregoriadis G et al. Cell. Mol. Life Sci. 2000, 57, 1964-1969) Dextran sulfate is a glucan (polysaccharide) complex consisting of units of glucose molecules, each of which contains approximately two sulfate groups. Dextran Sulfate is prepared
    50 by sulfation of dextran and subsequent purification by surplus procedures known to a person skilled in the art Heparin is a naturally occurring substance of the glycosaminoglycan family whose chemical structure comprises the repetition of 2-0 sulfoic acid disaccharide monomer units -L-iduronic and 2-deoxy · 2sulfamido - D-glucopyranosyl-6-0-sulfate. In the context of the present invention, it is possible to employ both the
    55 fractionated heparin as unfractionated. Traditional or unfractionated heparin is clearly distinguished from fractionated or low molecular weight heparin. The first one is a natural substance present in all vertebrates. Both types of heparin can be used in the form of a free base or in the form of salt, such as its sodium or calcium salt. Fractional or low molecular weight heparin is produced by chemical or enzymatic depolymerization of conventional heparins. Examples of this type of heparins are enoxaparin, pamaparin, dalteparin and nadroparin, as well as their salts such as sodium and calcium salts.
    The heparin derivatives can also be employed in the composition of the nanoparticular system of the present invention. These derivatives are known in the state of the art and originate as a result of the reactivity of the different functional groups present in the molecule. Thus, Nacetylated, O-decarboxylated, oxidized or reduced heparins are widely known. Chondroitin sulfate is a sulfated glucosaminoglycan (GAG) composed of a chain of sugars.
    10 alternates It is normally bound to proteins as part of a proteoglycan. In the context of the present invention, the term "chondroitin sulfate" includes all its different isomers and derivatives, as well as combinations thereof. For example, it is selected from the following substances and combinations thereof, summarized in formula IV
    or R0
    OR
    OR
    OR13
    HO OR OR 15 NH
    OH I Ac o e
    IV
    • Chondroitin A sulfate which is predominantly sulfated in carbon 4 of Nacethylgalactosamine sugar (GaINAc) and also known as 4-chondroitin sulfate (R13 = H, R14 = S03H and RI5 = H)
    20-Chondroitin B sulfate which is also called dermatan sulfate. This substance is composed of linear repeating units containing N-acetylgalactosamine and either L · iduronic acid or glucuronic acid, and each disaccharide can be sulphated once or sulfated twice. It is present mostly in the skin, but it is also found in blood vessels, cardiac valves, tendons and lungs
    25-Chondroitin C sulfate which is predominantly sulfated in carbon 6 of GalNAc sugar and
    which is also known as 6-chondroitin sulfate (RI3 = S03H, RI4 = H and RI5 = H);-chondroitin sulfate O which is predominantly sulfated in carbon 2 of glucuronic acidyen carbon 6 of GalNAc sugar and is also known as 2,6-chondroitin sulfate (RI3 = S03H,RI4 = H and R15 = S03H);
    Chondroitin E-30 sulfate which is predominantly sulfated in carbons 4 and 6 of GalNAc sugar and is also known as 4,6-chondroitin sulfate (RI3 = S03H, R14 = S03H and R1S = H); and where "e" represents the number of repetitions of the monomer, that is, its degree of polymerization
    The term "chondroitin sulfate" also includes organic and inorganic salts thereof. Generally such
    Salts are prepared, for example, by reacting the basic form of this compound with a stoichiometric amount of the appropriate acid in water or in an organic solvent or in a mixture of both. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Examples of inorganic salts include sodium, potassium, calcium, ammonium, magnesium, aluminum and lithium salts, and organic salts include, for example, ethylenediamine, ethanolamine, N, N-dialkylene salts.
    40 ethanolamine, triethanolamine, glucamine and basic amino acids. Preferably the salts are pharmaceutically acceptable. Keratane sulfate is a sulfated glucosaminoglycan similar to chondroitin sulfate in which the sulfate group is found in glucuronic acid. Specifically, it is made up of galactose and GlcNAc-6 sulfate, linked by a ~ -1, 4 bond.
    45 Carrageenan or carrageenan is made up of anhydrogalactose galactose and anfo units, sulfated or not, linked by alternate bonds · 1, 3 and -1.4. Depending on the degree of sulphation, the positions of the sulfate groups and the presence of anhydrogalactose groups, several types of carrageenan are distinguished, all included in the scope of the present invention. Glucomannan is a water-soluble polysaccharide of natural origin. The chemical structure of this compound
    50 consists of a linear polymer chain with a small proportion of ramifications. Specifically, it consists of o-mannose and O-glucose units linked by -1.4 bonds in a ratio of 1.6: 1, respectively. In a particular embodiment of the invention, the glucomannan used is a negatively charged glucomannan derivative selected from the phosphorylated, carboxymethyl and dicarboxy derivatives.
    glucomannan
    Gellan gum is a water-soluble polysaccharide of natural origin. The chemical structure of this compound
    It consists of a polymer chain consisting of units of a-L · rhamnosium, ~ -Glucuronic acid and two units
    5 of ~ -D-glucose. The polymer may be in a partially acetylated form. Depending on its degree of acetylation, gellan gum provides gels with different mechanical properties. In the context of the present invention, the term "gellan gum" includes all its different derivatives, as well as combinations thereof. Without wishing to be limited by theory, we think that the nanoparticles of the invention are homogeneous solid structures in which miRNAs They are adsorbed. This is contrary to the systems used to transport miRNAs that have been used so far. As already mentioned above, those that are now being used to perform dynamic tests of miRNAs use liposomes or vesicles, that is, lipid bilayer structures that enclose an aqueous phase inside. The miRNAs are bound to the solid structure formed by the sorbitol esters and the positively charged substance.
    The average size of the nanoparticles of the invention is between 1 and 999 nanometers. And they may have a negative or positive potential, depending on the components added, for example, whether or not they comprise a negatively charged substance, or the concentration of the added miRNA (s). According to a preferred embodiment, the nanoparticle of the invention has a positive potential between +1 and +100 mV. According to another preferred embodiment, the nanoparticle of the invention has a negative potential between -25 and -40 mV. The nanoparticles of the invention can induce other auxiliary substances, for example, a derivative of ethylene oxide, a compound in which a unit -CH2CH20- is repeated. Said ethylene oxide derivative may be a compound of formula R160 [CH2 '"CH2 + 0] rC (H) {R17) {R16), wherein R17 is a carbonyl or hydrogen group; R16 is an alkyl, alkenyl or alkynyl, from 2 to 24 carbon atoms; R16 is hydrogen or a group
    Alkyl of 1 to 6 carbon atoms; f is a value of between 1 and 100, for example, between 1 and 50, or between 1 and 24. Examples of ethylene oxide derivatives, without being limited thereto, are polyethylene glycol dodecyl ether (Brij 30), polyethylene glycol hexadecyl ether ( BriJ 56), polyethylene glycol 2-octadecyl ether (Brij 72), polyethylene glycol 8-octadecyl ether (Brij 78), polyethylene glycol 8-stearate (My ~ 45). 2-Hydroxyethyl octadecanoate (Myrj 52), ethylene glycol monostearate Ethylene oxide derivatives can be incorporated in proportions ranging between 0.1% and 20% by weight with respect to the total weight of the nanoparticle. Depending on the applications, the proportions may vary and be, for example, between 0.1% and 15% or between 5% and 15% or between 7 and 13% by weight, based on weight total nanoparticle
    Applications and miRNAs
    The other essential component of the nanoparticles of the invention is a miRNA or a mixture of miRNAs, which are usually found in proportions below 25% by weight with respect to the total weight of the nanoparticle. The proportion in which they are found in each case can be adjusted and can be, for example, between 0.01 and 10% or between 0.2% and 3% by weight with respect to the total weight of the nanoparticle. The miRNAs that are used in the nanoparticles of the invention are RNA molecules that typically comprise between 5 and 30 bases or between 15 and 25 bases. The miRNAs have multiple applications, and therefore also the nanoparticles of the invention, for example, in the Pharmaceutical, cosmetic or nutrition field. Thus, for example, the therapeutic application of various miRNAs in fields such as cancer (miRNA-20a, miR-29, miR-652 miR-34a, miR-16), diabetes or neurodegenerative diseases is currently being studied. One of the fields in which the use has been most developed
    45 of the miRNAs is cancer. Thus, a preferred embodiment is the nanoparticles of the invention for use in the treatment of a cancer that is selected from the group consisting of colorectal, malignant pleural mesothelioma, liver, pancreas, colon and liver and lung metastases. The term "treatment" or "treat" herein means administering the nanoparticles of the invention to prevent, reduce or eliminate one or more of the symptoms or causes or effects (metastases) of a disease or condition. It also covers the prevention, reduction or elimination of the sequelae of said disease or condition, or of the side or adverse effects caused by another medication used. It also covers the administration to maintain health in subjects at risk of suffering from such disease. The term "reduce" is understood as any improvement in the patient's situation, well measured by subjective parameters (for example, patient perception) or objectives (measurement of physiological, biochemical parameters,
    55 histopathological, microbiological-analytical). For example, the nanoparticles of the invention for use in the treatment of liver metastases is an embodiment of the invention, and it has been found that it is possible to reduce or eliminate the metastases and effects of said liver metathesis that often accompanies the colorectal cancer, which also constitutes an embodiment of the invention. In one embodiment the nanoparticles of the invention comprise miR-20a, miR-29, miR-652 or mixtures thereof, preferably miRNA-20a, for use in the treatment of colorectal cancer and its associated liver metastasis. Another aspect of the The invention is a method for the treatment of an individual in need of treatment comprising the administration of a therapeutically effective amount of nanoparticles of the invention. In the sense used in this description "therapeutically effective amount · refers to the amount of active ingredient calculated to produce the desired effect and will generally be determined, among other reasons, by the characteristics of the active ingredient used and the therapeutic effect that goes to be obtained In a particular embodiment, the dose of active ingredient administered to a subject for the treatment or prophylaxis of the aforementioned conditions is in the range of 10.10 to 1010 mgfkg of body weight, usually between 10.3 and
    10'10 mgl kg or between 10.2 and 50 mglKg of body weight Thus, the nanoparticles of the invention can form pharmaceutical compositions together with a pharmaceutically acceptable excipient. In fact, it is preferred that all components be pharmaceutically acceptable. The term "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not normally produce an allergic or similar unwanted reaction, such as gastric discomfort, dizziness and the like, when administered to a human being. Preferably, such as used herein, the term "pharmaceutically acceptable" means approved by a federal government or state regulatory agency or listed in the US Pharmacopoeia or other pharmacopoeia generally recognized for use in animals and more particularly in humans The term "excipient" refers to a diluent, adjuvant, or vehicle with which the drugs are administered.
    15 nanoparticles of the invention. They are substances that, for example, are added to the active substances or their associations to serve as a vehicle, enable their preparation and stability, modify their organoleptic properties or determine the physical-chemical properties of the drug and its bioavailability. Such pharmaceutical vehicles may be sterile liquids, such as water or oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Preferably water or saline solutions of aqueous solution and aqueous solutions of dextrose and glycerol are used as vehicles, particularly for injectable solutions. Suitable pharmaceutical vehicles are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The pharmaceutical composition or medicament may be in any form suitable for administration to humans and / or animals, preferably humans, including infants, children and adults and may
    25 be prepared by standard procedures known to those skilled in the art. The medicament can be prepared by standard procedures known to those skilled in the art, for example, reflected in "Pharmaceutics: The Science of Dosage Forms, second edition, Aullon, ME (ed.) Churchil1 UVingstone, Edinburgh (2002);" Encyclopedia of Phannaceutical Technology ", second edition, Swarbrick, J. and Boylan JC (eds.), Marcel Dekker, Inc. New York (2002);" Modern Pharmaceutics ", fourth edition, Banker GS and Rhodes
    C.T. (eds.) Marcel Dekker, Inc. New York 2002 and "The Theory and Practice of Industrial Pharmacy ·, Lachman
    L., Lieberman H. and Kanig J. (eds.), Lea & Febiger, Philadelphia (1986). The respective descriptions are incorporated herein by reference and are part of the description. The composition of the medication may vary depending on the route of administration. Non-limiting illustrative examples of said pharmaceutical dosage forms of the pharmaceutical composition of the invention include formulations.
    Oral or oral (liquids, solution, suspension, emulsion, gel, paste, powder, oral lyophilisate, tablets, capsules, pills, emulsions); sublingual formulations; ophthalmic formulations; ear formulations (otics); topical formulations; nasal formulations; rectal formulations; vaginal formulations; intrauterine formulations; inhalation or pulmonary formulations; parenteral formulations, for example, injectable formulation, for example, for intravenous injections or for subcutaneous injections.
    Preparation Procedure
    Another advantage of the present invention is the ease with which nanoparticles are prepared,
    procedure that does not require injection or homogenization In general terms the procedure
    comprises (i) the step of adding an organic solution comprising an organic solvent and an ester of
    45 sorbitan on an aqueous solution; (ii) the step of evaporating the solvent and water; and {iii} an optional incubation step. The positively charged substance can be incorporated into the organic solution or the aqueous solution. On the other hand, the miRNA (or miRNAs) is incorporated {n), either during stage (i) as part of the aqueous solution, either in the incubation stage (iii), or in both stages (i) and (iii). In the event that no miRNa is incorporated during stage (i), it is necessary to proceed to the incubation {stage (iii »to incorporate the miRNAs (or miRNAs) Thus, an alternative is a procedure comprising (i) the stage of adding a solution comprising an organic solvent, a sorbitan ester and a positively charged substance on an aqueous solution comprising a miRNA; and (ii) the step of evaporating the solvent and the water.Another alternative comprises (i) the step of adding a solution comprising an organic solvent, a sorbitan ester and a charged substance
    Positively over an aqueous solution; (ii) the step of evaporating the solvent and water; and (iii) the step of incubating the nanoparticle resulting from step (ii) in the presence of a miRNA. Suitable procedures are described, for example, in W02013 / 068625 A detailed description is made below which will be complemented by the examples. The organic phase is preferably added under stirring of the aqueous phase. The components that the system can additionally comprise, such as a negatively charged substance, can be added to the organic phase or the aqueous phase, depending on the characteristics of the substance incorporated into the system. Thus, in a particular embodiment, the aqueous solution further comprises a negatively charged substance.
    Alternatively, the additional components may be incorporated at a later stage, for example, step (iii) of incubation of the dispersion of nanoparticles formed with a solution comprising any of these substances. Alternatively, it is possible to obtain pegylated or modified nanoparticles with ethylene oxide derivatives
    5 These pegylated or modified nanoparticles with ethylene oxide derivatives can be prepared in a single step and also have the advantage that no chemical reaction is necessary in order to anchor the ethylene oxide chains to the nanoparticle surface. Thus, in another particular embodiment the organic phase further comprises an ethylene oxide derivative, for example, one of formula R160 [CHz-CH2-0] rC (H) (R H) (R1S) as described above. For the preparation of the nanoparticles of the invention it is usually preferable that the solvent of the organic phase is a hydromiscible solvent, for example, an aliphatic alcohol, such as ethanol, easy to evaporate, more harmless and stable against the components of the nanoparticles. The concentrations of the different components is not critical. For example, the somitan ester is dissolved in the organic phase at a concentration that can be between 0.1 and 10 mg / ml, or between 2 and 7 mg / ml. On the other hand,
    The positively charged substance may be in a concentration of between 0.01 and 5.0 mg / mL, for example, between 0.1 and 2.0 mgfmL preferably between 0.2 and 0.5 mgfmL. The negatively charged substance may be in a concentration between 0.01 and 5.0 mg / mL, for example, between 0.05 and 2.0 mg / mL preferably between 0.1 and 0.3 mg / mL The mixture of the aqueous and organic phases can be done at room temperature or by heating one or both phases. It has been found that the nanoparticles of the invention endure lyophilization and other dehydration processes without degradation. Therefore, the process may comprise an additional stage of total or partial dehydration (lyophilization or drying, respectively). In this way it is possible to preserve them during storage so that they retain their initial characteristics and reduce the volumes of
    25 product to be handled. The lyophilization or drying process leads, respectively, to a totally or partially dehydrated product. In case of dehydration, the process comprises an additional step in which the partially dehydrated or lyophilized nanoparticles are regenerated. In this way it is possible to dehydrate the nanoparticles to obtain a more stable product during storage and subsequently regenerate or recover the nanoparticles by means of a re-suspension process in an aqueous medium. Thus, a final aspect of the invention is directed to obtainable nanoparticles as described above. The components described above can be combined so that in each case the resulting nanoparticle of the invention adapts to the specific situation, and the present invention encompasses different combinations of esters
    35 somitan, positively charged substances, miRNAs, as well as negatively charged substances and other optional components if used. For example, one embodiment of the present invention is a nanoparticle comprising (i) one or more miRNAs (for example, miR-20a, miR-29, miR-652 or mixtures thereof); (ii) a sorbitan ester of formula J, as defined above; (iii) a positively charged substance of formula (R 'O) p (R ll) (R 12) NR9, as described above; and (iv) a negatively charged polysaccharide. In another embodiment, the present invention comprises (i) one or more miRNAs (eg, miR-20a, miR-29, miR-652 or mixtures thereof); (ji) a sorbitan ester of formula 1, as defined above; (iii) a positively charged substance that is selected from the group consisting of CTAB, BZC, oleylamine and mixtures thereof; and (iv) a negatively charged polysaccharide. Another embodiment of the present invention is a nanoparticle comprising (i) one or more miRNAs; (ii) an ester of
    45 sorbitan of formula 1, as defined above; (iii) a positively charged substance of
    Fnula (Rl0) P (Rll) (R12) NR9, as described above; and (iv) a negatively charged polysaccharide wherein at least one of its monomers comprises a group -COOH or -S03H. Another embodiment of the present invention is a nanoparticle comprising (i) one or more miRNAs; (ii) a sorbitan ester; (iii) a positively charged substance; and (iv) a positively charged polysaccharide wherein at least one of its monomers comprises a -COOH or -S03H group. Another embodiment of the present invention is a nanoparticle comprising (j) one or more miRNAs; (ii) a sorbitan ester of formula 1, as defined above; (jii) a positively charged substance of phonula (R 10) p (R 1 ') (R 12) NR 9, as described above; and (iv) a negatively charged polysaccharide. The above combinations and others that are not explicitly described are also part of the scope of protection
    It should be borne in mind that, in addition to the miRNA, the nanoparticles of the invention may comprise other components such as, for example, other active ingredients of interest, for which it would be sufficient to add them, to the organic solution or to the aqueous solution or both during the preparation of the nanoparticle It would also be possible to add them during the additional step (iii) of incubation.
    EXAMPLES For the description of some of the following examples, reference is made to results obtained by the following techniques · The size of the nanoparticles was determined by the technique of photon correlation spectroscopy (PCS) and using a Zeta Sizer (Zeta Sizer, Nano Series, Nano-ZS, Malvern Inslruments, RU), obtaining the average population size and polydispersion index_ The procedure involves diluting the samples in Milli-Q water to a 1:19 ratio. Each analysis was carried out at 25Q C with a detection angle of 173Q
    •
    The zeta potential of the nanoparticles was detected by the laser dispersion anemometry technique
    5 (LDA) using a Zeta Sizer (Zeta Sizer, Nano series, Nano-ZS, Malvern Instruments, RU.). The process comprises diluting the samples in millimolar KCI solution. The efficacy of the association of miRNAs with nanoparticles was determined by the agarose gel electrophoresis technique. The process comprises preparing a 2% agarose gel in TAE buffer (Tris-Acetate-EDTA, 40 mM Tris, 1% acetic acid, 1 mM EDTA), pH 8 with SYBR®. As loading substance
    10 gel staining of gold and glycerol nucleic acids was used. A potential difference of 25 mV was applied for 30 minutes and free miRNA was used as a control. As used in the following examples, the following polymers were purchased from different commercial establishments: hyaluronic acid (Bioibérica, Spain), chondroitin sulfate (Calbiochem, USA). Span® 80 and oleylamine were purchased from Sigma (Spain). The different miRNAs used are
    15 acquired from Exiqon (Denmark). The other products indicated in the examples below were purchased from Sigma (Spain)
    Example 1: Proof of concept In v va of the therapeutic effect in terms of cynical regulation of liver metastases provided
    Preparation of nanoparticles To prepare the nanoparticles, a solution of sorbitan monooleate (Span® 80, SP80) and oleylamine (OA) in 3 ml of ethanol (organic phase) was prepared at a concentration of 6.6 and 0.33 mgfml respectively Next, this organic phase was added to 6 ml of a stirred aqueous phase containing chondroitin sulfate
    25 (CS) at a concentration of 0.125 mgfml, thereby resulting in the spontaneous formation of NPs. The ethanol was finally removed under reduced pressure on a rotary evaporator. The miRNA was included in the aqueous phase at a concentration of 8.33 IJg / ml during the preparation of the nanoparticles. For this purpose, miR-20a was selected. These proportions correspond to percentages by weight with respect to the total nanoparticle weight of 91.67% SP80, 4.63% OA, 3.47% CS and 0.23% miR-20a
    Characterization of the nanoparticles The size and zeta potential of the nanoparticles was measured by photon correlation spectroscopy (photon corre / ation spectroscopy) and laser Doppler anometry (Iaser Dopp / er anemometry), respectively
    Formulation size (n m)Pdl~ Potential
    SP80-0A-CS 115.9 ± 15.60.073-37.5 ± 1.5
    SP80-0A-CS-miR20 (50 IJgfml miR-20a) 144, H 1.70.062-31, 9 ± 2.1
    Table 1. Physico-chemical characteristics of the nanoparticles of the invention
    Efficiency of the association between the miRNA and the nanoparticle The efficiency of the charge of the microRNA to the nanoparticles was confirmed by the agarose gel electrophoresis technique, as illustrated in Figure 1.
    Animal handling · All the experiments described in this work have been carried out in accordance with Spanish and European laws regarding the care of animals for experimentation. Animal handling and experimental methods of our laboratory have been analyzed and approved by the Animal Experimentation Committee of
    45 the University of the Basque Country, Spain. Every effort was made to minimize the number of animals used and their suffering. C57 BU6NCrl mice (females, B weeks old) were obtained from Charles River Laboratories España, S.A. The mice were housed in the Biological Resources Unit of the University of the Basque Country and kept in a temperature controlled environment (21 ± 1 QC), relative humidity of 55 ± 5%, light / dark cycle of 08hOO- 20hOO and they were supplied with conventional mouse feed
    50 yagua ad / ibitum
    Model of liver metastases and clinical evaluation: A model of liver metastasis was developed by injecting in mice a c26 murine colorectal cancer cell line (200,000 cells / animal). The animals were anesthetized with isofluorane (5% and 1.5% maintenance) and the cells were injected into the spleen. 55 After the intervention, the incisions were sutured. Animals were treated on day 3 after tumor inoculation and subsequently every 3 days until day 21 with a total dose of 25 IJg of miRNA per animal. The mice were separated into 5 different groups depending on the treatment. Group 1 was treated with placebo (glucose); group 2, according to the invention, was treated with SP80-0A-CS nanoparticles associated with
    miR-20a with a fluorescent marker (6-carboxyfluorescein) (FAM); group 3 was treated with free or naked miR-20a (without any specific vehicle); group 4 was treated with SP80-0A-CS nanoparticles associated with a miRControl (this miR does not attack any mRNA); Group 5 was treated with blank SP80-0A-CS nanoparticles (no associated mRNA). On day 21 after inoculation the animals were sacrificed and observed
    5 Jos livers and were subsequently processed for histological analysis
    Histology: After cervical dislocation, the liver was rapidly removed and fixed in paraformaldehyde (4% PFA) in PBS, overnight at 4Q C. The fixed livers were processed for paraffin inclusion. Be
    10 mounted serial sections of 10 J.lm thick in five parallel series and processed for hematoxylin eosin. Microscopic images were captured on a Zeiss microscope. Some liver sections were observed 48 hours after the last injection. Sections were incubated with anti-mannose receptor marker and Alexa 593 anti-mouse antibody. The green marker of miR20a (FAM) was observed inside the endothelial cells marked in red, confirming the endothelial attack or
    15 the specific distribution to sinusoid endothelial cells that provide the nanoparticles to miR-20a. After macroscopic evaluation of the livers, the therapeutic effect was confirmed in terms of clinical regulation of liver metastases in the group of animals treated with SP80-0A-CS associated with miR-20a (Figure 2). Figure 2 were stained with hematoxylin-eosin and the occupied area was quantified
    20 for the tumor under the microscope. The different groups were related to the control group (treated with glucose as a placebo) using the T-test. Statistically significant differences (p <0.05) were indicated with a • Likewise, the group treated with SP80-0A-CS nanoparticles associated with miR-20a and blank SP80-0A-CS nanoparticles (without mRNA) were compared associated) and the statistically significant difference (p <0.05) was indicated with a +. As can be seen in Figure 3, the area occupied by the
    The tumor was surprisingly smaller in mice treated with SP80-0A-CS nanoparticles associated with miR-20a (according to the invention) than in mice treated with miR-20a alone. This confirms an improvement impossible to anticipate a priori of nanoparticles according to the invention in the vehiculization of miRNAs in vivo. Moreover, the therapeutic effect provided by these nanoparticles in terms of clinical regulation of liver metastases is surprising.
    Example 2: Lyophilization The developed nano particles were successfully evaluated for their ability to maintain association with miRNA after a lyophilization process.
    权利要求:
    Claims (20)
    [1]

    A nanoparticle comprising (i) between 60% and 99% by weight, with respect to the total weight of the nanoparticle, of a sorbitan ester; (ii) a positively charged substance; and (iii) a miRNA
    [2]
    2. The nanoparticle according to claim 1, characterized in that said positively charged substance comprises an ammonium or amine group attached to one, two or three moieties that are independently selected from the group consisting of C, -C40 alkyl, C2-C40 alkenyl, CZ-C40 alkynyl
    [3]
    3. The nanoparticle according to any of the preceding claims, characterized in that said positively charged substance has the formula (RIO), (Rll) (R12) NR9, wherein
    each of Rl0, R11 and R12 is independently selected from the group consisting of -H, Cl alkylC4, C2-C4 alkenyl, C2-C4 alkynyl, and C7-C15 phenylalkyl;R9 is selected from the group consisting of C, -C4 alkyl or, CZ-C4Q alkenyl, C2-C40 alkynyl;pes001,
    15 yen where it also comprises a counterion in the case where p is 1.
    The nanoparticle according to any one of the preceding claims, characterized in that said positively charged substance is selected from the group consisting of oxylamine, benzalkonium chloride and cetyl trimethyl ammonium bromide.
    [5]
    5. The nanoparticle according to any of the preceding claims, characterized in that said sorbitan ester comprises a compound of formula I
    Formula I where each of RS, R6, R7 and Re is independently selected from the group consisting of -H, - (C = O)
    Cl-C40 alkyl, - (C = O) - CZ-C40 alkenyl, (C = O) -C2, C40 -alkynyl, with the proviso that at least one of R5, R6, R7Y Re is not -H ; each of a, b, c and d is independently a number between O and 100.
    The nanoparticle according to claim 5, characterized in that said alkyl group is linear and comprises between 2 and 20 carbon atoms. The nanoparticle according to any of claims 5 or 6, characterized in that said alkyl group has 9, 11, 13, 15 or 17 carbon atoms. The nanoparticle according to any of claims 5, 6 or 7, characterized in that said alkenyl group is linear and comprises between 4 and 25 carbon atoms
    [9]
    9. The nanoparticle according to any of claims 5, 6, 7 or 8, characterized in that said group
    35 alkenyl has the flanule - (CHz) n-CH = CH- (CHz) rrr-CH3, where n is an integer between 1 and 10, and m is an integer between 1 and 10
    [10]
    10. The nanoparticle according to claim 9, characterized in that n is 7 and m is 7.The nanoparticle according to any of claims 5 to 10, characterized in that a, b, c and d are O
    [12]
    12. The nanoparticle according to any of claims 5 to 10, characterized in that the sum of a, b, c and
    40 d is between 10 and 30 13 The nanoparticle according to any of claims 5 to 12, characterized in that R6, R7 and Re are -H
    [14]
    14. The nanoparticle according to any one of claims 1 to 5, characterized in that said sorbitan ester has the formula 11
    or
    HO
    HO OH
    Formula 11whereR5 is selected from the group consisting of - (C = O) -alkyl el-G.Il) and - (C = O) -alkyl C¡-C40; Y5 b is a number between O and 100.
    The nanoparticle according to claim 14, characterized in that b is O; and RS has the flange - (CH2) nCH = CH- (CH2) m-CH3, where n is an integer between 1 and 10, and m is an integer between 1 and 10.
    . The nanoparticle according to any of the preceding claims, characterized in that it has a positive potential between +1 and +100 mV.
    [17]
    17. The nanoparticle according to any of claims 1 to 15, characterized in that it comprises a negatively charged substance. . The nanoparticle according to claim 17, characterized in that said negatively charged substance comprises a polysaccharide whose repetitive unit has the formula [X-Y- (Z) n] wherein n is Oo 1; X, Y Y Z
    15 are each independently selected from the group consisting of monosaccharides, disaccharides and polysaccharides; with the proviso that at least one of X, Y, and Z comprises an acidic sugar and wherein the groups X, Y, and Z bind to each other through -O-glycosidic bonds
    [19]
    19. The nanoparticle according to claim 18, characterized in that said acid sugar is selected from the
    group formed by aldonic acids. ulosonic acids uronic acids aldaric acids and mixtures thereof
    [20]
    20. The nanoparticle according to claim 18, characterized in that Y comprises an acid sugar.
    [21 ]
    twenty-one . The nanoparticle according to claim 20, characterized in that Y comprises a uronic acid. 22 The nanoparticle according to any of claims 19 or 21, characterized in that said acid
    uronic has the formula 111OR
     Or 0-
    HO
     R 'RR ' 
    Formula 111
    wherein each of R ', R2 and R3 is independently selected from the group consisting of -H, -OH, -0-, OR4, N (H) -R4 Y -0-803, with the proviso that at least one of R ', R2 and R3 be -0-,
    Where -0- is the oxygen atom that forms the glucosidic bond, and where R4 is selected from the group consisting of C, -C4 alkyl, C2-C4 alkenyl, CZ-C4 alkynyl, (C = O) - C, -C4 alkyl, - (C = O} -CZ-C4 alkenyl, - (C = O) -C2-C4 alkynyl.
    The nanoparticle according to claim 17, characterized in that said negatively charged substance is a polysaccharide comprising one or more carboxylic groups. The nanoparticle according to claim 23, characterized in that said negatively charged substance comprises glucuronic acid in its repetitive structure.
    The nanoparticle according to any one of claims 17 to 24, characterized in that said negatively charged substance is selected from the group consisting of hyaluronic acid, chondroitin sulfate and xanthan gum
    The nanoparticle according to any of claims 17 to 25, characterized in that it has a negative potential between -25 and -40 mV
    The nanoparticle according to any of the preceding claims, wherein said miRNA is selected from the group consisting of miR-20a, miR-29, miR-652 miR-34a, miR-16 and mixtures thereof.
    [28]
    28. The nanoparticle according to claim 27, characterized in that said miRNA is miR-20a. 29 A pharmaceutical composition comprising the nanoparticle as defined in any of the
    claims 1 to 28, and a pharmaceutically acceptable excipient
    [30]
    30 Use of the nanoparticleso andhowbedefineinany of the claimsto28, for the
    manufacture of a medicine
    31 Use of the nanoparticle suchYhowbedefineinany of the claimsto28, forthe
    manufacture of a medicament for the treatment of an indication that is selected from the group consisting of cancer, diabetes, neurodegenerative diseases, respiratory system diseases, metabolic disorders and vascular diseases.
    [32]
    32. Use according to claim 31 for the treatment of cancer.33 Use according to any of claims 31 or 32 for the treatment of colorectal cancer
    [34]
    3. 4. Use according to any of claims 31, 32 or 33 for the treatment of liver metastases.
    [35]
    35 A process for the preparation of the nanoparticle as defined in any one of claims 1 to 28, characterized in that it comprises (i) the step of adding an organic solution comprising an organic solvent and a sorbitan ester over an aqueous solution ; wherein said organic solution or said aqueous solution or both comprise a positively charged substance; (ii) the stage of evaporating organic solvent and water; and (iii) the optional step of incubating the nanoparticle resulting from step (ii) in the presence of other substances; wherein a miRNA is incorporated (a) during stage (i) as part of the aqueous solution, (b) in the incubation stage (iii), or in both stages (i) and (ii)
    [36]
    36. The process according to claim 35, characterized in that said aqueous solution comprises a negatively charged substance
    [37]
    37. The process according to any of claims 35 or 36, characterized in that said organic solvent is totally or partially soluble in water.
    [38]
    38. The process according to any of claims 35, 36 or 37, characterized in that said solvent is an alcohol.
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    ES2636646B1|2018-08-03|NEW VEHICLES FOR THE TRANSFECTION OF miRNAs
    同族专利:
    公开号 | 公开日
    ES2636646B1|2018-08-03|
    WO2017174847A1|2017-10-12|
    US20190111002A1|2019-04-18|
    EP3441074A1|2019-02-13|
    EP3441074B1|2021-11-24|
    EP3441074A4|2019-11-13|
    US10888529B2|2021-01-12|
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    ES201630417A|ES2636646B1|2016-04-05|2016-04-05|NEW VEHICLES FOR THE TRANSFECTION OF miRNAs|ES201630417A| ES2636646B1|2016-04-05|2016-04-05|NEW VEHICLES FOR THE TRANSFECTION OF miRNAs|
    PCT/ES2017/070205| WO2017174847A1|2016-04-05|2017-04-04|NOVEL VEHICLES FOR THE TRANSFECTION OF miRNAS|
    EP17778723.1A| EP3441074B1|2016-04-05|2017-04-04|Novel vehicles for the transfection of mirnas|
    US16/091,071| US10888529B2|2016-04-05|2017-04-04|Vehicles for the transfection of miRNAs|
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