Nanovesicles and their use in nucleic acid delivery

Abstract

The present invention relates to nanovesicles comprising a sterol and a non-lipid cationic surfactant, such as myristalkonium chloride, wherein the sterol comprises DC-cholesterol. The present invention also relates to pharmaceutical compositions comprising the present invention, and to the use of the present invention as a delivery system and as a bioimaging and theranostic tool. Furthermore, the present invention also relates to nanovesicles or pharmaceutical compositions for use as drugs, particularly for use in the treatment of cancer.

Description

[Technical Field]

[0001] This application claims the benefit of European Patent Application No. 19382372.1, filed May 13, 2019.

[0002] The present invention relates generally to the field of nanovesicles useful for the delivery of nucleic acids, particularly small RNA molecules. The present invention provides, inter alia, nanovesicles, processes for preparing these nanovesicles, and their use in the treatment of diseases such as cancer (e.g., neuroblastoma). [Background technology]

[0003] RNA therapy is an emerging field with a number of promising targets across the entire transcriptome, including small RNAs, particularly small interfering RNAs (siRNAs) and microRNAs (miRNAs) (Non-Patent Document 1). RNA-based therapy could be an alternative for chemotherapy-resistant tumors, but in vivo administration remains a challenge in this field due to the rapid clearance and degradation of small RNAs in the bloodstream.

[0004] Nanovesicles have been the subject of much research due to their potential use for encapsulating nucleic acids and drugs and their clinical applications. Liposomes are the most studied nanovesicles, but in recent years, interest in nonliposomal lipid nanovesicles has grown. Quatosomes are stable unilamellar nanovesicles with uniform morphology. They contain a quaternary ammonium surfactant, such as cetrimonium bromide (CTAB), myristalkonium chloride (MKC), or cetylpyridinium chloride (CPC), and a sterol, such as cholesterol or β-sitosterol, in a defined molar ratio (Non-Patent Document 2). Compressed fluid-based techniques, such as the Depressurization of an Expanded Liquid Organic Solution-Suspension (DELOS-SUSP) method, have been used to fabricate quatosomes (Non-Patent Document 2; Patent Document 1).

[0005] Although nanovesicles are expected to be useful in nucleic acid delivery, they pose a problem with intracellular release of their contents, as nanovesicles may be taken up into endosomes and degraded in lysosomes, preventing the release of the nanovesicle cargo contents into the cytoplasm. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. WO2006 / 079889 [Non-patent literature]

[0007] [Non-Patent Document 1] Bumcrot D et al. Nat Chem Biol 2006,2:711-719 [Non-patent document 2] Grimaldi N.et al.Chem Soc Rev 2016,45:6520-6545 Summary of the Invention [Problem to be solved by the invention]

[0008] From what is known in the art, there remains a need to find nucleic acid delivery methods that effectively release useful nucleic acids into the cytoplasm through endosomal escape for diseases that use nucleic acids as therapeutic agents, such as cancer diseases. [Means for solving the problem]

[0009] The present inventors have developed a nucleic acid delivery tool that allows the transported nucleic acid to carry out its activity in the cytosol. This tool is a quatsome (QS) (hereinafter referred to as "the nanovesicles of the present invention" or "the QS of the present invention"), which contains a non-lipid cationic surfactant (e.g., MKC) and DC-cholesterol (in a specific case, 100% DC-Chol) at a molar ratio of, for example, 1:1.

[0010] The QS of the present invention are small unilamellar vesicles less than 100 nm in size, have low polydispersity, are spherical, and have high colloidal stability over time (see Figures 1-4). Furthermore, the QS of the present invention are pH sensitive, allowing for a buffering effect (see Figure 5).

[0011] Surprisingly, it was discovered that DC-Chol formed nanovesicles in all formulations tested when combined with nonlipid cationic surfactants, whereas cholesterol or other cholesterol derivatives did not necessarily form nanovesicles. (Figure 6 shows that Chol-VS and a nonlipid cationic surfactant (CTAB) formed ribbons, and Figure 6C shows that ribbon-like nanostructures were favorably formed in water containing 10% etOH cholesterol and the nonlipid cationic surfactant MKC.)

[0012] The inventors have used the nanovesicles of the present invention for siRNA and miRNA delivery in neuroblastoma cells, with surprising results in both expression of the nucleic acids and their targets.

[0013] Compared with QS containing other sterols, the QS of the present invention exhibits high RNA complexation efficiency (see Figure 8) and high cell viability when complexed with miRNA (see Figure 9). Surprisingly, the QS of the present invention is the only QS that also enables the expression of miRNA during delivery (see Figure 10), and was able to regulate the expression of the target of this miRNA (miR-323a-5p) in neuroblastoma cells at the mRNA level (see Figure 11) and protein level (see Figure 12). Quatosomes of the present invention using 100% DC-Chol as a sterol (referred to as "QS4" in the examples below) were optimal for miRNA delivery (see Figures 11 and 12). However, quatosomes of the present invention containing approximately 50% DC-Chol as a sterol (referred to as "QS3" in the examples below) also exhibited slower miRNA release from QS (see Figure 13). The most effective QS4-sRNA complex for the above effects in neuroblastoma cells was found to have a mass ratio of miRNA to QS4 of 13.5·10 -2 and 20.24·10 -2 The complexes QS4-miR-323a-5p(V) and QS4-miR-323a-5p(VI) were obtained from the QS4-miR-323a-5p complexes (see Table 7 and Figures 15-16). Transfection of miR-323a-5p using the QS4-miR-323a-5p complex of the present invention reduced cell proliferation in neuroblastoma cell lines, demonstrating a similar effect compared to Lipofectamine 2000 (registered trademark) (see Figures 17-18). Furthermore, the QS4-miR-323a-5p complex of the present invention also enabled siRNA transfection using siCCND1 in neuroblastoma cells (see Figures 19-22).

[0014] From the data provided below, it is noteworthy that the QS of the present invention can be used as a nucleic acid delivery system for diseases that can be treated with nucleic acids, such as cancer, and for the specific example, neuroblastoma.

[0015] Furthermore, the nanovesicles of the present invention have been demonstrated to protect nucleic acid cargo from RNAse A degradation (Figure 25).

[0016] QS of the present invention can be efficiently functionalized, for example, with fluorescent molecules for in vitro and in vivo tracking of these particles (see Example 5, Figure 23 for functionalization with Dil), with targeting units such as peptides or antibodies to facilitate "selective" delivery of biomolecules at specific target sites, or with stealth polymers such as poly(ethylene glycol) (PEG) to improve blood circulation time (Cabrera I, et al. 2013 Nano Letters, 2013, 13(8), 3766-3774). Functionalization of QS with Dil did not alter miRNA binding or delivery and reduced neuroblastoma growth to the same extent as unfunctionalized QS-miRNA complexes (see Figure 24).

[0017] Biodistribution analysis of QS4 combined with miR-control or miR-323a-5p showed that 24 hours after a single dose, miR-323a-5p expression was increased in the liver, lung, spleen, kidney, and subcutaneous tumors (150-fold, 66,000-fold, 15,000-fold, 570-fold, and 125-fold, respectively) compared to the QS4-miRNA control (see Figure 26). No gross signs of toxicity or adverse side effects were observed.

[0018] Thus, a first aspect of the present invention relates to nanovesicles comprising a sterol and a non-lipid cationic surfactant, wherein the sterol comprises DC-cholesterol (DC-Chol).

[0019] A second aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of nanovesicles according to the first aspect of the present invention and a pharmaceutically acceptable excipient or vehicle.

[0020] A third aspect of the present invention relates to nanovesicles according to the first aspect of the invention or pharmaceutical compositions according to the second aspect of the invention as a delivery system.

[0021] The nanovesicles of the first aspect of the invention or the pharmaceutical composition of the second aspect of the invention may be used, for example, in the treatment of diseases in humans using nucleic acids as therapeutic agents for the treatment of such diseases.

[0022] A fourth aspect of the present invention relates to a nanovesicle according to the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention for use as a medicament.

[0023] A fifth aspect of the present invention relates to nanovesicles according to the first aspect of the invention or pharmaceutical compositions according to the second aspect of the invention for use in the treatment of a non-infectious disease, preferably in the treatment of cancer.

[0024] A sixth aspect of the present invention relates to the use of nanovesicles according to the first aspect of the invention as a bioimaging tool.

[0025] The QS can be prepared by the CO2-based DELOS-SUSP method (International Application No. WO2006079889), which ensures robustness and reproducible scale-up of the QS, allowing for the preparation of sufficient quantities of nanomedicine for both preclinical and clinical trials.

[0026] A seventh aspect of the present invention relates to a process for producing nanovesicles of the first aspect of the present invention using the DELOS-SUSP method.

[0027] An eighth aspect of the present invention relates to a kit comprising nanovesicles according to the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention.

[0028] A ninth aspect of the present invention relates to the use of nanovesicles according to the first aspect of the invention as a theranostic tool.

[0029] A tenth aspect of the present invention relates to nanovesicles according to the first aspect of the present invention as pH buffering agents. [Brief explanation of the drawings]

[0030] [Figure 1] Physicochemical properties of the above-mentioned quatosome (QS) systems formed by self-assembly of quaternary ammonium surfactants (MKC) with various sterols (Chems, cholesterol, or DC-Chol). Hydrodynamic diameter and surface charge density of the above-mentioned QS systems measured by DLS technique 1 week (A) or 2 months (B) after Delos-SUSP preparation. Graphs represent the mean ± SD of three independent experiments. [Figure 2] Physicochemical properties of the above-mentioned quatosome (QS) systems formed by self-assembly of quaternary ammonium surfactants (MKC) with various sterols (Chems, cholesterol, or DC-Chol). Hydrodynamic diameter and surface charge density of the above-mentioned QS systems measured by DLS technique 1 week (A) or 2 months (B) after nanovesicle purification by diafiltration. Graphs represent the mean ± SD of three independent experiments. [Figure 3] Narrow particle size distributions of various QS systems measured by DLS after (A) preparation of DELOS-SUSP or (B) diafiltration. Graphs represent the mean ± SEM of three independent experiments. [Figure 4] Representative high-resolution cryo-TEM images of QSs with varying sterol composition: QS0 (A), QS1 (B), QS2 (C), QS3 (D), QS4 (E), QS5 (F), QS6 (G), and QS7 (H). Scale bar, 200 nm. [Figure 5] Buffering capacity of QS at various % of pH-sensitive sterol DC-Chol. The graph shows the pH change with acidic HCl concentrations from 0.01 μM to 3 μM. [Figure 6]High-resolution representative cryo-TEM images of aqueous Chol / Chol-VS / CTAB mixtures with various compositions: CS-VS1: (32% Chol-VS / 68% Chol):CTAB; CS-VS2: (49% Chol-VS / 51% Chol):CTAB; CS-VS3: (66% Chol-VS / 34% Chol):CTAB; CS-VS4: (74% Chol-VS / 26% Chol):CTAB; CS-VS5 (100% Chol-VS / 0% Chol):CTAB). A) High-resolution representative cryo-TEM images of the CS-VS1 system taken 14 and 40 days after preparation. B) High-resolution representative cryo-TEM images of the CS-VS2, CS-VS3, CS-VS4, and CS-VS5 systems analyzed 14 days after sample preparation. C) High-resolution cryo-TEM image showing CS-CH composed of (100% Chol / 0% DC-Chol):MKC nanostructures formed in water with a 1:1 molar ratio of sterol to surfactant containing 10% EtOH 7 days after sample preparation by DELOS-SUSP. [Figure 7] Morphology and layering of QS-miRNA complexes. High-resolution cryo-TEM images depicting various formulations of QS-miRNA complexes with varying miRNA-to-QS mass ratios (III (A, D, G, J, and M); V (B, E, H, K and N); VI (C, D, I, L and O), various QS systems: QS0(A)-C, QS1(D-F), QS2(G-I), QS3(J)-L), and QS4(M-O). Scale bar, 200 nm. [Figure 8] Complexation efficiency of QS4 with miRNA through electrostatic interactions. Gel electrophoresis of miR-323a complexes with QS4 at various miRNA-to-QS mass ratios listed in Table 7 (lanes 2–9) and a standard calibration of naked miRNA (lanes 11–14). [Figure 9] High cell viability of QS and QS-miRNA complexes in a chemotherapy-resistant NB cell line (SK-N-BE(2)). Proliferation studies measuring the IC50 after 24 h of incubation of QS (A) with the QS1-4-miR-control complex (B). Means ± SEM from two experiments are plotted. [Figure 10] miR-323a-5p expression levels in SK-N-BE(2) cells transfected with naked miRNA (50 nM), MKC-miR-323a-5p micelles at the miRNA-to-MKC mass ratio (I), and QS-miR-323a-5p complexes at the above-mentioned miRNA-to-QS mass ratios ((III), (IV), (V), and (VI), see Table 7). MiRNA expression levels were measured by qPCR. Graphs represent the mean ± SEM of three independent experiments. *P<0.05, *p<0.01, *p<0.001. [Figure 11] Regulation of miR-323a direct targets after transfection with QS-miRNA complexes. miRNA-direct target expression 48 hours after transfection of miR-323a-5p or miR-control (50 nM) with naked miRNA, MKC micelles, and the QS system described above in SK-N-BE(2) cells. Graphs represent the mean ± SEM of three independent experiments. *P<0.05*, *p<0.01**, *p<0.001***. All QS-miRNA complexes are listed in Table 7. [Figure 12] Regulation of miR-323a direct targets at the protein level after transfection with QS-miRNA complexes. Quantification of representative band intensities of the above-mentioned proteins in NB cells 72 hours after transfection with the above-mentioned QS-miRNA complexes. Histograms represent quantification of band intensity signals mean ± SEM from three independent experiments. *P<0.05*, p<0.01**, p<0.001***. All QS-miRNA complexes are listed in Table 7. [Figure 13] miRNA release from the QS3 or QS4 surface after overnight incubation with NB cells. The graph shows the FRET ratio of DilQS-miR-control Cy5 complexes using various QS formulations after overnight transfection into SK-N-BE(2) neuroblastoma cells. The composition of all QS-miRNA complexes is shown in Table 7. [Figure 14]Increased miR-323a-5p expression levels after transfection with QS4-miR-323a-5p complexes. Expression levels of miR-323a-5p in SK-NBE(2) were measured by qPCR 48 hours after transfection with QS4-miR-323a-5p complexes and QS4-miR-control complexes, both at the above-mentioned miRNA-to-QS mass ratios (V, VI, and VIII). Graphs represent the mean ± SEM of three independent experiments. *P<0.05*, *p<0.01**, *p<0.001***. All QS-miRNA complexes are listed in Table 7. [Figure 15] Modulation of miR-323a-5p direct targets at the mRNA expression level 48 hours after transfection with QS4-miR-323a-5p complexes at the above-mentioned miRNA-to-QS mass ratios (V, VI, and VIII) in NB cells. Graphs represent quantification of the mean ± SEM of three independent experiments. *P<0.05*, *p<0.01**, *p<0.001***. All QS-miRNA complexes are listed in Table 7. [Figure 16] Modulation of miR-323a-5p direct and indirect targets at the protein level after transfection with QS4-miR-323a-5p complexes at the above-mentioned miRNA-to-QS mass ratios (V, VI, and VIII) in NB cells. Histograms represent quantification of the mean ± SEM of three independent experiments. *P<0.05*, p<0.01**, p<0.001***. All QS-miRNA complexes are shown in Table 7. [Figure 17] Decreased proliferation of SK-N-BE(2) cells after transfection with QS4-miR-323a-5p complexes at the above-mentioned miRNA-to-QS mass ratios (I, III, and IV). A proliferation experiment comparing miR-323a-5p complexed with QS4 with miRNA-control (50 nM) was performed in NB cells 96 hours after transfection. Graphs represent the mean ± SEM of three independent experiments. *P<0.05*, *p<0.01**, *p<0.001***. All QS-miRNA complexes are listed in Table 7. [Figure 18]Cell proliferation analysis in SK-N-BE(2) cells after transfection with QS4-miR-323a-5p complexes compared to Lipofectamine 2000®. A proliferation experiment comparing miR-323a-5p complexed with QS4 or liposomes (i.e., Lipofectamine 2000) with miRNA-control (50 nM) was performed in NB cells 96 hours after transfection. Graphs represent the mean ± SEM of three independent experiments. *P<0.05*, *p<0.01**, *p<0.001***. All QS-miRNA complexes are shown in Table 7. [Figure 19] Modulation of CCND1 direct targets at the mRNA expression level 48 hours after transfection with QS4-siCCND1 complexes at the above-mentioned siRNA-to-QS mass ratios (V, VI, and VIII) in NB cells. The graph represents the mean ± SEM of three independent experiments. *P<0.05*, p<0.01**, p<0.001***. All QS-miRNA complexes are shown in Table 7. [Figure 20] Modulation of CCND1 direct and indirect target modifications at the protein level after transfection with QS4-siCCND1 complexes (V, VI, and VIII) in NB cells. Histograms represent quantification of the mean ± SEM of three independent experiments. *P<0.05*, p<0.01**, p<0.001***. All QS-miRNA complexes are shown in Table 7. [Figure 21] Decreased proliferation of SK-N-BE(2) cells after transfection with QS4-siCCND1 complexes (V, VI, and VIII). Proliferation experiments were performed on NB cells 96 hours after transfection, comparing siCCND1 complexed with QS4 with siRNA control (50 nM). Graphs represent the mean ± SEM of three independent experiments. *P<0.05*, *p<0.01**, *p<0.001***. All QS-miRNA complexes are shown in Table 7. [Figure 22]Cell proliferation analysis in SK-N-BE(2) cells after transfection with QS4-siCCND1 complexes compared to Lipofectamine2000®. Proliferation experiments were performed in NB cells 96 hours after transfection comparing siCCND1 complexed with QS4 with siRNA control (50 nM). Graphs represent the mean ± SEM of three independent experiments. *P<0.05*, *p<0.01**, *p<0.001***. All QS-miRNA complexes are shown in Table 7. [Figure 23] Physicochemical characterization of the above-mentioned quatosome (QS4) system, formed by self-assembly of a quaternary ammonium surfactant (MKC) with DC-Chol sterol and functionalized with a Dil fluorophore (Dil-QS4) or a PEG stealth polymer (PEG-QS4), composed of 10% Chol-PEG / 90% DC-Chol:MKC. Hydrodynamic diameter and surface charge density of the above-mentioned QS system were measured by DLS technique one week after purification. Graph represents the mean ± SEM of three independent experiments. [Figure 24] Cell proliferation analysis of SK-N-BE(2) cells transfected with DilQS4-miR-323a-5p complexes and simple QS4-miR-323a-5p complexes. A proliferation experiment comparing miR-323a-5p complexed with DilQS4 or simple QS4 with miRNA control (50 nM) was performed in NB cells 96 hours after transfection. Graphs represent the mean ± SEM of three independent experiments. *P<0.05*, *p<0.01**, *p<0.001***. All QS-miRNA complexes are shown in Table 7. [Figure 25]QS4 protects miR-323a-5p from degradation by RNAse A. Gel electrophoresis of miRNA protection after QS4 complex formation in the presence of RNAse A. QS4 was added in lane 2, and the QS4-miRNA complex was added at a dose (V) (lanes 3–8). Naked miRNA was added as a negative control (lanes 9–14). RNAse A (25 μg / mL) complex treatment was performed for 30 min, 1 h, 2 h, or 4 h (lanes 5–12). SDS (0.25%) decomplexation was performed after complex formation (lane 4), after RNAse A treatment (lanes 5–12), and with naked miRNA (lane 14). [Figure 26] Biodistribution analysis. DilQS4-miR-323a-5p complexes (2 mg / kg miRNA and 10 mg / kg QS4) were intravenously injected into athymic nude mice. 24 hours later, miR-323a-5p expression was analyzed by qPCR. Compared with mice injected with DilQS4-miR-control, miR-323a-5p accumulated in subcutaneous neuroblastoma tumors, lungs, spleen, kidneys, and liver. *P<0.05*, *p<0.01**, *p<0.001***. The QS-miRNA complex preparation protocol is shown in Table 11. DETAILED DESCRIPTION OF THE INVENTION

[0031] Detailed Description of the Invention All terms used herein in this application are to be understood in their ordinary meaning as known in the art unless otherwise specified. Other, more specific definitions of certain terms used in this application are set forth below and are intended to be applied uniformly throughout the specification and claims, unless another expressly set forth definition provides a broader definition.

[0032] DC-cholesterol, CAS number 137056-72-5, is also known as DC-Chol, cholesteryl N-(2-dimethylaminoethyl)carbamate, or 3β-{N-[2-(dimethylamino)ethyl]carbamoyl}cholesterol or 3-(N-(N',N' dimethylaminoethane)carbamoyl)cholesterol) or (C32-H56-N2-O2) or (cholest-5-en-3-ol(3beta)-,(2-(dimethylamino)ethyl)carbamate) or (3beta-(N-(N',N' dimethylaminoethane)carbamoyl)cholesterol).

[0033] Non-lipid cationic surfactants include, but are not limited to, non-lipid cationic quaternary ammonium surfactants. The cationic surfactants of the present invention are not lipids.

[0034] Non-lipid quaternary ammonium surfactants are quaternary ammonium salts in which one nitrogen substituent is a long-chain alkyl group. Non-lipid quaternary ammonium surfactants are water-soluble and self-assemble to form micelles above the critical micelle concentration (cmc). Conversely, lipid quaternary ammonium surfactants self-assemble to form other structures such as vesicles, planar bilayers, and reverse micelles. The quaternary ammonium surfactants of the present invention are not lipids.

[0035] In an embodiment of the first aspect of the present invention, the non-lipid cationic quaternary ammonium surfactant is selected from the list consisting of myristalkonium chloride (MKC), cetyltrimethylammonium bromide (CTAB), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), cetyltrimethylammonium chloride (CTAC), benzethonium chloride (BZT), stearalkonium chloride, cetrimide, benzyldimethyldodecylammonium chloride, and combinations thereof.

[0036] In an embodiment of the first aspect of the present invention, the non-lipid cationic quaternary ammonium surfactant is myristalkonium chloride (MKC).

[0037] Myristalkonium chloride (MKC), CAS number 139-08-2, is also known as benzyldimethyltetradecylammonium chloride or myristyldimethylbenzylammonium chloride or N-benzyl-N-tetradecyldimethylammonium chloride or N,N-dimethyl-N-tetradecylbenzenemethanaminium chloride or tetradecylbenzyldimethylammonium chloride.

[0038] In another embodiment of the first aspect of the present invention, the non-lipid cationic quaternary ammonium surfactant is cetyltrimethylammonium bromide (CTAB).

[0039] A first aspect of the present invention relates to nanovesicles comprising a sterol and a non-lipid cationic surfactant, wherein the sterol comprises DC-cholesterol (DC-Chol).

[0040] In an embodiment of the first aspect of the present invention, the sterol is DC-Chol, e.g., , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.

[0041] In an embodiment of the first aspect of the present invention, the percentage of DC-Chol to total sterols is at least 20%, alternatively at least 47%, alternatively at least 90%, alternatively 100%.

[0042] In an embodiment of the first aspect of the present invention, the sterol is a mixture of DC-Chol and cholesterol, or a mixture of DC-chol and a cholesterol derivative. For example, the cholesterol derivative includes polyethylene glycol (PEG). For example, the cholesterol derivative is Chol-PEGn-X, where "n" is the length of the PEG chain (e.g., n=0 or at least 1), and "X" is -SH, -OH, -CHO, -OCH3, -NH2, -NH, -CH3, -N3, -COOH, -maleimide, a peptide, an antibody, or a sugar. Here, the peptide may be HSYWLRS peptide (SEQ ID NO: 22) (e.g., sequence: YSHSHSYWLRSGGGC (SEQ ID NO: 35)), GD2 mimetic binding peptide (e.g., sequence: RCNPNMEPPRCWAAEGD (SEQ ID NO: 36) or VCNPLTGALLCSAAEGD (SEQ ID NO: 37)), neuropeptide Y (e.g., sequence: MLGNKRLGLSGLTLALSLLVCLGALAEAYPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRYGKRSSPETLISDLLMRESTENVPRTRLEDPAMW (SEQ ID NO: 38)), P75 neurotrophin receptor (e.g., sequence: CENLYFQSGSMAFIPYFAR) (SEQ ID NO: 39), rabies virus glycoprotein (Rabies virus The glycoprotein (RVG) peptide (e.g., sequence: YTIWMPENPRPGTPCDIFTNSRGKRASNG (SEQ ID NO: 40) or KSVRTWNEIIPSKGCLRVGGRCHPHVNGGG) (SEQ ID NO: 41), a dopaminergic peptide (e.g., sequence: CCYHWKHLHNTKTFL) (SEQ ID NO: 42), an RGD-peptide, and a GD2 antibody. An example of a sugar may be D-glucose or a glucosamine derivative.

[0043] In an embodiment of the first aspect of the present invention, the nanovesicles are non-liposomal lipid nanovesicles.

[0044] In another embodiment of the first aspect of the present invention, the nanovesicles are quatosomes comprising 100% DC-Chol as the sterol and MKC in a molar ratio ranging from 10:1 to 1:5.

[0045] In an embodiment of the first aspect of the invention, the nanovesicles are quatosomes comprising 100% DC-Chol as sterols and MKC in a ratio of 1:1. In another embodiment of the first aspect of the invention, the nanovesicles are quatosomes comprising 100% DC-Chol as sterols and MKC in a ratio of 1:2 and 2:1.

[0046] In an embodiment of the first aspect of the present invention, the nanovesicles are spherical, unilamellar, uniformly sized and stable.

[0047] In the state of the art, there are well-known methods to characterize the nanovesicles of the invention, for example by their electrical potential Z. The size of the nanovehicles can be measured by any method known to the expert, such as dynamic light scattering (DLS), mass spectrometry, small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM) or high-resolution transmission electron microscopy (HR-TEM).

[0048] For example, to characterize the nanovesicles of the present invention, the protocol disclosed in Danaei, M.; et al. "Impact of Particle Size and Polydispersity Index on the Clinical Applications of Lipidic Nanocarrier Systems" Pharmaceuticals 2018, 10, 57 is followed.

[0049] In the present invention, the term "spherical" refers to a diameter of 20 to 500 nm, for example, 50 to 300 nm.

[0050] The term "uniform size" refers to nanovesicles having a polydispersity index (PDI) of 0.1 to 0.5, such as 0.1 to 0.3. The stability of the nanovesicles of the present invention can be measured by dynamic light scattering (DLS). For example, the stability of the nanovesicles of the present invention over time can be measured by DLS and refers to a hydrodynamic diameter remaining below 300 nm and a PDI within the range of 0.1-0.3.

[0051] In an embodiment of the first aspect of the present invention, the nanovesicles have an average diameter of less than 300 nm, a PDI of 0.1 to 0.3, and are stable for up to at least 2 months.

[0052] In an embodiment of the first aspect of the present invention, the nanovesicles comprise nucleic acids, ie small RNAs such as miRNA, siRNA or shRNA.

[0053] In an embodiment of the first aspect of the invention, the nucleic acid is inside the nanovesicle. In another embodiment of the first aspect of the invention, the nucleic acid is outside the nanovesicle.

[0054] The term "small RNA" refers to RNAs less than 200 nucleotides in length. They are typically non-coding RNA molecules that are modulators of gene expression, such as microRNAs (miRNAs) or small interfering RNAs (siRNAs).

[0055] In an embodiment of the first aspect of the present invention, the nanovesicles comprise nucleic acids with tumor suppressor function.

[0056] In an embodiment of the first aspect of the present invention, the miRNA is selected from the group consisting of hsa-miR-323a-5p, hsa-miR-497, has-miR-380-5p, hsa-miR-892b, hsa-miR-654-5p, hsa-miR-885-3p, hsa-miR-193a-3p, hsa-miR-661, hsa-miR-491-3p, hsa-miR-193b-5p, hsa-miR-3150a-3p, hsa-miR-744-5p, hsa-miR- iR-326, hsa-miR-665, hsa-miR-185-3p, hsa-miR-34b-5p, hsa-miR-138-2-3p, hsa-miR-4440, hsa-miR-450b-3p, hsa-miR- 1180, hsa-miR-3140-3p, hsa-miR-4291, hsa-miR-30b-3p, hsa-miR-541-3p, hsa-miR-483-5p, hsa-miR-4292, hsa-miR-124- 3p, hsa-miR-1207-5p, hsa-miR-193b-3p, hsa-miR-221-5p, hsa-miR-3913-3p, hsa-miR-5095, hsa-miR-891b, hsa-miR-127 5, hsa-miR-299-3p, hsa-miR-149-3p, hsa-miR-132-5p, hsa-miR-509-3-5p, hsa-miR-3677-3p, hsa-miR-876-3p, hsa-miR- 940, hsa-miR-4655-5p, hsa-miR-555, hsa-miR-342-5p, hsa-miR-3181, hsa-miR-3154, hsa-miR-5585-3p, hsa-miR-708-5p, hsa-miR-3135a, hsa-miR-4664-3p, hsa-miR-4289, hsa-miR-135a-3p, hsa-miR-522-5p, and any combination thereof.

[0057] In an embodiment, the above-mentioned miRNAs are identified by their identification numbers in the public database miRBase (as of April 30, 2019): MIMAT0004696, MIMAT0002820, MIMAT0000734, MIMAT0004918, MIMAT0003330, MIMAT0004948, MIMAT0000459, MIMAT0003324, MIMAT0004765, MIMAT0004767, MIMAT000 0734, MIMAT0015023, MIMAT0004945, MIMAT0000756, MIMAT0004952, MIMAT0004611, MIMAT0000685, MIMAT0004596, M IMAT0018958, MIMAT0004910, MIMAT0026735, MIMAT0015008, MIMAT0016922, MIMAT0004589, MIMAT0004920, MIMAT00 04761, MIMAT0016919, MIMAT0000422, MIMAT0005871, MIMAT0002819, MIMAT0004568, MIMAT0019225, MIMAT0020600, MIMAT0004913, MIMAT0005929, MIMAT0000687, MIMAT0004609, MIMAT0004594, MIMAT0004975, MIMAT0018101, MIMAT0 004925, MIMAT0004983, MIMAT0019721, MIMAT0003219, MIMAT0004694, MIMAT0015061, MIMAT0015028, MIMAT0022286, MIMAT0004926, MIMAT0015001, MIMAT0019738, MIMAT0016920, MIMAT0004595, MIMAT0005451, and any combination thereof.

[0058] In an embodiment of the first aspect of the invention, the miRNA is hsa-miR-323a-5p. In another embodiment of the first aspect of the invention, the miRNA is SEQ ID NO: 1.

[0059] In an embodiment of the first aspect of the present invention, the siRNA is selected from the list consisting of siCCND1, siCHAF1A, silNCENP, siKIF11, siCDC25A, siFADD and siBCL-XL.

[0060] In an embodiment of the first aspect of the present invention, the above-mentioned siRNA is an siRNA that silences the expression of the following genes according to their identification numbers in the public database GenBank (as of April 30, 2019): gene ID 3832 (KIF11, kinesin family member 11), gene ID 3619 (INCENP, inner centromere protein), gene ID 10036 (CHAF1A, chromatin assembly factor 1 subunit A), gene ID 993 (CDC25A, cell division cycle 25A), gene ID 8772 (FADD, Fas associated via death domain), gene ID 595 (CCND1, cyclin D1), and gene ID 598 (BCL-XL, BCL2 like 1 isoform).

[0061] In an embodiment of the first aspect of the present invention, the siRNA as mentioned above is selected from the list consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and any combination thereof.

[0062] In an embodiment of the first aspect of the present invention, the siRNA is SEQ ID NO:2 and / or SEQ ID NO:3, alternatively SEQ ID NO:4 and / or SEQ ID NO:5, alternatively SEQ ID NO:6 and / or SEQ ID NO:7, alternatively SEQ ID NO:8 and / or SEQ ID NO:9, alternatively SEQ ID NO:10 and / or SEQ ID NO:11, alternatively SEQ ID NO:12 and / or SEQ ID NO:13, alternatively SEQ ID NO:14 and / or SEQ ID NO:15.

[0063] In an embodiment of the first aspect of the invention, the siRNA is siCCND1. In another embodiment of the first aspect of the invention, the siRNA is siCCND1 of sequence SEQ ID NO: 12.

[0064] In an embodiment of the first aspect of the present invention, the miRNA to QS mass ratio is 1×10 -2 ~300×10 -2 and in another embodiment, 1×10 -2 ~100×10 -2 and in another embodiment, 1×10 -2 ~90×10 -2 and in another embodiment, 2×10 -2 , 3×10 -2 , 4×10 -2 , 5×10 -2 , 6×10 -2 , 7×10 -2 , 8×10 -2 , 9×10 -2 , 10×10 -2 , 20×10 -2 , 30×10 -2 , 40×10 -2 , 50×10 -2 , 60×10 -2 , 70×10 -2 , 80×10 -2 , 81×10 -2 , 82×10 -2 , 83×10 -2 , 84×10 -2 , 85×10 -2 , 86×10 -2 or 87 x 10 -2 is.

[0065] In an embodiment of the first aspect of the present invention, the nanovesicles are further conjugated to a member selected from the group consisting of a fluorophore, a radiopharmaceutical, a peptide, a polymer, an inorganic molecule, a lipid, a monosaccharide, an oligosaccharide, an enzyme, an antibody or antibody fragment, an antigen, and any combination thereof.

[0066] In an embodiment of the first aspect of the present invention, the fluorophore is a carbocyanine fluorophore.

[0067] In another embodiment of the first aspect of the present invention, the carbocyanine fluorophore is 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate.

[0068] In another embodiment of the first aspect of the present invention, the radiopharmaceutical comprises: 131 1-labeled metaiodobenzylguanidine (MIBG).

[0069] In another embodiment of the first aspect of the invention, the nanovesicles are conjugated to a hydrophilic polymer (a "stealth" polymer) that prevents the opsonization process. In another embodiment of the first aspect of the invention, the polymer is polyethylene glycol (PEGn).

[0070] In an embodiment of the first aspect of the present invention, the nanovesicles are conjugated to a tumor-targeting peptide, such as WHWRLPS (SEQ ID NO: 16) peptide, an NGR-containing peptide, an RGD peptide, an aminopeptidase A (glutamyl aminopeptidase, APA)-binding peptide, or a peptide capable of recognizing cancer cells, such as neuroblastoma cells, and / or tumor-associated endothelial cells.

[0071] In an embodiment of the first aspect of the present invention, the NGR-containing peptide is peptide SEQ ID NO: 17 (NGRGGVRSSRTPSDKYC), SEQ ID NO: 18 (CNGRCGVRSSRTPSDKY) or SEQ ID NO: 19 (GNGRGGVRSSRTPSDKY).

[0072] RGD peptides (containing an Arg-Gly-Asp motif) are peptides generally described in the art as peptides capable of interacting with integrins present in cell membranes, and are of particular interest in cell adhesion studies, both between cells and between cells and different tissues or basement membranes.

[0073] Aminopeptidase A (glutamyl aminopeptidase, APA) is a transmembrane cell surface protein that is overexpressed in angiogenic blood vessels and perivascular cells of human tumors. In an embodiment of the first aspect of the invention, the APA-binding peptide is a peptide comprising the sequence CPRECES (SEQ ID NO: 20). In another embodiment of the first aspect of the invention, the APA-binding peptide is the peptide CPRECESARSSRTPSDKY (SEQ ID NO: 21).

[0074] In an embodiment of the first aspect of the present invention, the tumor-targeting peptide is an HSYWLRS-containing peptide (SEQ ID NO: 22), such as, for example, YSHSHSYWLRSGGG (SEQ ID NO: 23), RALKYSHSHSYWLRSGGG (SEQ ID NO: 24) or YSHSHSYWLRSGGGC (SEQ ID NO: 35).

[0075] In an embodiment of the first aspect of the present invention, the tumor targeting peptide is conjugated to PEG.

[0076] A second aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of nanovesicles according to the first aspect of the present invention and a pharmaceutically acceptable excipient or vehicle.

[0077] As used herein, the phrase "therapeutically effective amount" refers to an amount of a compound (i.e., nanovesicles of the present invention) sufficient, when administered, to prevent the onset of, or alleviate to some extent, one or more symptoms of, a disease of interest. Of course, the specific dose of a compound administered in accordance with the present invention will be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and similar considerations.

[0078] The phrase "pharmaceutically acceptable excipient or carrier or vehicle" refers to a pharmaceutically acceptable material, composition, or vehicle. Each component must be pharmaceutically acceptable in the sense of being compatible with the other components of the pharmaceutical composition. Each component must also be suitable for use in contact with the tissues or organs of humans or animals without undue toxicity, irritation, allergic response, immunogenicity, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0079] A third aspect of the present invention relates to nanovesicles of the first aspect of the invention or pharmaceutical compositions of the second aspect of the invention as a delivery system. This aspect may be modified to the use of nanovesicles of the first aspect of the invention or pharmaceutical compositions of the second aspect of the invention as a delivery system.

[0080] In an embodiment of the third aspect of the present invention the delivery system is a drug delivery system.

[0081] In an embodiment of the third aspect of the invention, the delivery system is a drug delivery system for gene therapy and / or epigenetic therapy, or for miRNA transfection or siRNA transfection.

[0082] In an embodiment of the third aspect of the invention, the delivery system is a nucleic acid transfecting agent.

[0083] A fourth aspect of the present invention relates to a nanovesicle according to the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention for use as a medicament.

[0084] The fourth aspect of the invention may be modified as the use of the nanovesicles of the first aspect of the invention or the pharmaceutical composition of the second aspect of the invention for the manufacture of a medicament.

[0085] A fifth aspect of the present invention relates to a nanovesicle according to the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention for use in the treatment of cancer.

[0086] In an embodiment of the fifth aspect of the invention the cancer is neuroblastoma.

[0087] The fifth aspect of the invention may be modified as the use of nanovesicles of the first aspect of the invention or pharmaceutical compositions of the second aspect of the invention for the manufacture of a medicament for the treatment of cancer diseases, such as neuroblastoma, or as a method for the treatment or prevention of cancer diseases, such as neuroblastoma, comprising administering a therapeutically effective amount of nanovesicles of the first aspect of the invention together with a pharmaceutically acceptable carrier or excipient to a subject, including a human, in need thereof.

[0088] The drug may be provided in a form suitable for parenteral, dermal, oral, epidural, sublingual, nasal, intrathecal, bronchial, lymphatic, rectal, transdermal, or inhalation administration. A form suitable for parenteral administration refers to a physical state that allows its injectable administration, i.e., preferably in a liquid state. Parenteral administration can be performed by intramuscular, intraarterial, intravenous, intradermal, subcutaneous, or intraosseous administration, but is not limited to these types of parenteral administration routes. Forms suitable for oral administration are selected from the list including, but not limited to, drops, syrups, decoctions, elixirs, suspensions, ready-to-use suspensions, drinkable vials, tablets, capsules, granules, stamps, pills, tablets, lozenges, electuaries, or lyophilized preparations. Forms suitable for rectal administration are selected from the list including, but not limited to, suppositories, rectal capsules, rectal dispersions, or rectal ointments. Forms suitable for transdermal administration are selected from the list including, but not limited to, transdermal patches or iontophoresis.

[0089] In an embodiment of the fourth and fifth aspects of the invention, the medicament is provided in a form adapted for intravenous administration. In another embodiment of the fourth and fifth aspects of the invention, the medicament is provided in a form adapted for oral administration.

[0090] In an embodiment of the fourth or fifth aspect of the invention the medicament is administered twice a week.

[0091] In a fourth alternative embodiment of the fifth aspect of the invention, the agent is administered at least every 6, 8, 12, 24, 48 hours. In a fourth alternative embodiment of the fifth aspect of the invention, the agent is administered at least once a week or twice a week.

[0092] In an embodiment of the fourth and fifth aspects of the invention, the medicament comprises a therapeutic amount of nanoparticles of the first aspect of the invention for administration to mice, e.g., 10-30 μM miRNA, in another example 15-20 μM miRNA, and in yet another example 17.7 μM miRNA (equivalent to 0.25 mg / mL and 2 mg / kg in mice). In another embodiment of the fourth and fifth aspects of the invention, the medicament comprises a therapeutic amount of nanoparticles of the first aspect of the invention for administration to mice, e.g., 0.2-3 mg / kg miRNA, in another example 0.3-2 mg / kg miRNA, and in yet another example 0.27 μM miRNA.

[0093] Advantageously, the nanovesicles of the first aspect of the invention can be easily functionalized with fluorescent dyes (e.g., as described in Ardizzone et al., SMALL, 2018, 14) for observation by, for example, super-resolution microscopy. These fluorescent nanovesicles, when bound to fluorescent microRNAs, exhibit a fluorescence resonance energy transfer (FRET) signal that can be used to track the cellular internalization and intracellular distribution of QS-miRNAs. The nanovesicles of the first aspect of the invention can be used as a bioimaging tool to track the internalization and delivery of nucleic acids.

[0094] The nanovesicles of the present invention may be labeled, for example, with dyes, functionalized with targeting ligands for site-specific labeling, and ultimately deliver therapeutic agents (e.g., miRNA and / or siRNA).

[0095] Thus, a sixth aspect of the present invention relates to the use of nanovesicles according to the first aspect of the present invention as a bioimaging tool.

[0096] In an embodiment of the sixth aspect of the invention, the nanovesicles of the first aspect of the invention are used as bioimaging tools to follow the internalization and delivery of nucleic acids (eg miRNA or siRNA).

[0097] "Bioimaging tools" should be understood in accordance with this description as reagents used in imaging techniques used in the field of biology to track cells or certain compartments of specific tissues. Examples of bioimaging tools include chemiluminescent compounds, fluorescent and phosphorescent compounds, X-ray or alpha, beta, or gamma ray emitting compounds, etc.

[0098] The nanovesicles of the first aspect of the present invention can be formed, for example, by self-assembly of DC-Chol and a non-lipid cationic surfactant (ie, MKC).

[0099] The nanovesicles of the first aspect of the present invention can be formed by a variety of techniques, including ultrasonication (US), thin film hydration (THF), and a one-step scalable method using CO2-expanded solvents, called decompression suspension of expanded organic solutions (DELOS-susp) (International Application No. WO2017147407; Cano-Sarabia M et al. Langmuir 2008, 24, 2433-2437; Elizondo E et al. Nanomed. 2012, 7, 1391-1408).

[0100] A seventh aspect of the present invention relates to a process for producing nanovesicles of the first aspect of the present invention using the DELOS-SUSP method.

[0101] In an embodiment of the seventh aspect of the present invention, the DELOS-SUSP method comprises: a) preparing an aqueous solution of a non-lipid cationic surfactant (i.e., MKC); b) dissolving DC-Chol in an organic solvent and expanding the solution using a compressed fluid (CF); c) synthesizing nanovesicles by decompressing the solution obtained in step b) in the solution obtained in step a); Includes.

[0102] In another embodiment of the seventh aspect of the present invention, the DELOS-SUSP method comprises: a) providing an aqueous solution; b) dissolving DC-Chol and a non-lipid cationic surfactant (i.e., MKC) in an organic solvent and expanding the solution using a compressed fluid (CF); c) synthesizing nanovesicles by decompressing the solution obtained in step b) in the aqueous solution of step a); Includes.

[0103] In an embodiment of the seventh aspect of the present invention, the method comprises the steps of: In step b), dissolving DC-Chol and a water-insoluble organic dye in an organic solvent and expanding the solution using a compressed fluid (CF); In step c), synthesizing fluorescent nanovesicles by decompressing the solution obtained in step b); Further includes:

[0104] Another aspect of the present invention is also nanovesicles obtainable by the method of the seventh aspect of the present invention.

[0105] An eighth aspect of the present invention relates to a kit comprising the nanovesicles of the first aspect of the invention or the pharmaceutical composition of the second aspect of the invention. The kit may also include instructions for delivery of the nucleic acid contained in the nanovesicles of the first aspect of the invention.

[0106] The kit may further comprise additional means for visualizing the nanovesicles.

[0107] Also part of the invention is the use of a kit according to the eighth aspect of the invention for an application described in any of the other aspects of the invention above or below.

[0108] Also part of the invention is a kit comprising a device for the release of nanovesicles from the first aspect of the invention or a pharmaceutical composition from the second aspect of the invention, and also comprising nanovesicles from the first aspect of the invention or a pharmaceutical composition from the second aspect of the invention.

[0109] Also part of the invention is a device for the release of nanovesicles from the first aspect of the invention or from a pharmaceutical composition of the second aspect of the invention comprising nanovesicles.

[0110] The nanovesicles of the first aspect of the invention can simultaneously diagnose, image, and treat a target diseased area with precise spatiotemporal control of dosage while monitoring the therapeutic efficacy of the treatment. Thus, a ninth aspect of the invention relates to the use of nanovesicles of the first aspect of the invention as a theranostic tool.

[0111] According to the present invention, the nanovesicles of the first aspect of the invention exhibit pH buffering capacity (see Figure 5). Thus, a tenth aspect of the invention relates to nanovesicles of the first aspect of the invention or pharmaceutical compositions of the second aspect of the invention as pH buffering agents.

[0112] Another aspect of the present invention is the use of nanovesicles according to the first aspect of the invention or the pharmaceutical composition according to the second aspect of the invention as an antibacterial or antifungal agent.

[0113] The antibacterial effect of the nanovesicles of the first aspect of the invention or the pharmaceutical composition of the second aspect of the invention may be exerted via perturbation of the bacterial plasma membrane, for example, leading to bacterial cell lysis. The antibacterial effect may be measured by methods known to those skilled in the art, such as biofilm models, the Alamar Blue assay for measuring bacterial viability, crystal violet staining, etc., and may be demonstrated in Gram-positive or Gram-negative bacteria, for example known model pathogens such as Staphylococcus aureus, Bacillus subtilis, Escherichia coli, etc.

[0114] The antibacterial activity can be measured by methods known to those skilled in the art, for example against Aspergillus niger or Candida albicans.

[0115] Throughout the description and claims, the word "comprises" and variations of that word are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprises" encompasses the case of "consisting of." Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the present invention. The following examples and drawings are provided by way of illustration and are not intended to limit the invention. Reference signs placed in connection with the drawings and within parentheses in the claims are intended only to aid in understanding the claims and should not be construed as limiting the scope of the claims. Furthermore, the present invention encompasses all possible combinations of specific preferred embodiments described herein. [Example]

[0116] 1. Quatosome Synthesis

[0117] 1.1 Synthesis and physicochemical properties of quatosomes

[0118] Materials and Methods Cholesten-3β-ol (Choi, purity 95%; #A0807; CAS number: 57-88-5) and sodium hydroxide (NaOH, purity ≥ 98.0%) were obtained from PanReac (Castellar del Valles, Spain). Cholesteryl N-(2-dimethylaminoethyl)carbamate (DC-Chol, purity ≥ 98%; #92243) and cholesteryl hemisuccinate (Chems, purity ≥ 98%; #C6512; CAS number: 1510-21-0) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0119] Benzyldimethyltetradecylammonium chloride (MKC; purity ≥99%; #262393) was obtained from AttendBio Research SL (Santa Coloma de Gramenet, Spain). Cetyltrimethylammonium bromide (CTAB, molecular biology grade ultra) was purchased from Fluka-Aldrich. 1,1'-Dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (Dil) was obtained from Thermofisher. Ethanol was purchased from Teknochroma (Sant Cugat del Valles, Spain). Polyethylene glycol derivative of cholesterol (mPEG-CLS; mPEG chain: 1000; #MF001095-1K) was purchased from BioChemPEG (Watertown, MA 02472, USA). Carbon dioxide (purity 99.9%) was purchased from Carburos Metalicos SA (Cornella de Llobregat, Spain). All chemicals were used without further purification, and all solutions were prepared using pretreated Milli-Q water (Millipore Iberica, Madrid, Spain).

[0120] Lipofectamine2000 (#11668019) was purchased from ThermoFisher Scientific (Waltham, MA, USA).

[0121] Human synthetic miRNA mimics, Dy547-labeled-miR-control-1 (#CP-004500-01; Table 1 ) and hsa-miR-323a-5p (#CP-301085-01; Table 1 ), were obtained from the current Dharmacon Inc (Lafayette, CO, USA).

[0122] Control siRNA (5'GUAAGACACGACUUAUCGC3') (SEQ ID NO: 25) and siCCND1 (5'CCUACGAUACGCUACUAUAUU3') (SEQ ID NO: 12) were purchased from Sigma (Table 2).

[0123] [Table 1]

[0124] [Table 2]

[0125] siRNA was received lyophilized from the supplier and resuspended in water for later use at the desired concentration.

[0126] QS synthesis Quatosomes (QS) were constructed with sterols such as Choi, DC-Chol, or Chems and highly positively charged nonlipid cationic surfactants such as MKC and CTAB. Various QSs were prepared by adjusting the ratio of Chol to the modified sterol (DC-Chol or Chems).

[0127] QS0: (0% Chol / 100% ChemChem):MKC; QS1: (100% Chol / 0% DC-Chol):MKC; QS2: (91% Chol / 9% DC-Chol):MKC; QS3: (53% Chol / 47% DC-Chol):MKC; QS4: (0% Chol / 100% DC-Chol):MKC; QS5: (90.5% Chol / 9.5% DC-Chol):CTAB; QS6: (51% Chol / 49% DC-Chol):CTAB; QS7: (0% Chol / 100% DC-Chol):CTAB. All QSs were prepared with various sterols and surfactants (MKC or CTAB) at a molar ratio of 1:1, except for QS1, which was prepared at a molar ratio of 1:3. Furthermore, we prepared QS4 membranes functionalized with Dil by inserting Dil into them. We also functionalized PEG-QS4 by replacing some DC-Chol molecules with PEG, achieving a final composition of QS4:(10% Chol-PEG / 90% DC-Chol):MKC at a molar ratio of 1:1.

[0128] QS was prepared using a method using CF (Ferrer-Tasies et al. Langmuir. 2013 Jun 4;29(22):6519-28). Briefly, sterols such as Choi, Chems, or DC-Chol or their derivatives (see Table 3) were dissolved in EtOH (V) at 313-318 K for 10 min. EtOH The organic phase was then added to the vessel at operating temperature (Tw=311 K) and atmospheric pressure. CO2 was then added to the vessel at high pressure (Pw=11.5 MPa), 311 K, and a predetermined CO2 mole fraction (X CO2 A volume-swollen solution of lipids was obtained with CO2-swelling solution (V = 0.6). After 1 hour of homogenization, this CO2-swollen solution was added to the non-lipid cationic surfactants (i.e., MKC or CTAB) listed in Table 3 (V H20 =8.3V EtOH ), the working pressure (Pw) was reduced to atmospheric pressure to obtain uniform unilamellar nanovesicles in the form of a QS system. The method may be carried out in continuous or batch mode.

[0129] To prepare quatosomes functionalized with fluorescent dyes, such as water-insoluble organic dyes, this method further involves the addition of sterol and EtOH (V EtOH The method involves adding the Dil fluorophore (70 μM) to the organic phase formed by the addition of Dil and then swelling the lipid solution with Dil by adding CO2 at high pressure (Pw = 11.5 MPa), 311 K, and a predetermined CO2 mole fraction (XCO2 = 0.6). After 1 hour of homogenization, this CO2-swollen solution is swollen with a non-lipid cationic surfactant (i.e., MKC or CTAB) from Table 3 (V H20 =8.3V EtOH ), the pressure was reduced from the working pressure (Pw) to atmospheric pressure, and uniform fluorescent unilamellar nanovesicles were obtained.

[0130] [Table 3]

[0131] After 1 week of stabilization, all samples were purified by diafiltration using a KrosFlo® Research III TFF system (Spectrum Labs, Repligen Corporation, Waltham, MA, USA) using a MicroKros filter column with size exclusion mPE (molecular weight cutoff 100 KDa, surface area 20 cm). 2 The sample was diafiltered using MilliQ water to remove ethanol and excess material not encapsulated in QS, leaving the QS in a MilliQ aqueous medium.

[0132] Nanovesicles containing 100% DC-Chol and MKC as sterols at ratios of 1:2 and 2:1 were also prepared (data not shown).

[0133] Preparation of miRNA-QS complexes: a) Depending on the miRNA / QS loading required, the corresponding volume in μL of QS (see Table 7 for in vitro experiments and Table 11 for in vivo experiments) was added to a new Eppendorf. b) The corresponding volume in μL of miRNA (depending on the required final concentration of miRNA; i.e., 2.5 μL (stock concentration 20 μM) to achieve a final miRNA concentration of 2.5 μM in in vitro experiments, and 42.6 μL (stock concentration 100 μM) of miRNA to achieve a final miRNA concentration of 21.3 μM in in vivo experiments) (see Tables 7 and 11) was added to the QS solution. c) The complexes were diluted with 1x PBS to ensure mixing, avoid aggregation, and maintain constant miRNA concentration among the various QS-miRNA complexes. d) After pipetting up and down twice (less than 5 min incubation), complexes were formed. e) If necessary, the formed complexes were added to cells.

[0134] Physicochemical properties

[0135] Dynamic Light Scattering The particle size, polydispersity, and surface charge density of the QS were evaluated using dynamic light scattering (DLS) techniques with a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). Hydrodynamic diameter and polydispersity index (PDI) from three replicate measurements were obtained using 4 mW incident He-Ne laser light with homodyne detection, a wavelength of 633 nm, and a fixed detector angle of 173°. Samples were measured at 298 K immediately after production without modification or dilution. In addition, Z potential was measured at 298 K in a DTS1070 disposable collapsible capillary cuvette by another noninvasive backscattering technique using the Zetasizer Nano ZS. Reported values ​​were the mean ± standard deviation (SD) of hydrodynamic diameter or Z potential ± standard deviation (SD) across samples. Experiments were performed at least three times.

[0136] The stability of QS over time was measured by DLS at 1 week, 2 weeks, 1 month, 3 months, 6 months, and 1 year after sample preparation or purification. QS was considered stable over time if its hydrodynamic diameter remained below 300 nm and its PDI remained within the range of 0.1–0.3 for up to 2 months.

[0137] Cryo-TEM Cryo-TEM images were acquired at 200 KV using a JEOL JEM 2011 transmission electron microscope (JEOL, Tokyo, Japan). Samples were placed on holey carbon grids or copper grids coated with a holey polymer film and then frozen in liquid ethane. A Gatan 626 cryotransfer system was inserted into the microscope. Images were recorded using a Gatan Ultrascan US1000 CCD camera and analyzed with the Digital Micrograph 1.8 program.

[0138] pH buffering capacity The buffering capacity of QS was measured by acid-base titration. Briefly, QS was at a final concentration of 5 mg / mL in aqueous solution. The resulting solution was adjusted to pH 9 with sodium hydroxide (0.01 M). A titration curve was performed by stepwise addition of 10 μL aliquots of hydrochloric acid (0.01 M). After addition, the pH was measured using a pH meter (Hanna Instruments, Woonsocket, RI, USA) until a pH of 2 was reached.

[0139] result: QS were prepared with different sterol and surfactant compositions (QS0: (0% Chol / 100% Chems):MKC; QS1: (100% Chol / 0% DC-Chol):MKC; QS2: (91% Chol / 9% DC-Chol):MKC; QS3: (53% Chol / 47% DC-Chol):MKC; QS4: (0% Chol / 100% DC-Chol):MKC, QS5: (90.5% Chol / 9.5% DC-Chol):CTAB; QS6: (51% Chol / 49% DC-Chol):CTAB; QS7: (0% Chol / 100% DC-Chol):MKC). l):CTAB. DLS measurements (see Table 4) and cryo-TEM images (Figure 4) revealed that all the obtained quatosomes (QS) were uniformly sized, spherical, and unilamellar nanovesicles. DLS measurements (see Figure 3) revealed a unimodal size distribution with an average size of approximately 100 nm. In addition, QS exhibited low polydispersity and high colloidal stability over time, as only slight variations in size distribution were observed for all QS (Figures 1 and 2). The positive charge from QS ensured high complexation efficiency with the negative charge of small RNA (sRNA) through electrostatic interactions.

[0140] [Table 4]

[0141] Furthermore, QS containing a high concentration of DC-Chol in the composition exhibited pH-sensitive behavior, maintaining a constant pH under acidic conditions. QS4 ((0% Chol / 100% DC-Chol):MKC) had the highest buffering capacity (Figure 5).

[0142] 2. Colloidal Structure

[0143] 2.1-Colloidal structures containing cholesterol, Chol-VS, and CTAB in aqueous media

[0144] Materials and Methods: Cholesten-3β-ol (Choi, 95% purity; #A0807) was obtained from PanReac (Castellar del Valles, Spain).

[0145] Cetyltrimethylammonium bromide (CTAB, molecular biology grade ultra) was purchased from Fluka-Aldrich. Cholest-5-ene, 3-[2-(ethenylsulfonyl)ethoxy]-, (3b)-(Chol-VS), was synthesized and characterized. Ethanol was purchased from Teknochroma (Sant Cugat del Valles, Spain). Carbon dioxide (99.9% purity) was purchased from Carburos Metalicos SA (Cornella de Llobregat, Spain). All chemicals were used without further purification, and all solutions were prepared using pretreated Milli-Q water (Millipore Iberica, Madrid, Spain).

[0146] Synthesis of cholest-5-ene, 3-[2-(ethenylsulfonyl)ethoxy]-, (3β)-(Chol-VS)

[0147] To a solution of cholesterol (300 mg, 0.77 mmol) in THF (20 mL) was added divinyl sulfone (0.12 mL, 1.16 mmol) and potassium tert-butoxide (9 mg, 0.077 mmol). The reaction mixture was magnetically stirred at room temperature for 1 hour. An Amberlita IR 120H was then added, and magnetic stirring was continued for another 30 minutes. After filtration, the solvent was evaporated under reduced pressure. TLC of the crude product indicated the presence of cholesterol. To the resulting crude product, acetic anhydride (8 mL) and pyridine (4 mL) were added, and the new reaction mixture was maintained at room temperature for 16 hours. Acetylation of the crude product allowed for the isolation of compound Chol-VS. Evaporation under reduced pressure afforded the crude product, which was purified by column chromatography (ethene-hexane 1:2) to give compound Chol-VS as a solid (204 mg, 52%).

[0148] MP 133-135°C; [α]D -19(c1, chloroform);v max (KBr) / cm -1 :3409, 1461, 1373, 1319, 1115, and 1052; 1 H-NMR (CDCI3, 400MHz): δ6.75 (dd, 1H, J=16.7 and 9.9Hz), 6.40 (d, 1H, J=16.7Hz), 6.07 (d, 1H, J=9.9Hz), 5.35 (brs, 1H), 3.88 (t, 2H, J=5.6Hz), 3.2 3 (t, 2H, J=5.6Hz), 3.19 (m, 1H), 2.35~1.84 (several meters, 7H), 1.56~0.95 (several meters, 21H) , 0.99(s, 3H), 0.92(d, 3H, J=6.4Hz), 0.86(d, 6H, J=6.6Hz), 0.67(s, 3H); 13 C-NMR (CDCI3, 125MHz): δ140.2, 138.0, 128.5, 122.1, 79.8, 61.5, 56.7, 56.1, 55.4, 50.1, 42.3, 39.7, 39.5, 38.8,37.0,36.8,36.2,35.8,31.9,31.8,28.2,28.1,28.0,24.3,23.8,22.9,22.5,21.0,19.3,18.7,11.8;C 31 H 52 O3SNa[M+Na] + HRMS (m / z) (FAB+) calculated: 527.3535; found: 527.3535.

[0149] Synthesis of colloidal structures Colloidal structures (CS) were constructed from sterols such as Chol and Chol-VS and highly positively charged quaternary ammonium surfactants such as CTAB at a molar ratio of 1:1 between sterols (Choi + Chol-VS) and CTAB surfactant.

[0150] Colloidal structures were prepared using a previously described method (Ferrer-Tasies et al. Langmuir. 2013 Jun 4;29(22):6519-28). Briefly, sterols or their derivatives, such as Chol or Chol-VS (see Table 5), were dissolved in EtOH at 308 K. The organic phase was then added to a vessel at the operating temperature (Tw = 308 K) and atmospheric pressure. CO2 was then added to obtain a volume-swollen solution of lipids at high pressure (Pw = 10 MPa), 311 K, and a predetermined CO2 mole fraction (XCO2 = 0.8). After 1 h of homogenization, the CO2-swollen solution was depressurized from the operating pressure (Pw) to atmospheric pressure in a continuous water flow containing the nonlipid cationic surfactant CTAB (see Table 5). Various colloidal structures were obtained depending on the ratio of Chol to Chol-VS.

[0151] [Table 5]

[0152] result: CS-VS were prepared with different sterol and surfactant compositions (CS-VS0: (0% Chol-VS / 100% Chol):CTAB; CS-VS1: (32% Chol-VS / 68% Chol):CTAB; CS-VS2: (49% Chol-VS / 51% Chol):CTAB; CS-VS3: (66% Chol-VS / 34% Chol):CTAB; CS-VS4: (74% Chol-VS / 26% Chol):CTAB; CS-VS5 (100% Chol-VS / 0% Chol):CTAB). DLS measurements (see Table 6) and cryo-TEM images (see Figure 6) revealed that the gradual replacement of cholesterol molecules in the equimolar mixture Chol:CTAB with a novel cholesterol molecule bearing vinyl sulfone (Chol-VS) led to different colloidal self-assembly behaviors.

[0153] [Table 6]

[0154] On the other hand, DLS measurements of the QS-VS systems from QS-VS2 to QS-VS4 did not meet the quality criteria due to the presence of large structures. Furthermore, all cryo-TEM images showed the coexistence of mainly thin, micrometer-long ribbons (nanoribbons) with a few monolayer spheres. On the other hand, in the cryo-TEM images of the complete replacement of Chol with the Chol-VS and CS-VS5 systems, no vesicular assemblies were formed, and only nanoribbon assemblies were found.

[0155] 2.2-Colloidal structures containing cholesterol and MKC

[0156] This system consisted of 100% Chol / 0% DC-Chol:MKC and was prepared in milliQ purified water containing 10% EtOH at a sterol to surfactant molar ratio of 1:1.

[0157] CS-CH was prepared using a method using CF (Ferrer-Tasies et al. Langmuir. 2013 Jun 4;29(22):6519-28). Briefly, sterol (Chol) was dissolved in EtOH (V EtOH ) at 313-318 K for 10 min. The organic phase was then added to the vessel at operating temperature (Tw = 311 K) and atmospheric pressure. CO2 was then added to the vessel at high pressure (Pw = 11.5 MPa), 311 K, and a predetermined CO2 mole fraction (X CO2 = 0.6) to obtain a volume-swollen solution of lipids. After 1 hour of homogenization, this CO2-swollen solution was diluted with a non-lipid cationic surfactant (i.e., MKC) (V H20 =8.3V EtOH The colloidal structure was obtained by depressurizing the aqueous solution containing HCl from the working pressure (Pw) to atmospheric pressure. This method was carried out in continuous or batch mode. Composition used for preparing the CS-CH system by the DELOS-SUSP method: The composition used for preparing the CS-CH system had Choi (0.070 M) in EtOH as the organic phase and MKC (0.008 M) in water as the aqueous phase. The membrane component concentrations were 5.6 mg / mL, and the %D-Chol / (Chol + D-Chol) was 0%.

[0158] result: The CS-CH system did not form nanovesicles. DLS measurements and cryo-TEM images (see Figure 6C) revealed that the self-assembly of cholesterol molecules with MKC in an equimolar mixture resulted in a different colloidal self-assembly behavior, preferentially forming nanoribbons. DLS measurements of the CS-CH system did not meet the quality criteria due to the presence of large structures: hydrodynamic diameter (D) (nm), D = 174.3 ± 33.2; polydispersity index (Pdl) = 0.56 ± 0.09. Moreover, all cryo-TEM images showed the coexistence of primarily thin, micrometer-long ribbons (nanoribbons) and a few monolayer spheres.

[0159] Example 3: QS-sRNA complex formation

[0160] Materials and Methods: QS-sRNA complexes were formed by mixing QS and small RNA (sRNA) at various sRNA-to-QS mass ratios (w / w), referred to as QS-sRNA loadings. For in vitro experiments, QS was first diluted with Depc-treated water (ThermoFisher; #750024) to achieve the desired concentrations, such as 3.98 mg / mL for QS0, 1.15 mg / mL for QS1, 1.76 mg / mL for QS2, 1.88 mg / mL for QS3, and 1.99 mg / mL for QS4. To form QS-sRNA complexes, 2.5 µL of sRNA was added to the appropriate volume (µL) of QS solution to achieve the desired sRNA-to-QS mass ratio (w / w) and maintain a constant sRNA concentration (see Table 7). To achieve a constant final concentration of sRNA (2.5 µM), the QS-sRNA complex was diluted with 1x PBS to reach the desired final volume (20 µL) and then mixed by pipetting up and down twice (less than 5 min of incubation). The resulting QS-sRNA complex was generated by ionic interactions between the positive charges on the surface of QS and the negative charges on the sRNA. Depending on the QS composition, various sRNA-to-QS mass ratios (w / w) were calculated.

[0161] [Table 7] JPEG0007774254000008.jpg174170 JPEG0007774254000009.jpg90170

[0162] Gel electrophoresis: Agarose electrophoresis gels were prepared with 2.5% agarose and 0.005% ethidium bromide in Tris / Acetate / EDTA (1x TAE). After 5 minutes of incubation, QS-sRNA complexes were prepared and added to each well of the gel in PBS loading buffer (2.5% glycerol). To separate miRNA from QS, 0.25% SDS was added to specific wells. Gel electrophoresis was performed at 120 V for 1 hour. Electrophoresis images were captured using a Gel Doc XR+ System (Biorad, Hercules, CA, USA).

[0163] result: The QS-miRNA complexes exhibited various morphologies depending on the QS composition and the mass ratio of QS to miRNA. 0-2 is a QS that indicates a bundle structure. 3-4 On the other hand, a high miRNA-to-QS mass ratio resulted in larger aggregates than a low miRNA-to-QS mass ratio (see Figure 7).

[0164] QS0 could not complex miRNA with 100% efficiency, even when the amount of miRNA added per QS was low (e.g., I and II). The complexation efficiency was directly proportional to the increase in the DC-Chol composition of the QS. Therefore, QS1 (0% DC-Chol) was significantly higher than QS1. 2-4 However, the conjugation efficiency was lower than that of QS 2-4 showed 100% complex formation already upon addition of QS-miRNA(VI), whereas QS1 showed complete complex formation upon addition of QS-miRNA(IV).

[0165] Example 4: Cell viability study

[0166] Materials and Methods: cell culture SK-N-BE(2) cells were obtained from the Public Health England Culture Collections (Salisbury, UK) and stored in liquid nitrogen. After resuscitation, SK-N-BE(2) cells were cultured in Iscove's modified Dulbecco's medium (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) South America Premium, 1% insulin-transferrin-selenium supplement (Life Technologies, Thermo Fisher Scientific), 100 U / mL penicillin, 100 μg / mL streptomycin (Life Technologies, Thermo Fisher Scientific), and 5 μg / mL plasmocin (InvivoGen, San Diego, CA, USA). All cultures were maintained at 37°C in a saturated atmosphere of 95% air and 5% CO2. SK-N-BE(2) cells were routinely tested for mycoplasma contamination.

[0167] For in vitro experiments, SK-N-BE(2) neuroblastoma cells were reverse transfected with QS-sRNA complexes in complete cell culture medium (IMDM, 10% heat-inactivated fetal bovine serum (FBS) South America Premium (Biowest, Nuaille, France)) without antibiotics. After overnight incubation, the medium was replaced with IMDM supplemented with 10% FBS and antibiotics.

[0168] Cell viability assay To test the cytotoxicity of QS or QS-miRNA complexes, SK-N-BE(2) cells were cultured in 96-well plates at 18 × 10 3Cells were seeded at 6 replicates / well and treated with QS (0.7 μg / mL–52 μg / mL; Table 8) or reverse transfected with 2.5 μM miR-control Dy547 (having the same sequence as el microRNA control 1 disclosed in Table 1; Dharmacon Inc, Lafayette, CO, USA) complexed with QS at various miRNA-to-QS mass ratios (QS-miRNA loading) (I–VIII) to achieve a final miRNA concentration of 50 nM.

[0169] [Table 8]

[0170] result: QS 1-4 showed high survival rates (80-90%) even at low miRNA-to-QS mass ratios (QS-miRNA addition amounts), such as QS-miRNA(III). 1-4 -miRNA is not complexed with QS due to the shielding of the positive charge from QS by the negative charge of miRNA. 1-4 The survival rate was higher than that of the control group (Fig. 9).

[0171] Example 5: Expression of miRNA and siRNA using QS and function of QS

[0172] Materials and Methods: For the efficacy assay of QS-sRNA complexes, SK-N-BE(2) cells were plated in a 96-well plate at 9 × 10 3Cells were seeded at 6 replicates per well and reverse transfected with a final sRNA concentration of 50 nM. QS-sRNA complexes were formed as described in Example 1. At 24 or 96 hours posttransfection, cells were fixed with 1% glutaraldehyde (Sigma-Aldrich) and stained with 0.5% crystal violet (Sigma-Aldrich). Crystals were dissolved in 15% acetic acid (Fisher Scientific, Hampton, NH, USA), and absorbance was measured at 590 nm using an Epoch microplate spectrophotometer (Biotek, Winooski, VT, USA). The effect of QS-sRNA complexes on cell viability was normalized to mock-transfected cells.

[0173] Quantitative real-time PCR (qPCR) Total RNA, including small RNAs, was extracted using the miRNeasy Mini Kit (Qiagen, Las Matas, Spain). mRNA was reverse transcribed (0.5 μg total RNA) using the Taqman RT Kit (#4366596; Applied Biosystems, Thermo Fisher Scientific), and mature miRNA expression analysis was quantified using the Taqman microRNA Assay (#4440047; Applied Biosystems, Thermo Fisher Scientific) according to the manufacturer's recommendations. cDNA was quantified by standard RT-qPCR techniques using 2X Power SYBR Green Master Mix (Applied Biosystems, Thermo Fisher Scientific) on an ABI700 SDS instrument. Gene expression was normalized to the L27 housekeeping gene for mRNA and the RNU-44 small RNA for miRNA analysis (#4427975). Primer sequences are listed in Tables 9 and 10, respectively. Relative fold-change gene expression was quantified using the Taqman microRNA Assay (#4440047; Applied Biosystems, Thermo Fisher Scientific) according to the manufacturer's recommendations. (-ΔΔCT)This was performed using the method (Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25:402-408).

[0174] [Table 9]

[0175] [Table 10]

[0176] RNU44 was commonly used as a housekeeping gene to normalize the small RNA content of the analyzed samples. qPCR for hsa-miR-323a-5p and RNU44 was performed according to the manufacturer's instructions. The TaqMan MicroRNA Assay used target-specific stem-loop primers for hsa-miR-323a-5p and RNU44 during cDNA synthesis to generate templates for real-time PCR.

[0177] Western blot Protein extracts were obtained in 1x RIPA buffer (ThermoFisher Scientific) supplemented with 1x EDTA-free complete protease inhibitor cocktail (Roche, Sant Cugat del Valles, Spain). Protein concentration was quantified using the Lowry assay (DC Protein Assay, Bio-Rad). 30 μg of protein was prepared in 1x RIPA buffer containing 1x loading buffer and 1x sample reducing agent and subjected to NuPAGE 4-12% Bis-Tris gel electrophoresis at 150 V for 1 h at RT. The gel was transferred to an iBlot Gel Transfer Stacks PVDF membrane (Life Technologies, ThermoFisher Scientific) at 110 V for 1 h 30 min at 4°C. The membranes were incubated with blocking solution (Tris-buffered saline with 5% bovine serum albumin in Tween-20 (TBS-T)) for 1 h at RT, followed by overnight incubation at 4 °C with the primary antibodies listed above: anti-CCND1 (1:1000 Cell Signaling; ab 134175), anti-CHAF1A (1:1000 Cell Signaling; #5480S), p27 (1:1000 Cell Signaling; #3686), and phosphorylated Rb (pRB) (1:1000 Cell Signaling; #8516). The membranes were then incubated for 1 h 30 min with peroxidase-conjugated secondary antibodies, including anti-rabbit IgG-peroxidase antibody raised in goat (1:10,000, Sigma-Aldrich; #A0545). Anti-actin HRP (1:40,000 Santa Cruz; sc-1616) was used as a loading control. The membrane was finally developed using an EZ-ECL chemiluminescence detection kit (Biological Industries, Kibbutz Beit-Haemek, Israel). Quantification of Western blots was performed using ImageJ3. The intensity of each analyzed protein band was normalized to the intensity of actin.

[0178] Confocal microscopy imaging SK-N-BE(2) cells were seeded onto 8-well Nunc Lab-Tek chamber slides (Thermofisher, USA) 48 hours before imaging. After incubating the cells with QS4-Dil-miRNA(Cy5) complexes for 30 minutes, the cell medium was replaced to remove any uninternalized complexes. The miRNA used was the same miRNA control 1 used previously, but functionalized with Cy5 instead of Dy547 at the 5' end of the sense strand of the microRNA. QS4-Dil( Dil QS4) was prepared as described above in Example 1. Confocal images were acquired using an LSM800 microscope (Zeiss, Germany) after 2 minutes, 30 minutes, or at the indicated times for overnight incubation. Bright-field images were acquired using a 488 nm laser. The Dil and Cy5 fluorophores were excited using 530 nm and 633 nm lasers, respectively, and their signals were collected from 550-620 nm and 640-750 nm, respectively. The Dil and Cy5 signals were collected in two separate channels and processed to eliminate crosstalk between them. Images of the complexes were processed to obtain the FRET ratio over time. For FRET ratio graphs, each system was expressed as the mean ± SEM of technical triplicates.

[0179] statistical analysis Unless otherwise stated, values ​​represent the mean ± SEM values ​​of the mean of three independent experiments. Statistical significance was determined by unpaired two-tailed Student's t-test (GraphPad Prism Software, USA). * means p<0.05; ** means p<0.01, *** means p<0.001.

[0180] result: miRNA: To increase the expression level of miR-323a in SK-N-BE(2) cells, it was necessary to transfect the miRNA with the QS system, because naked miRNA or miRNA complexed with MKC micelles failed to increase the expression level of the miRNA by qPCR (Figure 10).

[0181] miR-323a target modification by qPCR and Western blot (Figures 11-12): Only transfection of miR-323a-5p with QS4 reduced miR-323a target expression (CHAF1 A and CCND1) at the RNA level (Figure 11) and protein level (Figure 12). 1-2 allowed miRNA internalization but not miRNA release.

[0182] QS3 reduced miR-323a target expression (CHAF1A and CCND1) at the mRNA level. QS4 consistently modified miR-323a-5p targets at both the mRNA and protein levels, whereas QS3 reduced only CHAF1A and CCND1 mRNA upon addition of QS3-miRNA (VI). These results are explained by the difference in the amount and time required for miRNA release from QS3 compared with QS4. In the QS3-miRNA complex, miRNA was released at a slower pace than in the QS4-miRNA complex (see Figure 13).

[0183] Transfection of QS-miR-323a complexes increased the expression level of miR-323a-5p even when miRNA was present in high amounts ((V-VIII)) of QS4 (Figure 14). miR-323a-5p transfected with QS4 at moderate miRNA-to-QS mass ratios (such as QS4-miRNA(V) and (VI)) modified the expression of direct miR-323a targets, such as CCND1 and CHAF1A, at the mRNA and protein levels. However, high miRNA-to-QS mass ratios (such as QS4-miRNA(VIII)) did not modify miR-323a targets.

[0184] Furthermore, miR-323a-5p transfected with QS4 at various loadings (miRNA to QS mass ratios), such as QS4-miRNA (V) and (VI), modified indirect targets of miR-323a, such as phosphorylated Rb (pRb) and p27, at the mRNA (Figure 15) and protein levels (Figure 16A, CHAF1A; Figure 16B, CCND1; Figure 16C, pRb; Figure 16D, p27).

[0185] Overexpression of the QS4-miR-323a-5p complex reduced SK-N-BE(2) cell proliferation after 96 hours when the miRNA was transfected with QS4 at miRNA-to-QS mass ratios such as (V) and (VI) (Figure 17).

[0186] High levels of miR-323a-5p inhibited cell cycle progression by eliminating CCND1 expression and preventing pRB phosphorylation, and increased p27 levels helped inhibit the function of the CDK4 / 6 complex.

[0187] Furthermore, proliferation analysis of SK-N-BE(2) cells after transfection with the QS4-miR-323a-5p complex (QS4-miRNA(V)) compared to Lipofectamine2000® showed similar results (p<0.001 compared to control). ***A proliferation experiment was performed in NB cells 96 hours after transfection comparing miR-323a-5p bound to QS4 or liposomes (i.e., Lipofectamine 2000) with miR-control (50 nM) (Figure 18).

[0188] siRNA: siCCND1 transfected with QS4 at various QS4-siRNA dosages (siRNA to QS mass ratios), such as (V) and (VI), reduced CCND1 expression at the mRNA (Figure 19) and protein levels (Figure 20A). However, high QS4-siRNA dosages (siRNA to QS mass ratios), such as dosage (VIII), did not reduce CCND1 expression as well as other dosages of QS4-siRNA (siRNA to QS mass ratios), such as (V) and (VI).

[0189] Furthermore, siCCND1 transfected with QS4 at the loadings (siRNA to QS mass ratio) (V) and (VI) modified indirect targets of CCND1, such as pRb and p27, at the mRNA or protein level (see Figures 20B and 20C, respectively). CCND1 depletion not only had a general effect on cell proliferation, but also best reflected miR-323a-5p overexpression on the reduction of phosphorylated Rb (pRb) levels and p27 accumulation.

[0190] Overexpression of the QS4-siCCND1 complex reduced SK-N-BE(2) cell proliferation after 96 hours when QS4-siRNA was transfected at the added doses (siRNA to QS mass ratio) (V) and (VI) (Figure 21).

[0191] Furthermore, cell proliferation analysis of SK-N-BE(2) cells after transfection with QS4-siCCND1 complex (QS4-siRNA(V)) compared to Lipofectamine2000® showed similar results (p<0.001 compared to control). ***A proliferation experiment was performed comparing siCCND1 complexed with QS4 to siControl (50 nM) in NB cells 96 hours after transfection (Figure 22).

[0192] Functionalization research: As described in Example 1, QS4 was functionalized with Dil fluorophores, or 10% of the DC-Chol sterol in the nanovesicle membrane was replaced with Chol-PEG. 1000 It was replaced with polymer QS4-Dil( Dil QS4) and PEG-QS4-functionalized QS exhibited similar size (30–70 nm), spherical shape, colloidal stability, and a similar positive surface charge to QS4 (see Figure 23). QS4-miRNA and QS4-(Dil)-miRNA complexes exhibited similar morphologies at the same QS vs. miRNA loading.

[0193] QS4, with or without Dil / PEG functionalization, Dil Even at high miRNA-to-QS mass ratios (additional QS-miRNA), such as (VIII) for QS4 and (VI) for PEG-QS4, complete complexation of miRNA was achieved with 100% efficiency. Furthermore, decomplexation of QS-miRNA with SDS enabled nearly 100% release of miRNA from QS.

[0194] Confocal imaging of live SK-N-BE(2) cells showed that the QS4-(Dil)-miR-control (miR-control 1) complex was internalized into SK-N-BE(2) cells as quickly as 30 min after QS4-(Dil)-miR-control transfection.

[0195] FRET ratio experiments: QS4-miRNA complexes remained stable during internalization in cell culture media. cy5 ) and QS4-Dil exhibit high FRET efficiency due to the attachment of QS4-Dil-miRNA(Cy5).

[0196] The miRNA was incubated overnight at a slow pace in cell culture medium.Dil It was released from QS3 but not from QS4 within 2 hours. Cy5 and Dil QS4 is a miR-control Cy5 and Dil Separation from QS4 showed a low FRET specific efficiency (see Figure 13).

[0197] The miRNA was released from QS4-Dil after overnight incubation in cell culture medium. The miRNA(Cy5) and QS4-Dil showed low FRET efficiency due to the separation of miRNA(Cy5) and QS4-Dil.

[0198] In addition, the amount of addition (VI) Dil Overexpression of QS4-miR-323a-5p reduced SK-N-BE(2) cell proliferation after 96 hours with efficacy comparable to or greater than that of simple QS4-miR-323a-5p.

[0199] Dil See FIG. 24 for cell proliferation analysis of SK-N-BE(2) cells transfected with the QS4-miR-323a-5p complex and the simple QS4-miR-323a-5p complex.

[0200] The QS3-miRNA and QS4-miRNA complexes may function at miRNA-to-QS mass ratios (IV) to (VII), depending on the cell type and cell confluence. Furthermore, the QS3-miRNA complex required more than 48 hours, i.e., 72 hours, to induce miRNA target modification at the protein level.

[0201] Example 6: miRNA protection from RNAse A degradation after QS4 complex formation

[0202] Materials and Methods: Agarose electrophoresis gels of QS4-miRNA complexes were prepared as previously described. To confirm the ability of QS to protect miRNAs from RNAse A degradation compared to naked miRNAs, both the complexes (Figure 25; lanes 5-8) and naked miRNAs (Figure 25; lanes 9-12) were treated with 25 μg / mL of RNAse A for 30 min, 1 h, 2 h, and 4 h in a 310K water bath. Selected complexes (Figure 25; lanes 4-8) and naked miRNAs (Figure 25; lanes 9-13) were then treated with 0.25% SDS to ensure the release of miRNAs that would not be degraded by RNAse A. All preparations were then loaded onto a gel in PBS-added buffer (0.008% glycerol). Finally, gel electrophoresis was performed and images were captured. Agarose gel experiments were performed twice, and representative images are shown.

[0203] result: The QS4 formulation demonstrated the ability to protect miRNA from ribonuclease-mediated degradation after 4 h of RNAse A incubation at supraphysiological conditions (>1 μg / mL) (Figure 25). After the addition of SDS, the miRNA was not degraded by RNAse A, was released from QS4, and could be detected on an agarose gel. Thus, QS4 protects miR-323a-5p from degradation (lanes 5–8) and potentially extends the half-life of the miRNA in vivo circulation compared to naked miRNA, which has a short half-life in circulation (30 min; lanes 9–12).

[0204] Example 7: In vivo experiments: in a xenograft mouse model Dil Tissue biodistribution of QS4:miRNA complexes

[0205] Materials and Methods: QS was prepared as described in the previous section. For in vivo experiments, to form QS-sRNA complexes, an appropriate volume of QS4 was added to a new Eppendorf tube to achieve final QS4 concentrations of 2.7 mg / mL and 1.8 mg / mL for each 200 μL injection for volumes (V) and (VI), respectively. Next, 42.6 μL of sRNA was added to the appropriate volume (μL) of QS solution to achieve the desired sRNA-to-QS mass ratio (w / w) and maintain a constant sRNA concentration (see Table 11). To achieve a constant final sRNA concentration (i.e., 21.3 μM), the QS-sRNA complex was diluted with 1x PBS to reach the desired final volume (i.e., 200 μL) and then vigorously mixed by vortexing and pipetting up and down twice (incubation time: less than 5 min). The resulting QS-sRNA complex was generated by ionic interactions between the positive charges on the surface of QS and the negative charges on the sRNA. The QS mass and sRNA mass were calculated for various sRNA to QS mass ratios (w / w).

[0206] [Table 11]

[0207] SK-N-BE(2) cells (5 × 10) in 300 μL of PBS:Matrigel (1:1). 6 ) was injected into the right flank of 6-8 week-old female athymic nude Foxn1 mice (n = 3 mice / condition). Tumor volumes were measured every 2-3 days. When tumors reached approximately 100-200 mm 3 Once the mice were treated, they were randomized into two groups: 2 mg / kg miR-control (n=3) or DilMice were injected with miR-323a-5p conjugated with QS4 (n = 3). 24 hours later, the liver, lungs, brain, spleen, kidneys, and tumors were removed and weighed. The tissues were homogenized using a Bead-Ruptor12 (Omni International; Georgia, USA) homogenizer (5 mA for 20 seconds, 2-3 cycles until complete homogenization), and total RNA was extracted using a previously described protocol. Mature miRNA expression analysis was quantified by qPCR as previously described. Results are plotted as the mean ± SEM of three independent mice.

[0208] result: Dil QS4 formulation suppressed the expression of miR-323a-5p in lung, spleen, kidney, liver, and subcutaneous neuroblastoma tumors in mice. Dil The QS4-miR-miR showed a potency of increasing expression by 150-, 66000-, 15000-, 570-, 150- and 125-fold, respectively, compared to the control (see FIG. 26).

[0209] No gross signs of toxicity or adverse side effects were observed.

[0210] References Patent documents WO2006079889 WO2017147407 Non-patent literature Bumcrot D et al. Nat Chem Biol 2006, 2:711-719. Grimaldi N. et al. Chem Soc Rev 2016, 45:6520-6545. Cabrera I, et al. 2013 Nano Letters, 2013, 13(8), 3766-3774. Ferrer-Tasies et al. Langmuir. 2013, 29(22):6519-28 Livak KJ, Schmittgen TD. Methods 2001; 25: 402-408. Cano- Sarabia M et al. Langmuir 2008, 24:2433-2437. Elizondo E et al. Nanomed. 2012, 7:1391- 1408. Ardizzone et al, SMALL, 2018, 14 (16) DOI: 10.1002 / smll.201703851. Danaei, M.; et al. “Impact of Particle Size and Polydispersity Index on the Clinical Applications of Lipidic Nanocarrier Systems” Pharmaceutics2018, 10, 57.

[0211] For completeness, various aspects of the present invention are presented in the following numbered sections. Item 1. Nanovesicles comprising a sterol and a non-lipid cationic surfactant, wherein the sterol comprises DC-cholesterol (DC-Chol). Item 2. The nanovesicle described in item 1, wherein the non-lipid cationic surfactant is of the quaternary ammonium type. Item 3. The nanovesicle of item 2, wherein the non-lipid cationic quaternary ammonium surfactant is selected from the list consisting of myristalkonium chloride (MKC), cetyltrimethylammonium bromide (CTAB), cetyltridinium chloride (CPC), benzalkonium chloride (BAC), cetyltrimethylammonium chloride (CTAC), menzethonium chloride (BZT), stearalkonium chloride, cetrimide, benzyldimethyldodecylammonium chloride, and any combination thereof. Item 4. The nanovesicles described in item 3, wherein the nanovesicles are quatosomes in which the non-lipid cationic quaternary ammonium surfactant is MKC and the sterol is 100% DC-Chol, the molar ratio of which is preferably 1:1. Item 5. Nanovesicles described in any one of items 1 to 4, which are spherical, unilamellar, uniformly sized, and stable. Section 6. Nanovesicles according to any one of sections 1 to 5, comprising a nucleic acid, preferably miRNA, siRNA and / or shRNA. Section 7. The miRNAs are hsa-miR-323a-5p, hsa-miR-497, has-miR-380-5p, hsa-miR-892b, hsa-miR-654-5p, hsa-miR-885-3p, hsa-miR-193a-3p, h sa-miR-661, hsa-miR-491-3p, hsa-miR-193b-5p, hsa-miR-3150a-3p, hsa-miR-744-5p, hsa-miR-326, hsa-miR-665, hsa-miR-185-3p, hsa -miR-34b-5p, hsa-miR-138-2-3p, hsa-miR-4440, hsa-miR-450b-3p, hsa-miR-1180, hsa-miR-3140-3p, hsa-miR-4291, hsa-miR-30b-3p, h sa-miR-541-3p, hsa-miR-483-5p, hsa-miR-4292, hsa-miR-124-3p, hsa-miR-1207-5p, hsa-miR-193b-3p, hsa-miR-221-5p, hsa-miR-3913- 3p, hsa-miR-5095, hsa-miR-891b, hsa-miR-1275, hsa-miR-299-3p, hsa-miR-149-3p, hsa-miR-132-5p, hsa-miR-509-3-5p, hsa-miR-3677 -3p, hsa-miR-876-3p, hsa-miR-940, hsa-miR-4655-5p, hsa-miR-555, hsa-miR-342-5p, hsa-miR-3181, hsa-miR-3154, hsa-miR-5585-3p, 7. The nanovesicle of claim 6, wherein the siRNA is selected from the list consisting of hsa-miR-708-5p, hsa-miR-3135a, hsa-miR-4664-3p, hsa-miR-4289, hsa-miR-135a-3p, hsa-miR-522-5p, and any combination thereof, or the siRNA is selected from the list consisting of siCCND1, siCHAF1A, silNCENP, siKIF11, siCDC25A, siFADD, siBCL-XL, and any combination thereof. Item 8. The nanovesicle of item 6 or 7, further conjugated to a member selected from the group consisting of a fluorophore, a peptide, a polymer, an inorganic molecule, a lipid, a monosaccharide, an oligosaccharide, an enzyme, an antibody or antibody fragment, an antigen, and any combination thereof. Section 9. A pharmaceutical composition comprising a therapeutically effective amount of the nanovesicles of any one of sections 1 to 8 and a pharmaceutically acceptable excipient or vehicle. Item 10. Nanovesicles according to any one of items 1 to 8 or pharmaceutical compositions according to item 9 as a delivery system. Item 11. Nanovesicles according to any one of items 1 to 8 or pharmaceutical compositions according to item 9 for use as a medicament. Item 12. Nanovesicles according to any one of items 1 to 8 or pharmaceutical compositions according to item 9 for use in the treatment of human diseases, preferably in the treatment of cancer. Item 13. The nanovesicle or pharmaceutical composition for use according to item 12, wherein said cancer is neuroblastoma. Item 14. Use of the nanovesicles described in any one of items 1 to 8 as a bioimaging tool. Item 15. A process for producing nanovesicles according to any of items 1 to 8 using the DELOS-SUSP method.

Claims

1. A nanovesicle comprising a sterol and a non-lipid cationic surfactant, wherein the sterol comprises DC-cholesterol (DC-Chol), and the percentage of DC-Chol to the sterol is at least 47 mol %.

2. The nanovesicle of claim 1 , wherein the nanovesicle is a non-liposomal nanovesicle.

3. 3. The nanovesicle of claim 1, wherein the sterol is a mixture of DC-chol and cholesterol or a mixture of DC-chol and a cholesterol derivative.

4. The nanovesicle of claim 3 , wherein the cholesterol derivative comprises polyethylene glycol (PEG).

5. 5. The nanovesicle of claim 1, wherein the non-lipid cationic surfactant is of the quaternary ammonium type.

6. 6. The nanovesicle of claim 5, wherein the non-lipid cationic quaternary ammonium surfactant is selected from the list consisting of myristalkonium chloride (MKC), cetyltrimethylammonium bromide (CTAB), cetyltridinium chloride (CPC), benzalkonium chloride (BAC), cetyltrimethylammonium chloride (CTAC), menzethonium chloride (BZT), stearalkonium chloride, cetrimide, benzyldimethyldodecylammonium chloride, and any combination thereof.

7. 7. The nanovesicle of claim 6, wherein the nanovesicle is a quatosome in which the non-lipid cationic quaternary ammonium surfactant is MKC or CTAB.

8. 8. The nanovesicle of claim 7, wherein the nanovesicle is a quatosome in which the non-lipid cationic quaternary ammonium surfactant is MKC and the sterol is 100 mol % DC-Chol.

9. 9. Nanovesicles according to any one of claims 1 to 8, which are spherical, unilamellar, uniformly sized and stable.

10. 10. The nanovesicles of claim 9, wherein the nanovesicles have an average diameter of less than 300 nm, a polydispersity index (PDI) of 0.1 to 0.3, and are stable for up to at least 2 months.

11. 11. A nanovesicle according to any one of claims 1 to 10, comprising a nucleic acid.

12. The nanovesicle of claim 11, wherein the nucleic acid is a miRNA, siRNA, and / or shRNA.

13. The miRNAs include hsa-miR-323a-5p, hsa-miR-497, has-miR-380-5p, hsa-miR-892b, hsa-miR-654-5p, hsa-miR-885-3p, hsa-miR-193a-3p, hsa-m iR-661, hsa-miR-491-3p, hsa-miR-193b-5p, hsa-miR-3150a-3p, hsa -miR-744-5p, hsa-miR-326, hsa-miR-665, hsa-miR-185-3p, hsa-miR -34b-5p, hsa-miR-138-2-3p, hsa-miR-4440, hsa-miR-450b-3p, hsa- miR-1180, hsa-miR-3140-3p, hsa-miR-4291, hsa-miR-30b-3p, hsa-m iR-541-3p, hsa-miR-483-5p, hsa-miR-4292, hsa-miR-124-3p, hsa-m iR-1207-5p, hsa-miR-193b-3p, hsa-miR-221-5p, hsa-miR-3913-3p, hsa-miR-5095, hsa-miR-891b, hsa-miR-1275, hsa-miR-299-3p, hsa- miR-149-3p, hsa-miR-132-5p, hsa-miR-509-3-5p, hsa-miR-3677-3p , hsa-miR-876-3p, hsa-miR-940, hsa-miR-4655-5p, hsa-miR-555, hs a-miR-342-5p, hsa-miR-3181, hsa-miR-3154, hsa-miR-5585-3p, hsa 13. The nanovesicle of claim 12, wherein the siRNA is selected from the list consisting of: hsa-miR-708-5p, hsa-miR-3135a, hsa-miR-4664-3p, hsa-miR-4289, hsa-miR-135a-3p, hsa-miR-522-5p, and any combination thereof; or wherein the siRNA is selected from the list consisting of: siCCND1, siCHAF1A, silNCENP, siKIF11, siCDC25A, siFADD, siBCL-XL, and any combination thereof.

14. 14. The nanovesicle of any one of claims 11 to 13, further conjugated to a member selected from the group consisting of a fluorophore, a peptide, a polymer, an inorganic molecule, a lipid, a monosaccharide, an oligosaccharide, an enzyme, an antibody or antibody fragment, an antigen, and any combination thereof.

15. 15. A pharmaceutical composition comprising a therapeutically effective amount of nanovesicles according to any one of claims 1 to 14 and a pharmaceutically acceptable excipient or vehicle.

16. Nanovesicles according to any one of claims 1 to 14 or pharmaceutical compositions according to claim 15 as a delivery system.

17. Nanovesicles according to any one of claims 1 to 14 or pharmaceutical compositions according to claim 15 for use as a medicament.

18. Nanovesicles according to any one of claims 1 to 14 or pharmaceutical compositions according to claim 15 for use in the treatment of human diseases.

19. 19. The nanovesicle or pharmaceutical composition of claim 18, wherein the human disease is cancer.

20. A method for producing nanovesicles according to any one of claims 1 to 14 using the DELOS-SUSP method.

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