NANOSYSTEM BASED ON MICRORNA FOR TREATING OBSESITY

Abstract

The present invention relates to compositions, methods and uses, consisting of a functionalised miRNA-based nanosystem for treating obesity, weight loss and/or reduction of localised fat, which comprises miR-21 or other derived or equivalent compounds such as modified polynucleotides, synthetic mimetic and/or isomiR of miR-21; and a carrier which comprises an optimised nanoparticle for effectively binding oligonucleotides, forming an adequate nanosystem for in vivo transfection and, in particular, for the in vivo release of genes in fatty tissue.

Claims

1. A functionalised nanosystem for transporting biologically active molecules comprising: (a) a biologically active molecule which is selected from the miR-21 microRNA, mimetic, isomiR, oligonucleotide molecule with more than 75% sequence similarity, compounds aimed at increasing the transcription or activity of miR-21 or a source thereof, or any of the combinations thereof; and (b) a pharmaceutically acceptable carrier comprising a nanoparticle and a transporter molecule capable of linking oligonucleotides.

2. The nanosystem according to the preceding claim, wherein the biologically active molecule and the nanoparticle are bound to the transporter molecule independently. Both bonds can be either a covalent bond or a non-covalent bond. Generally, by means of electrostatic interactions, hydrophobic interactions, surface adsorption, encapsulation or intercalated inside same.

3. The nanosystem according to any of the preceding claims, wherein the biologically active molecule and the nanoparticle are bound to the transporter molecule independently by means of non-covalent bond which can be: electrostatic interactions, hydrophobic interactions, surface adsorption, encapsulation or intercalated inside same.

4. The nanosystem according to any of the preceding claims, wherein the nanoparticle comprises a cationic metal core.

5. The nanosystem according to any of the preceding claims, wherein the cationic metal core is a cationic gold core.

6. The nanosystem according to any of the preceding claims, wherein the mean size of the nanoparticles is comprised between 1 and 20 nanometres.

7. The nanosystem according to claim 6, wherein the mean size of the nanoparticles is comprised between 3 and 7 nanometres and/or the mean size of the nanosystem is comprised between 30 and 80 nanometres.

8. The nanosystem according to any of the preceding claims, wherein the transporter molecule of the nanosystem is a cationic surfactant.

9. The nanosystem according to any of the preceding claims, wherein the transporter molecule is a Gemini cationic surfactant.

10. The nanosystem according to claim 9, wherein the Gemini surfactant is 16-3-16.

11. The nanosystem according to claim 10, wherein the mean size of the nanoparticle is comprised between 3 and 4 nanometres and/or the mean size of the nanosystem is comprised between 30 and 80 nanometres.

12. The nanosystem according to claim 9, wherein the Gemini surfactant is 16-Ph-16.

13. The nanosystem according to any of claim 12, wherein the mean size of the nanoparticle is comprised between 5 and 6 nanometres and/or the mean size of the nanosystem is comprised between 40 and 60 nanometres.

14. A composition comprising at least one nanosystem according to claims 1-13.

15. The composition according to the preceding claim, which is a pharmaceutical composition.

16. The nanosystem according to any of claims 1-13 or a composition according to claims 14-15, for use as a medicinal product.

17. The nanosystem or composition according to the preceding claim for preventing, delaying, mitigating, reversing, curing and/or treating a metabolic disease; wherein the metabolic disease is selected from the list consisting of: amyloidosis, cardiometabolic disease, dehydration, diabetes (type 1, type 2, diabetic foot ulcers, diabetic macular oedema, diabetic neuropathy, diabetic retinopathy, diabetic nephropathy, gestational diabetes, dyslipidaemia, hyperlipidaemia), glucose intolerance, hypercholesterolaemia, hyperglycaemia, hyperinsulinaemia, or insulin resistance, hyperkalaemia, hypoglycaemia, hypopotassaemia, lipodystrophy (lipoatrophy), metabolic syndrome, obesity, osteopenia, osteoporosis (including postmenopausal osteoporosis), phenylketonuria (PKU), hypersecretion of pituitary ACTH (Cushing's syndrome) and Pompe disease.

18. The nanosystem or composition according to the preceding claim, wherein the metabolic disease is selected from obesity, overweight and hyperinsulinaemia, or insulin resistance.

19. The nanosystem according to any of claims 1-13 or the composition according to claims 14-15 for preventing, delaying, mitigating, reversing, curing and/or treating a disease associated with body weight gain. More preferably, the disease associated with body weight gain is selected from the list consisting of hypothyroidism, Cushing's syndrome, hypogonadism, hypothalamic lesions, growth hormone deficiency, Prader-Willi syndrome, Bardet-Biedl syndrome, Cohen syndrome, MOMO syndrome, anxiety and depression; or any of the combinations thereof.

20. A kit-of-parts comprising: (a) a nanosystem according to claims 1-13 or a composition according to claims 14-15; and (b) a medicinal product which is selected from: (b1) a medicinal product associated with weight gain which is selected from the list consisting of: atenolol, carbamazepine, citalopram, clozapine, doxazosin mesylate, doxepin, escitalopram, fluvoxamine, gabapentin, gamma-hydroxybutyric acid, leuprolide, lithium, metoprolol, mirtazapine, nateglinide, nortriptyline, olanzapine, paroxetine, pioglitazone, propranolol, quetiapine, repaglinide, risperidone, terazosin, valproate and phenytoin; or (b2) a medicinal product for obesity approved in a national agency which is selected from the list consisting of: megestrol acetate, benzphetamine, caffeine, cathinone, cetilistat, clobenzorex, chlorphentermine hydrochloride, dexfenfluramine hydrochloride, diethylpropion hydrochloride, fenfluramine hydrochloride, phenmetrazine hydrochloride, phentermine hydrochloride, lorcaserin hydrochloride, mefenorex hydrochloride, sibutramine hydrochloride, dronabinol, phendimetrazine, fenfluramine, phenylpropanolamine, fenproporex, phentermine, fluoxetine, levocarnitine, levothyroxine sodium, mazindol, methamphetamine, methylcellulose, orlistat, phendimetrazine, rimonabant, Saxenda, sibutramine, amphetamine sulfate, phendimetrazine tartrate, “bupropion hydrochloride+naltrexone”, “phentermine hydrochloride+topiramate”, “levocarnitine+sibutramine” and “metformin+sibutramine”; or (b3) a medicinal product for obesity in the clinical development phase which is selected from the list consisting of: Adipotide, AKR-001, AM-833, AMG-598, beloranib, B1-456906, biotin, betahistine hydrochloride, lorcaserin hydrochloride, lorcaserin hydrochloride, efpeglenatide, G-3215, GMA-102, GT-001, GTS-21, HM-12525A, HM-15211, HSG-4112, LLF-580, MEDI-0382, MET-2, Miricorilant, NGM-313, NGM-386, NN-9277, NN-9423, NN-9536, NNC-01651562, NNC-01651875, Novdb-2, NovOB, pegapamodutide, REGN-4461, RZL-12, S-237648, S-237648, SAR-425899, SCO-792, setmelanotide, setmelanotide, tesofensine, TP-0101, VP-01, ZGN-1061, ZP-4982, (acarbose+orlistat), (leucine+sildenafil citrate), (leucine+metformin hydrochloride+sildenafil citrate) and (metoprolol+tesofensine).

21. The kit-of-parts according to the preceding claim for preventing, delaying, mitigating, reversing, curing and/or treating a metabolic disease, wherein the metabolic disease is selected from the list consisting of: amyloidosis, cardiometabolic disease, dehydration, diabetes (type 1, type 2, diabetic foot ulcers, diabetic macular oedema, diabetic neuropathy, diabetic retinopathy, diabetic nephropathy, gestational diabetes, dyslipidaemia, hyperlipidaemia), glucose intolerance, hypercholesterolaemia, hyperglycaemia, hyperinsulinaemia, or insulin resistance, hyperkalaemia, hypoglycaemia, hypopotassaemia, lipodystrophy (lipoatrophy), metabolic syndrome, obesity, osteopenia, osteoporosis (including postmenopausal osteoporosis), phenylketonuria (PKU), hypersecretion of pituitary ACTH (Cushing's syndrome) and Pompe disease.

22. The kit-of-parts according to claim 21, wherein the metabolic disease is selected from obesity, overweight and hyperinsulinaemia, or insulin resistance.

23. A method for synthesising a nanosystem according to claims 1-13 which comprises: (a) adding a Gemini surfactant as a stabilising agent in a hydrogen tetrachloroaurate tetrahydrate solution (HAuCl.sub.4.4H.sub.2O), (b) reducing hydrogen tetrachloroaurate tetrahydrate (HAuCl.sub.4.4H.sub.2O) by means of the controlled addition of a reducing agent, and (c) complexing with the biologically active molecule by means of the controlled addition of the miR-21 microRNA, mimetic, isomiR, oligonucleotide molecule with more than 75% sequence similarity, compounds aimed at increasing the transcription or activity of miR-21 or a source thereof or any of the combinations thereof; with excess polymer and stirring conditions being maintained.

24. The method according to the preceding claim, wherein the reducing agent is sodium borohydride (NaBH.sub.4).

25. The method according to any of claims 23-24, wherein the reducing agent is metered by means of fractionated dropwise addition.

26. The method according to any of claims 23-25, wherein vigorous stirring is performed between step (a) and step (b) in the absence of light for at least 3 minutes.

27. The method according to the preceding claim, wherein the vigorous stirring in the absence of light is performed for at least 4 minutes.

28. The method according to any of claims 23-27, wherein stirring is performed between step (b) and step (c) in the absence of light for at least 10 minutes.

29. The method according to the preceding claim, wherein the stirring in the absence of light is performed for at least 12 minutes.

30. The method according to any of claims 23-28, wherein gentle stirring is performed after step (c) for at least 20 minutes.

31. The method according to the preceding claim, wherein the gentle stirring is performed for at least 25 minutes.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0070] FIG. 1A shows the UV-vis spectrum for precursor Au@16-Ph-16.

[0071] FIG. 1B shows the UV-vis spectrum for precursor Au@16-3-16.

[0072] FIG. 1C shows the UV-vis spectrum for functionalised nanosystem Au@16-Ph-16/miR-21, R=0.124.

[0073] FIG. 1D shows the UV-vis spectrum for functionalised nanosystem Au@16-3-16/miR-21, R=0.378.

[0074] FIG. 1E shows the UV-vis spectrum for functionalised nanosystem Au@16-Ph-16/miR-21, R=0.093.

[0075] FIG. 1F shows UV-vis spectrum for functionalised nanosystem Au@16-3-16/miR-21, R=0.283.

[0076] FIG. 1G shows UV-vis spectrum for functionalised nanosystem Au@16-Ph-16/miR-21, R=0.062.

[0077] FIG. 1H shows the UV-vis spectrum for functionalised nanosystem Au@16-3-16/miR-21, R=0.189.

[0078] FIG. 2 shows TEM images corresponding to the synthesis of nanoparticles coated with cationic Gemini surfactant: (A) Au@16-Ph-16 and (B) Au@16-3-16.

[0079] FIG. 3 shows AFM topography images of Au@16-3-16/miR-21 adsorbed on APTES-modified mica, at different ratios of R. (A-B) R=0.378; (C-D) R=0.283; (E-F) R=0.189. Figures B, D and F correspond to the transverse analysis of heights along the indicated line corresponding to images A, C and E, respectively.

[0080] FIG. 4 shows AFM topography images of Au@16-Ph-16/miR-21 adsorbed on APTES-modified mica, at different ratios of R. (A-B) R=0.124; (C-D) R=0.093; (E-F) R=0.062. Figures B, D and F correspond to the transverse analysis of heights along the indicated line corresponding to images A, C and E, respectively.

[0081] FIG. 5A shows the formation and stability of the nanosystem Au@16-pH-16 in situ.

[0082] FIG. 5B shows the formation and stability of the nanosystem Au@16-pH-16 at 24 hours.

[0083] FIG. 5C shows the formation and stability of the nanosystem Au@16-pH-16 at 48 hours.

[0084] FIG. 5D shows the formation and stability of the nanosystem Au@16-pH-16 at 1 week.

[0085] FIG. 5E shows the formation and stability of the nanosystem Au@16-pH-16 at 2 weeks.

[0086] FIG. 5F shows the formation and stability of the nanosystem Au@16-pH-16 at 1 month.

[0087] FIG. 5G shows the formation and stability of the nanosystem Au@16-pH-16/miR-21, R=0.124, in situ.

[0088] FIG. 5H shows the formation and stability of the nanosystem Au@16-pH-16/miR-21, R=0.124, at 24 hours.

[0089] FIG. 5I shows the formation and stability of the nanosystem Au@16-pH-16/miR-21, R=0.124, at 48 hours.

[0090] FIG. 5J shows the formation and stability of the nanosystem Au@16-pH-16/miR-21, R=0.124, at 1 week.

[0091] FIG. 5K shows the formation and stability of the nanosystem Au@16-pH-16/miR-21, R=0.124, at 2 weeks.

[0092] FIG. 5L shows the formation and stability of the nanosystem Au@16-pH-16/miR-21, R=0.124, at 1 month.

[0093] FIG. 5M shows the formation and stability of the nanosystem Au@16-pH-16/miR-21, R=0.093, in situ.

[0094] FIG. 5N shows the formation and stability of the nanosystem Au@16-pH-16/miR-21, R=0.093, at 24 hours.

[0095] FIG. 5O shows the formation and stability of the nanosystem Au@16-pH-16/miR-21, R=0.093, at 48 hours.

[0096] FIG. 5P shows the formation and stability of the nanosystem Au@16-pH-16/miR-21, R=0.093, at 1 week.

[0097] FIG. 5Q shows the formation and stability of the nanosystem Au@16-pH-16/miR-21, R=0.093, at 2 weeks.

[0098] FIG. 5R shows the formation and stability of the nanosystem Au@16-pH-16/miR-21, R=0.093, at 1 month.

[0099] FIG. 5S shows the formation and stability of the nanosystem Au@16-pH-16/miR-21, R=0.063, in situ.

[0100] FIG. 5T shows the formation and stability of the nanosystem Au@16-pH-16/miR-21, R=0.063, at 24 hours.

[0101] FIG. 5U shows the formation and stability of the nanosystem Au@16-pH-16/miR-21, R=0.063, at 48 hours.

[0102] FIG. 5V shows the formation and stability of the nanosystem Au@16-pH-16/miR-21, R=0.063, at 1 week.

[0103] FIG. 5W shows the formation and stability of the nanosystem Au@16-pH-16/miR-21, R=0.063, at 2 weeks.

[0104] FIG. 5X shows the formation and stability of the nanosystem Au@16-pH-16/miR-21, R=0.063, at 1 month.

[0105] FIG. 6A shows the formation and stability of the nanosystem Au@16-3-16 in situ.

[0106] FIG. 6B shows the formation and stability of the nanosystem Au@16-3-16 at 24 hours.

[0107] FIG. 6C shows the formation and stability of the nanosystem Au@16-3-16 at 48 hours.

[0108] FIG. 6D shows the formation and stability of the nanosystem Au@16-3-16 at 1 week.

[0109] FIG. 6E shows the formation and stability of the nanosystem Au@16-3-16 at 2 weeks.

[0110] FIG. 6F shows the formation and stability of the nanosystem Au@16-3-16 at 1 month.

[0111] FIG. 6G shows the formation and stability of the nanosystem Au@16-3-16/miR-21, R=0.378, in situ.

[0112] FIG. 6H shows the formation and stability of the nanosystem Au@16-3-16/miR-21, R=0.378, at 24 hours.

[0113] FIG. 6I shows the formation and stability of the nanosystem Au@16-3-16/miR-21, R=0.378, at 48 hours.

[0114] FIG. 6J shows the formation and stability of the nanosystem Au@16-3-16/miR-21, R=0.378, at 1 week.

[0115] FIG. 6K shows the formation and stability of the nanosystem Au@16-3-16/miR-21, R=0.378, at 2 weeks.

[0116] FIG. 6L shows the formation and stability of the nanosystem Au@16-3-16/miR-21, R=0.378, at 1 month.

[0117] FIG. 6M shows the formation and stability of the nanosystem Au@16-3-16/miR-21, R=0.283, in situ.

[0118] FIG. 6N shows the formation and stability of the nanosystem Au@16-3-16/miR-21, R=0.283, at 24 hours.

[0119] FIG. 6O shows the formation and stability of the nanosystem Au@16-3-16/miR-21, R=0.283, at 48 hours.

[0120] FIG. 6P shows the formation and stability of the nanosystem Au@16-3-16/miR-21, R=0.283, at 1 week.

[0121] FIG. 6Q shows the formation and stability of the nanosystem Au@16-3-16/miR-21, R=0.283, at 2 weeks.

[0122] FIG. 6R shows the formation and stability of the nanosystem Au@16-3-16/miR-21, R=0.283, at 1 month.

[0123] FIG. 6S shows the formation and stability of the nanosystem Au@16-3-16/miR-21, R=0.189, in situ.

[0124] FIG. 6T shows the formation and stability of the nanosystem Au@16-3-16/miR-21, R=0.189, at 24 hours.

[0125] FIG. 6U shows the formation and stability of the nanosystem Au@16-3-16/miR-21, R=0.189, at 48 hours.

[0126] FIG. 6V shows the formation and stability of the nanosystem Au@16-3-16/miR-21, R=0189, at 1 week.

[0127] FIG. 6W shows the formation and stability of the nanosystem Au@16-3-16/miR-21, R=0.189, at 2 weeks.

[0128] FIG. 6X shows the formation and stability of the nanosystem Au@16-3-16/miR-21, R=0.189, at 1 month.

[0129] FIG. 7 shows microscopic images representative of all liver, lung, brain, spleen and kidney tissues; corresponding to hematoxylin and eosin staining. (A) Water, (B) Au@16-ph-16 (only), (C) Au@16-ph-16 miR21, (D) Au@16-3-16 (only), (E) Au@16-3-16 miR21.

[0130] FIG. 8 shows microscopic images representative of the histological sections of visceral white fats (VAT) and subcutaneous white fats (Inguinal). Hematoxylin and eosin staining.

[0131] FIG. 9 shows the CARS (Coherent Anti-Stokes Raman Scattering) images of different tissues of mice after 48 hours of treatment with Au@Gemini/miR-21 (R=0.30) and untreated control tissues. (A) PBS in control tissue: (B) Au@16-3-16/miR-21 in control tissue; (C) Au@16-Ph-16/miR-21 in control tissue; (D, G) Livers obtained from (D) Au@16-3-16/miR-21 and (G) Au@16-Ph-16/miR-21 mice; (E, H) Spleens obtained from mice treated with (E) Au@16-3-16/miR-21 and (H) Au@16-Ph-16/miR and (F, 1) SAT fat obtained from (F) Au@16-3-16/miR-21 and (1) mice treated with Au@16-Ph-16/miR.

[0132] FIG. 10 shows *<0.05 **<0.01 45% HFD Au@16-ph-16 with respect to 45% HFD miR-21 mimetic 0.2 ug+<0.05++<0.01 45% HFD Au@16-ph-16 with respect to 45% HFD miR-21 mimetic 0.3 ug and it changes in comparison with the baseline value (0), i.e., the mice in the control group show a significant weight gain with HFD in comparison with the baseline value 0 which is the initial weight of each mouse.

[0133] FIG. 11 shows microscopic images representative of the histological sections of interscapular white fat, visceral white fat, inguinal subcutaneous white fat and interscapular brown fat; with hematoxylin and eosin staining.

[0134] FIG. 12 shows gene expression analysis with messenger RNA extracted from inguinal subcutaneous adipose tissues (ISAT) and interscapular subcutaneous adipose tissues (int. SAT), as well as from interscapular brown adipose tissue of mice treated in vivo with the nanosystem Au@16-Ph-16-miR-21 and a control with Au@16-Ph-16 without miRNA. The gene expression of Ucp1, Tmem26, Prdm16, Pgc1-α and Vegf-A genes has been quantified by means of real-time PCR.

[0135] FIG. 13 shows protein expression analysis by means of immunohistochemical images of brown adipose tissue, inguinal subcutaneous white adipose tissue and visceral white adipose tissue of mice treated in vivo with the nanosystem Au@16-Ph-16-miR-21 and a control with Au@16-Ph-16 without miRNA. Antibodies specific for the UCP-1 protein (labelled in red), for the TMEM26 protein (labelled in green), for the DNA content with 4′,6-diamino-2-phenylindole or DAPI (labelled in blue) and a labelling containing the three preceding ones, called MERGE, were used.

[0136] FIG. 14 shows electron micrograph images of the abundance of mitochondria in subcutaneous white fat (inguinal) of control mice and in mice treated with the nanosystem Au@16-pH-16-miR-21.

DETAILED DESCRIPTION OF EMBODIMENTS/EXAMPLES

Example 1. Characterisation and Verification of the Stability of the Nanosystem

[0137] The process for synthesising the nanosystems begins with the prior synthesis of Gemini surfactants and uses HAuCl.sub.4 to provide the gold content of the nanoparticle. First, 390 μl of an aqueous HAuCl.sub.4 solution with a concentration of 23 mM prepared in an aqueous solution were taken, to which there were added 30 ml of the Gemini surfactant 16-Ph-16 or 16-3-16 at the concentration of 4.Math.10.sup.−5 M and 4.Math.10.sup.−4 M, respectively. Said preparation was subjected to vigorous continuous stirring for 5 minutes in the absence of light, a clear bright yellow solution being obtained as a result. Next, 100 μl of an aqueous sodium borohydride solution 0.4 M are added dropwise, keeping the mixture under moderate stirring for 15 minutes in the dark. During this time interval, the mixture turns from yellow to a clear reddish colour. As a result, precursor gold nanoparticles called Au@16-Ph-16 and Au@16-3-16 were obtained, in this example at concentrations of 5.6.Math.10.sup.−8 M and 1.7.Math.10.sup.−7 M, respectively.

[0138] Starting from the synthesis of the precursor nanosystems, nanosystems functionalised with miRNA biomolecule in different proportions were prepared. Complexing with miR-21 was performed by working in polymer excess conditions at all times. Therefore, starting from a fixed amount of precursor nanosystem, C.sub.AU@m-sm=5.6.Math.10.sup.−9 M and 1.7.Math.10.sup.−8 M for Au@16-Ph-16 and Au@16-3-16, respectively, variable amounts of the biopolymer, C.sub.miR-21=4.5.Math.10.sup.−8, 6.0.Math.10.sup.−8 and 9.0.Math.10.sup.−8 were added. As a result, the following preparations corresponding to the following proportions in analytical concentration ratio, R=C.sub.AU@m-s-m/C.sub.miR-21 for each system, were obtained: R=0.124, 0.093 and 0.063 for Au@16-Ph-16/miR-21; and R=0.378, 0.283 and 0.189 for Au@16-3-16/miR-21. Stable Au@m-s-m/miR-21 type complexes were thereby obtained by continuous stirring, where a total of 30 minutes of gentle stirring at room temperature was required for obtaining a preparation volume of 30 ml. Next, 24 hours lapse before injection into test individuals, with the prior conditioning of the nanosystem at a temperature of 4° C. being fundamental. A moderate change to a slightly purplish tone is indicative of the stabilisation and formation of the resulting nanosystem functionalised with miR-21.

[0139] The stability of gold nanoparticles coated with a surfactant (16-Ph-16@AuNPs and 16-3-16@AuNPs), as well as nanoparticles coated with a Gemini surfactant and miR-21 (miR-21/16-Ph-16@AuNPs and miR-21/16-3-16@AuNPs) was evaluated by means of UV-visible spectroscopy, following the shape and wavelength of the maximum of the surface plasmon band over time.

[0140] The absorbance spectra were prepared using a CARY 500 SCAN UV-vis-NIR spectrophotometer (Varian). Data was collected every 2 nm with a standard 10 mm thick glass cell and the spectra were recorded in the wavelength range of 800 to 400 nm. The surface plasmon resonance exhibited by the nanosystems was shown as a strong absorption band in the visible region. Wavelength precision and spectral bandwidth were ±0.3 nm and 0.5 nm, respectively.

[0141] The absorption spectra of each sample were measured for 30 minutes, in the worst cases, and the position of the maximum surface plasmon was verified for at least one month in sterility conditions, with no noticeable changes being observed in the original position of the surface plasmon band or in the wavelength of the maximum absorption. Therefore, no evidence of aggregation was observed in any of the studied nanosystems.

[0142] The experiments were carried out in an aqueous solution at a fixed colloidal gold concentration of 5.6.Math.10.sup.−9 M and 5.6.Math.10.sup.−8 M for systems 16-Ph-16 and 1.7.Math.10.sup.−8 M and 1.7.Math.10.sup.−7 M for the corresponding gold nanoparticle systems 16-3-16, respectively. Furthermore, the spectra of the miR-21/16-Ph-16-AuNP and miR-21/16-3-16-AuNP complexes were prepared with different molar ratios (defined as R=C.sub.Au-Gemini/C.sub.miR-21): R=0.124, 0.093 and 0.063 for miR-21-16-Ph-16-AuNPs and R=0.378, 0.283 and 0.189 for miR-21-16-3-16-AuNPs.

[0143] The UV-visible graphs shown in FIGS. 1A-1H show that the position of the plasmon peak (λspr) of the nanosystems coated with Gemini surfactants (Au@16-3-16 and Au@16-Ph-16) is similar to a value of about 519 nm. According to the H. Wolfgang correlation, λspr=512+6.53.Math.exp (0.0216xd), where d is the diameter of the gold core, a λspr=519 nm corresponds to a AuNP having a core size of about 3.2 nm. It is of interest to point out that in the different Au@16-3-16/miR-21 preparations, no modification whatsoever was observed in the position of the maximum λspr, but an increase in absorbance intensities in reference to the corresponding Au@16-3-16 precursor was observed, which indicates the relative formation of Au@16-3-16-miR-21 complexes (see FIG. 1A). However, in the case of Au@16-Ph-16 nanosystems, a shift of about 2 nm in the position of maximum absorption is observed (λspr=521 nm) for preparations corresponding to R=0.124-0.093 and of 10 nm (λspr=529 nm) for R=0.063, all this together with a reduction in absorbance intensity since these nanosystems are modified with miR-21 at different proportions thereof (see FIG. 1B). The increase observed in the positions of maximum wavelength without significant modification in the SPR band demonstrates that the Gemini surfactant/miR-21 complexes are correctly formed and no significant aggregation processes have been produced.

[0144] Moreover, the different nanosystems were characterised to determine both the morphology and the size and charge of the resulting complex using different structural techniques and in solution. Specifically, TEM and AFM microscopy techniques were used for determining the size and shape of the complexes. It was therefore determined that nanoparticles Au@16-3-16 and Au@16-Ph-16 have an average size of 3.8±0.5 nm and 5.5±0.5 nm, respectively (FIG. 2). Moreover, the formation of the Au@16-3-16/miRNA and Au@16-Ph-16/miRNA complex (FIGS. 3 and 4) is proven. The transverse section analysis shows nanosystems having a thickness which matches the diameter of the nanoparticles obtained by means of TEM, while at the same time showing miRNA binding, with Au@16-3-16/miRNA and Au@16-Ph-16/miRNA complexes being obtained, the average sizes of which in the x-y direction are about 50 nm and 60 nm, respectively. The results obtained by means of the technique of scattering light in solution, DLS, confirms the results obtained by microscopy.

[0145] For TEM examination, a droplet (10 μl) of the aqueous gold nanoparticle solution was placed on a copper grid coated with a carbon film, which was then left to air dry for a few hours at room temperature. TEM analysis was carried out in a Philips CM electron microscope working at 200 kV, and the resulting images were analysed using the free ImageJ software.

[0146] AFM images were obtained with Molecular Imaging Picoscan 2500 (Agilent Technologies). Silicon cantilevers (model Pointprobe, Nanoworld) with a resonance frequency of about 240 kHz and a nominal force constant of 42 N/m were used. All AFM images were taken in the air and in the tapping mode, with a scan speed of about 0.5 Hz and data collection speed at 256×256 pixels.

[0147] Lastly, the dynamic light scattering (DLS) technique and Zeta potential measurements were used to evaluate the size distribution and charge of the precursor nanosystems and nanosystems functionalised with miR-21. For DLS measurements, Zetasizer Model ZS-90 (Malvern Instrument, Ltd., United Kingdom) equipment was used. The sample was illuminated with a laser with a fixed detection arrangement of 90° towards the centre of the area of the cell to analyse fluctuation in scattered light intensity. At least 5 size measurements were taken for each sample, and the relative error for the hydrodynamic diameter was calculated to be <5%. The results were obtained in terms of average hydrodynamic diameters, with the percentage of the different complexes obtained in solution being obtained. DTS1060 capillary polycarbonate cell was used, and the samples were introduced in molar ratios identical to the UV-visible tests.

[0148] The zeta potentials of the different samples reveal the formation of highly positively charged structures (Table 1), which allows these nanosystems to be potential vectors for transporting medicinal products to the cell.

TABLE-US-00001 TABLE 1 DLS size distribution and Zeta potential per number of free Au@Gemini nanoparticles and nanoparticles functionalised with miR-21 in different molar ratios in water (defined as R = C.sub.Au@Gemini/C.sub.miR-21). Au@Germini/miR-21 nanosystem C.sub.miR-21 = 4.5 .Math. 10.sup.−8 M C.sub.miR-21 = 6.0 .Math. 10.sup.−8 M C.sub.miR-21 = 9.0 .Math. 10.sup.−8 M Au@16-3-16 type R = 0.378 R = 0.283 R = 0.189 nanosystems (46 ± 5) mV (35.6 ± 1.1) mV (50.5 ± 1.7) mV (18 ± 3) nm; 8% (5.0 ± 0.7) nm (11 ± 4) nm (4.6 ± 1.4) nm 92% Au@16-Ph-16 type R = 0.124 R = 0.093 R = 0.063 nanosystems (43 ± 6) mV (35 ± 4) mV (31.4 ± 1.2) mV (22 ± 2) nm; 47% (10.9 ± 1.6) nm (13.3 ± 1.4) nm (6.5 ± 1.4) nm 53%

Example 2. Efficiency of the Nanosystem for Complexing miRNA

[0149] To verify the effective formation of different functionalised nanosystems UV-vis spectra were prepared as a function of time with respect to the nanosystems described in Example 1, and pertinent modifications in the surface plasmon band of the nanoparticle, as well as the stability thereof over time, were studied. FIGS. 5 and 6 show the tracking of the formation of the nanosystems functionalised with miR-21 from precursors Au@16-Ph-16 and Au@16-3-16, respectively. In the case of functionalised nanosystems having nanoparticle Au@16-Ph-16 as a precursor in different proportions of R (0.124, 0.093 and 0.063), the formation of the resulting nanosystem is clearly proven 24 hours after the end of the continuous stirring process (see Example 1), which is considered “in situ” or time zero preparation. In that sense, if the UV-vis spectra from the in situ mixture up to 24 hours of formation are compared, in the different preparations, a shift of the surface plasmon band towards blue, along with a clear reduction in band intensity (see FIGS. 5A-5X), are indicated. The observed hypochromic effect can be interpreted as a consequence of the neutralisation of phosphate groups of the miR-21 as it binds to the different positively and oppositely charged surfactants surrounding the precursor nanoparticle. The absence of significant changes in the position and shape of the band after 24 hours of mixing and up until at least one month of synthesis proves both the complete formation of the nanocomplex 24 hours after mixing and the stability thereof over time. On the other hand, if once stabilised, the position of the plasmon band of the precursor and functionalised nanosystems is compared, a bathochromic shift typical of intercalative processes is observed.

[0150] Similarly, the formation of functionalised nanosystems having as a precursor nanoparticle Au@16-3-16 in different proportions of R (0.378, 0.283 and 0.189) is also obvious 24 hours after the mixing process. In this case, the comparison of the spectra from in situ mixing up until t=24 hours shows an increase in surface plasmon band intensity, without any shift in the position of the band being shown (see FIGS. 6A-6X). This difference may be due to a change in the manner in which the surfactant binds to miR-21 which gives rise nanosystems having a more regular and dispersed structure, producing a hyperchromic effect in the absorption intensity of the plasmon band of the precursor Au@16-3-16.

Example 3. Toxic Effect of Treatment Using miRNA

[0151]

TABLE-US-00002 TABLE 2a Short-term effect of miR-21-Nano on the weight of mice. Mice were treated daily for 7 days by means of subcutaneous injections of 200 μl of miR-21-Nano and were weighed after 48 h. Weight before Weight after Type of Amount of treatment treatment Code nanoparticles MiR-21 (μg) (t = 0) (t = 48 h) A1 H.sub.2O DEPC 0 28.9 27.5 A2 Au@16-pH-16 0 26.1 25.5 A3 Au@16-pH-16 0.495 25.1 25.4 A4 Au@16-pH-16 0.22 25.5 25.8 AS Au@16-pH-16 0.11 24.3 24.4 A6 Au@16-3-16 0 29.9 29.2 A7 Au@16-3-16 0.495 26.6 26.3 A8 Au@16-3-16 0.22 30.4 28.8 A9 Au@16-3-16 0.11 27.3 26.6 S1 Physiological 0 26.7 25.6 saline solution DEPC: Diethyl pyrocarbonate

[0152] Tests were performed to rule out the possible toxic effect of the nanosystem (Au@Gemini/miR-21 nanoparticles). C57BL/6J mice were injected daily for 7 days with nanosystem miR-21 having different miR-21 concentrations. The injections were subcutaneous injections in the interscapular part of the mouse. One group of mice was then sacrificed after 48 hours to evaluate the acute effect of said complex (Table 2a), and another group was sacrificed after one month to evaluate the chronic effect (Table 2b). In addition to control groups with serum and water, another group of mice treated with miR-21 conjugated with in vivo-JetPEI® delivery reagent with an miR-21 concentration of 0.5 μg was included in the study.

TABLE-US-00003 TABLE 2b Long-term effect of miR-21-Nano on the weight of mice. Mice were treated daily for 7 days by means of subcutaneous injections of 200 μL of miR-21-Nano and weighed after 15 days and 30 days. Weight before Weight after Weight after Type of Amount of treatment treatment treatment Code nanoparticles miR-21 (μg) (t = 0) (t = 15 days) (t = 30 days) B1 H.sub.2O DEPC 0 25.7 26.7 27.2 B2 Au@16-pH-16 0 24.9 25.4 26 B3 Au@16-pH-16 0.495 26.4 27.2 27.3 B4 Au@16-pH-16 0.22 28 28.8 28.6 B5 Au@16-pH-16 0.11 26.7 27.5 27.1 B6 Au@16-3-16 0 27 28.7 28.6 B7 Au@16-3-16 0.495 29.2 29.7 30.5 B8 Au@16-3-16 0.22 27 28.3 28.6 B9 Au@16-3-16 0.11 24 26.9 27.3 S2 Physiological 0 26.8 28.3 27.9 saline solution 1 jetPei 0.5 24.1 27 27.5 2 jetPei 0.5 25.9 28.2 27.4 3 jetPei 0.5 26.2 27.7 27.9 4 jetPei 0.5 26 27.2 27.9 5 jetPei 0.5 25 25 27.2

[0153] With these tests, no alterations were observed in the normal coat, behaviour, activity, weight level of the mice, no sign of depression and no palpable node was detected. Furthermore, no mouse died before the end of the study.

[0154] Blood samples were analysed before the mice were sacrificed in order to perform the corresponding blood counts, and the values were compared with those of the reference group (Table 3), without any changes being observed.

TABLE-US-00004 TABLE 3a Short-term effect of miR-21-Nano on the blood composition of mice. Blood count corresponding to mice treated daily for 7 days by means of subcutaneous injections of 200 μl of miR-21-Nano and after 48 h. Au@ Au@ Au@ Au@ Au@ Au@ 16- 16- 16- 16- 16- 16- ph- ph- ph- 3- 3- 3- Physio- Au@ Au@ 16- 16- 16- 16- 16- 16- REF. Type of logical 16- 16- miR- miR- miR- miR- miR- miR- VAL- nano- H.sub.2O saline ph- 3- 21- 21- 21- 21- 21- 21- UES particles d.p.c. solution 16 16 0.5 0.2 0.1 0.5 0.2 0.1 (n = 4) Code A1 S1 A2 A6 A3 A4 A5 A7 A8 A9 RBC 9.21 9.33 10.06 9.08 10.03 8.98 10.01 8.69 8.14 10.05 9.84 ± (×10.sup.6/μl) 0.35 HEMAT- 42.7 42.7 45.2 41.3 45.5 40.2 45.2 38.9 36.4 45.4 45.95 ± OCRIT 1.34 (%) HEMO- 14.2 14.1 15.5 13.5 15.3 13 15 12.7 11.2 15.4 14.37 ± GLOBIN 0.67 (g/dl) MCV 46.4 45.5 44.9 45.5 45.4 44.8 45.2 44.8 44.7 45.2 46.75 ± (fl) 0.40 MCHC 33.2 33.2 34.3 32.6 33.6 32.3 32.6 32.6 30.8 33.9 31.2 ± (d/dl) 0.59 MCH 15.4 15.1 15.4 14.8 15.3 14.4 15 14.6 13.8 15.3 14.57 ± (pg) 0.23 RDW 14.4 12.7 12.9 13.7 11.8 13.9 12.8 13.9 14 13.8 13.82 ± (%) 0.36 WBC 1.62 2 1.98 1.75 1.13 2.64 1.48 4.4 1.88 3.54 3.54 ± (×10.sup.3/μl) 0.24 NEUTRO- 9.56 37.12 9.79 10.1 19.68 6.98 11.86 10.1 7.08 5.03 17.46 ± PHILS 2.98 (%) EOSIN- 0 0 0.49 0.17 1.71 0.72 1.6 0.31 0.93 0.89 0.71 ± OPHILS 0.16 (%) BASO- 0 0 0 0.46 0 0 0 0.52 0 0 0.29 ± PHLS 0.10 (%) LYMPHO- 89.31 62.44 88.12 88.88 76.38 90.04 85 84.44 90.42 89.7 78.58 ± CYTES 3.38 (%) MONO- 1.13 0.44 1.6 1.64 2.61 1.9 1.5 1.45 1.57 4.38 2.96 ± CYTES 0.89 (%) PLATE- 198 147 468 84 105 160 144 131 152 298 187.75 ± LETS 32.49 (×10.sup.3/μl) PLATE- 0.15 0.11 0.39 0.06 0.06 0.11 0.11 0.08 0.12 0.21 0.15 ± LET- 0.03 CRIT (%) MPV 7.6 7.5 8.4 7.2 5.9 7 7.7 6.6 7.6 7.1 8.32 ± (fl) 0.12

TABLE-US-00005 TABLE 3b Long-term effect of miR-21-Nano on the blood composition of mice. Blood count corresponding to mice treated daily for 7 days by means of subcutaneous injections of 200 μl of h miR-21-Nano and after 30 days. Au@ Au@ Au@ Au@ Au@ Au@ 16- 16- 16- 16- 16- 16- ph- ph- ph- 3- 3- 3- Physio- Au@ Au@ 16- 16- 16- 16- 16- 16- REF. Type of logical 16- 16- mIR- mIR- mIR- miR- miR- miR- VAL- nano- H.sub.2O saline ph- 3- 21- 21- 21- 21- 21- 21- UES particles d.p.c. solution 16 16 0.5 0.2 0.1 0.5 0.2 0.1 (n = 4) Code B1 S2 B2 B6 B3 B4 B5 B7 B8 B9 RBC 9.27 8.87 9.28 10 9.77 9.97 8.8 10.52 10.14 8.76 9.84 ± (×10.sup.6/μl) 0.35 HEMAT- 45.8 39.6 44.3 46.2 45 46.9 41.6 48.2 46.8 44.6 45.95 ± OCRIT 1.34 (%) HEMO- 14.6 13.6 15.4 15.2 14.6 15.2 14.6 15.3 15.3 14.9 14.37 ± GLOBIN 0.67 (g/dl) MCV 49.5 44.7 47.8 46.2 46.1 47.1 47.3 45.9 46.2 51 46.75 ± (fl) 0.40 MCHC 31.8 34.3 34.7 32.9 32.4 32.4 35 31.7 32.6 33.4 31.2 ± (d/dl) 0.59 MCH 15.7 15.3 16.5 15.2 14.9 15.2 16.5 14.5 15 17 14.57 ± (pg) 0.23 RDW 12.6 14 14.9 12.5 13.5 12.3 13.2 12.6 12.6 13.1 13.82 ± (%) 0.36 WBC 2.63 3.12 3.07 2.61 1.83 4.78 3.13 3.76 3.29 2.61 3.54 ± (×10.sup.3/μl) 0.24 NEUTRO- 21.5 29.82 25.2 17.66 14.4 14.24 24.15 21.46 13.49 21.2 17.46 ± PHILS 2.98 (%) EOSIN- 0.91 0.64 2.05 0.69 14.65 1.23 0.97 2.99 4.24 4.18 0.71 ± OPHILS 0.16 (%) BASO- 0.27 0.64 0.38 0.26 0.23 0.41 0.41 0.12 0.12 0.11 0.29 ± PHILS 0.10 (%) LYMPHO- 63.66 58.19 77.81 72.57 66.47 74.29 71.68 63.09 70.26 67.46 78.58 CYTES 3.38 (%) MONO- 13.66 10.71 2.56 8.82 4.25 9.83 2.79 12.34 11.89 7.05 2.96 ± CYTES 0.89 (%) PLATE- 162 857 812 384 191 792 618 667 827 351 187.75 ± LETS 32.49 (×10.sup.3/μl) PLATE- 0.11 0.71 0.67 0.32 0.15 0.67 0.55 0.53 07 0.28 0.15 ± LET- 0.03 CRT (%) MPV 7.3 8.3 8.3 8.5 8 8.5 8.9 8 8.5 8 8.32 ± (fl) 0.12

[0155] Morphological studies consisting of performing the hematoxylin and eosin histochemical study (FIGS. 7A-D) to assess possible anomalies at the tissue level were then performed. This allowed verifying, by means of optical microscopy, whether the different tissues present structural or morphological anomalies susceptible to pathology. To that end, several organs will be extirpated: liver, lung, brain, spleen, kidney, visceral fat and inguinal fat. The organs have not experienced any alteration during treatment, with the most significant modifications being observed at the inguinal adipose tissue level, in which the appearance of multilocular adipose tissue (corresponding to beige adipose tissue) with 7 days of treatment with nanosystems Au@16-Ph-16-miR-21 and Au@16-3-16-miR-21 stands out (FIG. 8).

[0156] To detect the possible accumulation of nanoparticles in tissues, Raman spectrometry in 10-micron tissue slices will be used. CARS (Coherent Antistokes Raman Spectroscopy) microscopy allowed obtaining high-resolution images in freshly extirpated tissues without the need for labelling or prior preparation with a chromophore, in turn allowing the spatial distribution of the specific component selected for mapping the sample in the selected image area to be detected (FIG. 9).

[0157] Liver and lung tissues freshly extirpated from mice were dissected into thin tissue slices of about 10 pm thick and then submerged in PBS and placed on a glass slide for examination. To carry out the technique, there two parallel lines of independent laser excitation (Ti: Sapphire laser, model Tsunami, Spectra Physics) will be used, generating pulses of between 3 and 13 picoseconds that must be very carefully overlapped in time and space, i.e., they must precisely strike the same point of the sample and furthermore pulsate simultaneously, so that they generate the suitable CARS signal upon striking the sample. The beams are optically coupled to a vertical microscope (Olympus, model BX61WI) with ultrafast galvanometric mirror scanning. By setting the pump energy at 727 nm (with a pulse width of about 4.5 ps and a microscope inlet power of 60 mW) and the energy of the stokes has been selected at 858 nm (with a pulse width of about 8 ps and a microscope inlet power 80 mW) for observing vibration of 2100 cm.sup.−1 (CARS signal at 631 nm), for detecting gold nanoparticles in tissues. It could be confirmed through these tests that the nanoparticles did not accumulate in tissues.

[0158] In vitro analyses and accumulation at the tissue level in animal models are fundamental in understanding the toxicity level of the nanosystems. As is known, gold nanoparticles improve the Raman anti-Stoke signal of neighbouring amino acids, so the application of a suitable control CARS technique can detect the presence of AuNPs which measure this effect. To verify the possible accumulation of functionalised gold nanoparticles in different organs of mice, ex vivo CARS microscopy experiments were performed in different freshly dissected tissues after 48 hours of treatment (see FIG. 9). Furthermore, some control experiments were carried out in situ by directly depositing gold nanoparticles and PBS buffer control tissues (see FIGS. 9A-C).

[0159] As expected, the CARS images of the PBS did not show a significant contrast in non-resonant condition (see FIG. 9A), whereas the CARS signal clearly improved in the presence of Au@16-Ph-16-miR-21 and Au@16-3-16-miR-21 nanoparticles in control tissue (see FIGS. 9A-C), showing improved bright spots. The localised improvement of the anti-Stroke Raman signal at an excitation wavelength of 858 nm was observed for extirpated livers, spleens, lungs, brains, kidneys and VAT and SAT fatty tissues of the miR-21-AuNP derivatives (see FIGS. 9D-1) in the highest concentration of CmiR-21 (R 0.189 and 0.062 for Au@16-3-16-miR-21 and Au@16-Ph-16-miR-21, respectively) and treated mouse AuNP precursors. As a result, the Raman signal was completely absent for all the studied tissues treated with coated and non-coated miR-21 nanosystems after 48 hours of treatment. In this sense, the evidence of the present invention indicates that nanoparticles do not accumulate in the main organs under study, and this indicates the absence of toxicity due to the possible accumulation effect of the nanosystems of the present invention. However, more experiments must be conducted to evaluate a complete toxicity characterisation.

[0160] All in all, the CARS images of the “control” did not show a significant contrast in non-resonant condition, whereas the CARS signals of AuNPs increased considerably, appearing as bright spots scattered across the images taken of samples treated with AuNPs. The improvement probably resulted from the substantial scattering of AuNPs and the high third-order polarisability of AuNPs. In conclusion, the data demonstrated that these nanosystems did not show any short-term and long-term toxic effect on treated mice, and that the gold nanoparticles do not accumulate in tissues.

Example 4. Study of the Effect of In Vivo Treatment with Nanosystems Au@16-Ph-16-miR-21 and Au@16-3-16-miR-21 on Weight and Fat Gain in a Mouse Model of Obesity Subjected to a High Fat Diet

[0161] Male C57BL/6J mice obtained from Jackson Laboratory were used in the study. C57BL/6J is an animal model that is widely used in obesity and diabetes type 2 study. The animals were housed individually and kept in photoperiods of 12 hours of light and 12 hours of darkness at 24° C. and 45%±5% humidity. During the 7 days of adaptation, the animals followed the normal diet used in the animal facility. The animals were then subjected to a high fat diet (HFD, 45% Kcal) for 53-55 days to generate the obese phenotype. The mice were weighed and treated 2 times a week for 6 weeks with 0.2 μg or 0.3 μg of miR-21 mimetic conjugated with nanosystems Au@16-Ph-16 or Au@16-3-16 in a final volume of 200 μl by means of subcutaneous injections (interscapular or inguinal). Adipose tissues (inguinal subcutaneous and interscapular, visceral and brown) and blood were extracted while sacrificing the mice.

[0162] Weight gain in mice subjected to treatment with nanosystems Au@16-Ph-16-miR-21 and Au@16-3-16-miR-21 was halted in comparison with controls; particularly, treatment with nanosystem 16-ph-16-miR-21, both with 0.2 μg and with 0.3 μg of miR-21, significantly reduced weight gain in mice subjected to the fat rich diet in comparison with the control (FIG. 10).

[0163] The study was complemented with a histological analysis and a gene and protein expression analysis in inguinal subcutaneous, interscapular and visceral white adipose tissue and interscapular brown fat originating from mice subjected to in vivo treatment with nanosystems Au@16-Ph-16-miR-21 and Au@16-3-16-miR-21.

[0164] On one hand, the histological images of the tissues, which were prepared with hematoxylin and eosin staining (FIG. 11), showed that in vivo treatment with nanosystems Au@16-Ph-16-miR-21 and Au@16-3-16-miR-21 has transformed part of white fat (made up of unilocular adipocytes) into beige fat (made up of multilocular adipocytes).

[0165] On the other hand, a gene expression analysis was performed with messenger RNA obtained from inguinal subcutaneous and interscapular adipose tissues, thermogenesis marker genes (UCP1 and PGC-1α), beige cell markers (Tmem26), and browning process regulating genes Vegf-A and Prdm16 by means of real-time PCR (FIG. 12). The data demonstrates that both thermogenesis and browning are significantly induced by means of treatment with 0.2 ug of nanosystem Au@16-Ph-16-miR-21.

[0166] A protein expression analysis was then performed by means of immunohistochemistry on inguinal subcutaneous white adipose tissue and visceral white adipose tissue with anti-UCP-1 protein antibodies (as a brown adipose tissue marker) and anti-TMEM26 antibodies (as a beige adipocyte marker) alone and in combination (merge) (FIG. 13). The immunohistochemistry images show the appearance of a strong signal in the inguinal subcutaneous white fat corresponding to UCP-1 protein and TMEM26 with treatment with nanosystem-miR-21 in comparison with the control (without miR-21). Furthermore, a double labelling with both antibodies revealed the co-localisation of adipocytes with a high expression of UCP-1 and TMEM26 only in the inguinal subcutaneous white fat from mice treated with nanosystem-miR-21 in comparison with the control (without miR-21).

[0167] The number of mitochondria in inguinal adipose tissue was also evaluated by means of electron microscope imaging (FIG. 14). These images show the scarcity of mitochondria in the subcutaneous white fat (inguinal) of control mice in comparison with an abundance of large, electron-dense organelles in the subcutaneous white fat (inguinal) of mice treated with nanosystem Au@16-pH-16-miR-21.

[0168] The data confirms that the effect of nanosystems Au@16-pH-16-miR-21 and Au@16-3-16-miR-21 (at a low concentration of miR-21 of 0.2 and 0.3 μg) on the reduction in weight gain would be mediated by the induction of browning in subcutaneous white adipose tissue and the activation of thermogenesis in both brown fat and subcutaneous white fat by means of the conversion of white adipocytes into beige adipocytes.