Nanosystem for use in capturing, directing and / or transporting biologically active molecules to adipose tissue

Abstract

The present invention relates to a nanosystem for use in capturing, directing and / or transporting biologically active molecules to adipose tissue, therefore serving to treat illnesses such as obesity and / or type 2 diabetes.

Description

[0001] Nanosystem for use in the capture, targeting and / or transport of biologically active molecules to tissue

[0002] ADIPOSE

[0003] FIELD OF INVENTION

[0004] The present invention belongs to the medical field. Specifically, it relates to a nanosystem for use in the capture, targeting, and / or transport of biologically active molecules to adipose tissue, thereby serving as a treatment for diseases such as obesity and / or type II diabetes.

[0005] STATE OF THE ART

[0006] Adipocytes are found throughout the body, mainly in subcutaneous, visceral and medullary depots such as white adipose tissue (WAT), brown adipose tissue (BAT) or more specialized tissues.

[0007] At the cellular level, mature adipocytes comprise only 11–40% of the total cell population in adipose tissue, which also contains preadipocytes, stem cells, macrophages, endothelial cells, and vasculature, collectively referred to as stromal vascular cells.

[0008] Due to this cellular diversity, one of the greatest challenges in drug delivery is specifically targeting adipose tissue.

[0009] Therefore, there is an unmet medical need to design strategies that allow the capture, targeting, and / or transport of biologically active molecules specifically to adipose tissue. This would have great potential to offer a wide variety of therapeutic and diagnostic imaging applications in related diseases such as obesity and associated metabolic diseases.

[0010] The present invention is directed at solving this technical problem and refers to a nanosystem for use in the capture, targeting and / or transport of biologically active molecules to adipose tissue.

[0011] DESCRIPTION OF THE INVENTION

[0012] Brief description of the invention

[0013] The present invention relates to a nanosystem for use in the capture, targeting and / or transport of biologically active molecules to adipose tissue. In particular, the inventors of the present invention show, as proof of concept, Example 1 and Example 2 relating to the synthesis of a lipophilic and cationic nanosystem with a high positive charge and formed by gold nanoparticles, coated with Gemini-type cationic surfactants and microRNA as an example of a biologically active molecule (Au@16-Ph-16 / mimic miR-X). The high overall positive charge that said nanosystem has on its surface makes it possible to specifically direct it to subcutaneous adipose tissue by creating a negatively charged electric field. However, the affinity for adipose tissue of the 16-Ph-16 surfactant that coats the nanoparticle not only comes from its high charge, but also from its activity at the surface level.Thus, previous studies have demonstrated how the micelle formation capacity of this surfactant is highly enhanced. This property, combined with its low micropolarity and high solubilizing power toward hydrophobic compounds, contributes to increasing its affinity for adipose tissue. The negative field was created using two methods: i) the creation of a low-intensity electric field using electroacupuncture and a voltage higher than the zeta potential of the nanosystem, which is also harmless to the subject being treated; and ii) laser thermal treatment. These two methods have made it possible to specifically trap and localize the nanosystem in a specific area of ​​the body.

[0014] Importantly, this optimized delivery and targeting of the nanosystem allows for the design of an effective and safe pharmacotherapy specifically targeted to localized areas of adipose tissue, preventing the nanosystem from reaching other organs or tissues where fat may be present. This strategy allows both the nanosystem to be directed toward a specific area of ​​the body with adipose tissue and to trap the nanoparticles in the application area, preventing them from escaping the target area, which in this case would be subcutaneous fat.

[0015] Thus, it is important to highlight that the nanosystem of the invention is designed in such a way that it causes the adipose tissue to specifically take it up. The fact of achieving a nanosystem that is specifically taken up by adipose tissue confers a relevant inventive leap to the invention because, unlike certain tissues or organs that have distinctive proteins on the cell surface which can be used to carry out drug targeting, adipose tissue lacks specific markers due to its diverse cellular composition, which includes adipocytes, a complex network of blood vessels, extracellular matrix and other cell types [K Onogi, AEMM Khalil, and S. Ussar, “Identification and characterization of adipose surface epitopes,” Biochem J, vol. 477, no. 13, pp. 2509-2541, Jul. 2020, doi: 10.1042 / BCJ20190462], This complexity makes it challenging to design targeting strategies that exclusively and efficiently deliver biologically active substances to adipose tissue, while avoiding off-target effects in other tissues.

[0016] The results obtained (see Example 2 and Figures 2 to 10) demonstrate a significant increase in the injected biologically active molecule only in the area where the nanosystem was injected and both the electric field and laser were applied. While in the other tissues, no significant modification in the levels of the biologically active substance tested (miR-21) was observed. In particular, Figures 11 to 15 show the analysis of the effect of the nanosystem on the Browning mediator parameters, demonstrating that the induction of the markers has been significantly detected in the left inguinal fat where the puncture was performed and where both conduction systems were applied: the laser and the electric field.

[0017] Therefore, the first aspect of the present invention refers to a nanosystem to be used in the capture, targeting and / or transport of biologically active molecules to adipose tissue comprising: a) a positively charged nanoparticle bound to a biologically active molecule and b) an irradiation system that confers a negative charge specifically on the adipose tissue, causing specific targeting and capture of the nanoparticle by the adipose tissue due to charge differences.

[0018] In a preferred aspect, the positively charged nanoparticle is bound to the biologically active molecule through cationic surfactants that enhance the uptake and stability of the biologically active molecule.

[0019] In a preferred aspect, the biologically active molecule is a nucleic acid, preferably miRNA.

[0020] In a preferred aspect, the biologically active molecule is selected from miR-21, miR-21 mimic, isomiR, oligonucleotide molecule with more than 75% sequence similarity with miR-21, compounds intended to increase the transcription or activity of miR-21 or any combination thereof.

[0021] In a preferred embodiment, the irradiation system that confers a negative charge comprises a heat source or an electric field generator, for example, a laser or electroacupuncture. In a preferred embodiment, the biologically active molecule and the nanoparticle are bound to the surfactant by a covalent or non-covalent bond.

[0022] In a preferred aspect, the biologically active molecule and the nanoparticle are independently bound to the surfactant by non-covalent bonding which may be: electrostatic interactions, hydrophobic interactions, surface adsorption, encapsulation or intercalation within the same.

[0023] In a preferred aspect, the nanoparticle comprises a metallic core.

[0024] In a preferred aspect, the metal core is a gold core.

[0025] In a preferred aspect, the average size of the nanoparticles is between 1 and 20 nanometers.

[0026] In a preferred aspect, the nanosystem is part of a pharmaceutical composition.

[0027] In a preferred aspect, the nanosystem is characterized in that its application comprises the subcutaneous injection of the nanoparticle a) and irradiation b) in the same area of ​​the injection.

[0028] In a preferred aspect, the biologically active molecule is selected from miR-21, miR-21 mimic, isomiR, oligonucleotide molecule with more than 75% sequence similarity with miR-21, compounds intended to increase the transcription or activity of miR-21 or any combination thereof.

[0029] In a preferred aspect, the nanosystem is used for the treatment of obesity and / or type II diabetes.

[0030] For the interpretation of the present invention, the following terms are defined below:

[0031] • The term "comprising" means "including," but not limited to, what follows the expression "comprising." Thus, the use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.

[0032] • The term “consisting of” means “including,” but is limited to, what follows the expression “consists of.” Thus, the expression “consists of” indicates that the listed elements are mandatory, and that no other elements may be present. • “Biologically active substance” refers to an active ingredient, active principle, active substance, or active component that possesses biological activity and can exert a therapeutic effect. miR-21 is used as a proof of concept in the present invention.

[0033] Description of the figures

[0034] Figure 1. The evolution of body weight in mice during 18 weeks of treatment using the following conduction methods: (A) laser application in the same area of ​​the puncture (5V for 25 seconds) (A), and (B) electroacupuncture application in the same area of ​​the puncture (0.7V for 25 seconds).

[0035] Figure 2. miR-21 expression levels in left inguinal fat (Au@16-ph-16 / miR21 mimic application area).

[0036] Figure 3. Expression levels of miR-21 in the right inguinal fat in mice treated with Au@16-ph-16 / miR21 mimic and with application of the two conduction systems.

[0037] Figure 4. Expression levels of miR-21 in visceral fat in mice treated with Au@16-ph-16 / miR21 mimic and with application of the two conduction systems

[0038] Figure 5. Expression levels of miR-21 in brown interscapular fat in mice treated with the nanosystem and with application of the two conduction systems.

[0039] Figure 6. Expression levels of miR-21 in the brain in mice treated with Au@16-ph-16 / miR21 mimic and with application of the two conduction systems.

[0040] Figure 7. Expression levels of miR-21 in the heart in mice treated with Au@16-ph-16 / miR21 mimic and with application of the two conduction systems.

[0041] Figure 8. Expression levels of miR-21 in the liver in mice treated with Au@16-ph-16 / miR21 mimic and with application of the two conduction systems.

[0042] Figure 9. miR-21 expression levels in the spleen in mice treated with Au@16-ph-16 / miR21 mimic and with application of the two conduction systems.

[0043] Figure 10. Expression levels of miR-21 in the kidney of mice treated with Au@16-ph-16 / miR21 mimic and with application of both conduction systems. Figure 11. Expression levels of browning and thermogenesis markers in left inguinal fat in mice treated with Au@16-ph-16 / miR21 mimic and with application of both conduction systems.

[0044] Figure 12. Expression levels of browning and thermogenesis markers in the right inguinal fat in mice treated with Au@16-ph-16 / miR21 mimic and with application of the two conduction systems.

[0045] Figure 13. Expression levels of browning and thermogenesis markers in brown interscapular fat in mice treated with Au@16-ph-16 / miR21mimic and with application of the two conduction systems.

[0046] Figure 14. Mitochondrial DNA expression levels in left inguinal fat in mice treated with Au@16-ph-16 / miR21 mimic and with application of the two driving systems.

[0047] Figure 15. Mitochondrial DNA expression levels in the right inguinal fat in mice treated with Au@16-ph-16 / miR21 mimic and with application of the two driving systems

[0048] Detailed description of the invention

[0049] The Examples included below serve as proof of concept to illustrate the invention without limiting its scope.

[0050] Example 1. Material and methods

[0051] Example 1.1. Mice and diet-induced obesity

[0052] C57BL / 6J mice (The Jackson Laboratory) were used to obtain the obese phenotype after feeding a high-fat diet (HFD 45% kcal, D12451; Research Diets, New Brunswick, NJ, USA) for 8 weeks. Mice were housed individually under appropriate temperature and humidity conditions and with free access to pelleted food. Body weight was monitored twice a week. All experimental protocols and procedures were approved by the Ethics Committee of the University of Malaga (CEUMA authorization number 91-2021 -A) and complied with the recommendations of the European Union (2010 / 63 / EU). G*Power software was used to determine the appropriate number of animals for this study. Example 1.2. Synthesis of Au@16-ph-16 precursor gold nanoparticles.

[0053] Gold nanoparticle-functionalized Gemini surfactants were prepared by direct synthesis based on the in situ reduction of hydrogen(III) tetrad oroaurate hydrate (HAuCU 4H2O) by the addition of NaBEL as a reducing agent and the appropriate Gemini surfactant as a stabilizing agent. It is well known that determining the ratio between the AuNPs / Gemini surfactant concentration and the critical micelle concentration (eme) of the surfactant is crucial for parameter stability and nanosystem size optimization. The [Gemini surfactant] / cmc ratio was set equal to 5, with a lower and upper limit of 1 and 20, respectively, being set as a necessary condition for obtaining stable monodisperse nanosystems. 390 pl of 23 mM HAuCl aqueous solution was added to 30 ml of 4 - 10' aqueous solutions. 5M of 16-ph-16 Gemini surfactant and stirred vigorously. The yellow solutions obtained were stirred for 5 minutes in the absence of light. Next, 100 pL of a freshly prepared 0.4 M aqueous NaBEL solution was added dropwise to the previously prepared solutions and the mixture was stirred moderately for 15 minutes in the dark, acquiring a reddish color. As a result, aqueous solutions of Au@16-ph-16 gold nanoparticles of concentrations 5.6- 10' were obtained. 8 M.

[0054] Example 1.3. Synthesis of Au@16-ph-16 / miR-21-mimicking gold nanoparticles

[0055] The Au@16-ph-16 / miR-21 mimic was synthesized as previously described (J Mol Liquid Volume 313, 1 September 2020, 113577, https: / / doi.Org / 10.1016 / j.molliq.2020.113577). Briefly, the synthesis of the nanosystems was stabilized with Gemini surfactants and then functionalized with miR-21 using an excess of the polymer. Formulations were explored for the nanosystem, with fixed CAu@msm = 5.6- 10' 9 M for Au@16-ph-16 and CmiR-21 variable = 4.5- 10' 8 M, 6.0- 10' 8M. As a result, different ratios R = CAu@msm / CmiR-21 were explored in each system: R = 0.124, 0.093, and 0.063 for the Au@16-ph-16 / miR-21 mimic. In this way, stable Au@16-ph-16 / miR-21 mimic complexes were obtained after 30 minutes of continuous stirring of the mixture at room temperature, and subsequent conditioning of the obtained samples at a temperature of 277.0 K for 24 hours. A moderate change in the color of the solution to a slightly purple hue was indicative of the stabilization of the complex. Example 1.4. In vivo treatment of mice with the Au@16-ph-16 / miR- 21 mimic nanosystem

[0056] After 8 weeks of HFD feeding, obese mice were separated into 4 subgroups: (1) a laser heating group (control), (2) an electric field group (Control), (3) a group of mice receiving subcutaneous injections of 200 μl of Au@16-ph-16 / miR-21 mimic 0.2 μg followed by laser, and (4) a group of mice receiving subcutaneous injections of 200 μl of Au@16-ph-16 / miR-21 mimic 0.2 μg followed by electroacupuncture.

[0057] Body weight was monitored twice weekly. Cumulative weight gain was calculated using the last pretreatment weight for each mouse as the baseline (0g) (Figure 1).

[0058] Example 1.5. Isolation of miRNAs from tissues and quantitative real-time PCR (qRT-PCR)

[0059] miRNA was extracted from tissues (80–100 mg) using the miRvana miRNA Isolation Kit (AM1561; Ambion, Spain) according to the manufacturer’s recommendations. Total RNA was quantified using a Nanodrop ND-2000 (Thermo Fisher Scientific, USA). The reverse transcription reaction was carried out in a total volume of 15 pL, containing 5 pL (5ng) of miRNA, 7 pL of the mixture consisting of RNase inhibitor, reverse transcriptase and buffer (Taqman microRNA reverse transcription, 4366597), and 3 pL of miRNA-specific primers, namely hsa-miR-21 (000397) or control miRNA assay, snoRNA-142 (001231). qRT-PCR was performed using the Agilent Mx3005P qPCR system and the specific Taqman probes indicated above in a total volume of 20 pl. The cycle threshold (Ct) was determined during amplification, and signals were normalized using snoRNA-142 as a reference gene.

[0060] Example 1.6. Isolation of mRNA from adipose tissue and quantitative real-time PCR (qRT-PCR)

[0061] Total RNA was isolated from 100 mg of adipose tissue using the Qiazol RNeasy Lipid Tissue Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's protocol. The RT reaction was performed using 20 U / mL reverse transcriptase, 03531287001; Roche), RNase inhibitor (03335399001; Roche), and deoxynucleoside triphosphate (11969064001; Roche). Then, qRT-PCR reactions were performed using 10 ng of cDNA and specific probes. Next, the Ct of each gene during amplification was determined, and then the specific signals were normalized using Tbp as a reference gene (Mm 00446973). The expression levels were calculated using the formula 2- Act The Taqman genes used in this study were: Ucpl (Mm 01244861), Tmem26 (Mm 01173641-ml), Pgc-la (Mm 01208835) and Prdml6 (Mm 00556-ml).

[0062] Example 2. Results

[0063] Figure 2 highlights that administration of the Au@16-ph-16 miR-21 mimic nanosystem, followed by application of one of the driving systems, either laser or electric field, to the left inguinal region, resulted in a significant increase in miR-21 levels in that specific area. However, no changes in miR-21 levels were observed in the other tissues (such as right inguinal fat, visceral fat, brown interscapular fat, kidney, spleen, heart, and brain), suggesting that the nanosystem did not reach other regions (Figures 3-10).

[0064] These findings were supported by analysis of the expression of browning and thermogenesis parameters in different fat depots, including the left inguinal fat (treatment site), the right inguinal fat, and the interscapular fat. This analysis revealed a significant induction of the genes Prdml6, Tmem26, PGCla, and VEGA exclusively in the left inguinal fat, whereas no change in the expression of these genes was observed in the other fat depots (Figures 11–15).

Claims

CLAIMS 1. Nanosystem for use in the capture, targeting and / or transport of biologically active molecules to adipose tissue comprising: (a) A positively charged nanoparticle bound to a biologically active molecule, and (b) An irradiation system that confers a negative charge specifically on adipose tissue, causing specific targeting and capture of the nanoparticle by adipose tissue due to charge differences.

2. Nanosystem for use according to claim 1, wherein the positively charged nanoparticle is bound to the biologically active molecule through cationic surfactants that improve the uptake and stability of the biologically active molecule.

3. Nanosystem for use according to any of the preceding claims, wherein the biologically active molecule is a nucleic acid, preferably miRNA.

4. Nanosystem for use according to any of the preceding claims, wherein the biologically active molecule is selected from miR-21, miR-21 mimetic, isomiR, oligonucleotide molecule with more than 75% sequence similarity with miR-21, compounds intended to increase the transcription or activity of miR-21 or any combination thereof.

5. Nanosystem for use according to any of the preceding claims, wherein the irradiation system that confers a negative charge comprises a heat input source or an electric field generator, for example: laser or electroacupuncture.

6. Nanosystem for use according to any of the preceding claims, wherein the biologically active molecule and the nanoparticle are linked to the surfactant by a covalent or non-covalent bond.

7. Nanosystem to be used, according to any of the preceding claims, wherein the biologically active molecule and the nanoparticle are bound to the surfactant independently by non-covalent bonding which may be: electrostatic interactions, hydrophobic interactions, surface adsorption, encapsulation or intercalation within the same.

8. Nanosystem for use according to any of the preceding claims, wherein the nanoparticle comprises a cationic metal core.

9. Nanosystem for use according to any of the preceding claims, wherein the cationic metal core is a cationic gold core.

10. Nanosystem for use according to any of the preceding claims, wherein the average size of the nanoparticles is between 1 and 20 nanometers.

11. Nanosystem for use according to any of the preceding claims, characterized in that it forms part of a pharmaceutical composition.

12. Nanosystem for use according to any of the preceding claims, characterized in that the method comprises subcutaneous injection of the nanoparticle a) and irradiation b) in the same injection area.

13. Nanosystem for use according to any of the preceding claims in the treatment of obesity and / or type II diabetes.

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