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    • 专利摘要:
    Nanosystems with complexes. The present invention relates to a nanosystem characterized by a complex and a cover, to the methods of obtaining, to complexes and their uses. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2738173A1
    申请号:ES201830729
    申请日:2018-07-19
    公开日:2020-01-20
    发明作者:Fernandez Maria Jose Alonso；Lobato Matilde Duran
    申请人:Universidade de Santiago de Compostela；
    IPC主号:
    专利说明:

    [0001]
    [0002] Nanosystems with complexes
    [0003]
    [0004] Technical sector
    [0005]
    [0006] The present invention relates to a nanosystem characterized by a complex and a cover, to the methods of obtaining, to complexes and their uses.
    [0007]
    [0008] Background of the invention
    [0009]
    [0010] The administration of biological molecules is one of the challenges in pharmaceutical formulation due to lability, inability to cross or interact with biological barriers and / or inadequate solubility of said active molecules. Some of the solutions that have been presented in the art are based on nanotechnology.
    [0011]
    [0012] There are bibliographic antecedents that show that nanosystems formed by lipids and polymers are capable of releasing insulin, but frequently they do not do so in a controlled way but that the release profile shows a strong initial burst (Santalices, I., Gonella, A., Torres , D., & Alonso, MJ, Advances on the formulation of proteins using nanotechnologies. Journal of Drug Delivery Science and Technology, 2017, 42, 115-180; Müller, RH, MaEder, K., & Gohla, S., Solid lipid nanoparticles (SLN) for controlled drug delivery-a review of the state of the art, European Journal of Pharmaceutics and Biopharmaceutics, 2000, 50 (1), 161-177).
    [0013]
    [0014] Thus, it is still necessary to provide nanosystems that are capable of protecting said active molecules after exposure to a biological environment and facilitating their transport through biological barriers, whether mucous, epithelial or cellular barriers. And also, that really control the release of active substances, such as insulin, avoiding the initial burst .
    [0015]
    [0016] Description of the invention
    [0017]
    [0018] The authors of the present invention have developed a nanosystem capable of protecting biological molecules from the degradation they suffer in biological fluids. But in addition, the nanosystems of the invention are capable of protecting said biological molecules as well. against degradation in the animal body such as pancreatic degradation, thus allowing permanence for a longer time in that place of action.
    [0019]
    [0020] Thus, in a first aspect the invention is directed to a nanosystem characterized by:
    [0021]
    [0022] (i) a complex comprising a) a polyamino acid or a polynucleotide characterized by having a negative charge and b) a metal, and
    [0023] (ii) a cover comprising at least one charged molecule,
    [0024] with the proviso that the net charge of the complex (i) is opposite to the charge of the shell molecule.
    [0025]
    [0026] A second aspect is directed to a process for obtaining the nanosystem of the first aspect of the invention, which comprises adding a solution of at least one ionizable or charged molecule, to a complex comprising a) a polyamino acid or a polynucleotide characterized by having negative charge, and b) a metal,
    [0027]
    [0028] with the proviso that the net charge of the complex is opposite to the charge of the ionizable or charged molecule.
    [0029]
    [0030] In a particular embodiment, the invention is directed to a process for the preparation of the complex described above, comprising:
    [0031]
    [0032] a) mixing a polyamino acid or a polynucleotide and a metal,
    [0033] b) when in step a) a polyamino acid is mixed, then modify the pH of the mixture to reach a pH equal to or greater than the isoelectric point of the polyamino acid,
    [0034] c) incubate the mixture at a temperature between 0 and 8 ° C, and
    [0035] d) disperse the mixture in a buffered medium.
    [0036]
    [0037] In a third aspect the invention is directed to the complex obtainable by the procedure described in steps a) to d). In another aspect the invention is directed to a complex comprising a protein and a transition metal and has an average size of less than 200 nm. In another aspect the invention is directed to a complex consisting of insulin and zinc and having an average size of less than 120 nm.
    [0038] In a fourth aspect the invention is directed to a pharmaceutical composition comprising a nanosystem or a complex as described in the present invention, and a pharmaceutically acceptable excipient.
    [0039]
    [0040] In a fifth aspect the invention is directed to a nanosystem or a pharmaceutical composition as described in the present invention, for use in medicine. In a particular embodiment, for use in the treatment and / or prevention of diseases associated with hormonal, metabolic or immunological disorders, inflammatory processes, degenerative diseases and tumors. More particularly, for use in the treatment of diabetes. More in particular, for oral administration.
    [0041]
    [0042] Description of the figures
    [0043] Figure 1 Transmission Electron Microscopy (TEM) images of Insulin-Zn nanocomplexes, revealing a cubic or orthopedic morphology.
    [0044] Figure 2 Change in ZP from negative to neutral values as the concentration of coating agents increases: 2A: coated by LAE and PEG st; 2B: covered by CTAB and PEG st.
    [0045] Figure 3 Transmission Electron Microscopy (TEM) images of coated Insulin-Zn nanocomplexes prepared in Example 4, revealing a cubic or orthopedic morphology.
    [0046] Figure 4 Characterization of the NMR system: 4A: schematic representation of the LAE and PEG st roof structure approach; 4B: H-NMR spectrum of uncoated nanocomplexes; 4C: PEG st! H-NMR spectrum; 4D: LAE * H-NMR spectrum; 4E: * H-NMR spectrum of nanocomplexes coated with LAE and PEG st; 4F: DOSY spectrum of coated nanocomplexes; 4G: waterLOGSY spectrum of coated nanocomplexes.
    [0047] Figure 5. Colloidal stability of Insulin-Zn nanocomplexes without and with SIF coating: 5A: uncoated nanocomplexes; 5B: coated by LAE and PEG st; 5C: covered by CTAB and PEG st.
    [0048]
    [0049] Figure 6. Insulin release in SIF from: 6A: the uncoated nanocomplexes; 6B: nanocomplexes coated by LAE and PEG st.
    [0050] Figure 7. Proteolytic study of coated nanocomplexes.
    [0051] Figure 8. Lyophilization of nanocomplexes coated with different concentrations of cryoprotectant (trehalose)
    [0052] Figure 9. Evolution in time of size and count rate of lyophilized and concentrated formulations x4 and x8 times maintained at room temperature.
    [0053] Figure 10. Distribution of radioactivity in organs after administration of 10A: nanocomplexes labeled with Technetium-99m in rats; 10B: control (Technetium-99m free). Figure 11. Subcutaneous administration (s.c.) in healthy rats of nanocomplexes, before and after lyophilization, compared with an insulin solution.
    [0054] Figure 12. Glucose reduction after intrajejunal administration (i.j.) in healthy rats.
    [0055] Figure 13. Glucose reduction after intrajejunal administration (i.j.) in diabetic rats.
    [0056]
    [0057] Detailed description of the invention
    [0058]
    [0059] The inventors have designed nanosystems characterized by:
    [0060]
    [0061] (i) a complex comprising a) a polyamino acid or a polynucleotide characterized by having a negative charge and b) a metal, and
    [0062] (ii) a cover comprising at least one charged molecule,
    [0063] with the proviso that the net charge of the complex (i) is opposite to the charge of the shell molecule.
    [0064]
    [0065] The complexes in the nanosystems of the invention have a net charge dependent on the components that form it, and thus, the net charge may vary according to the proportion in which said components are found. One skilled in the art knows methods to measure the net charge of the complex. According to the present invention, the net charge of the complex is the Zeta potential value measured by Doppler Laser anemometry. A particular description of how to measure said Zeta potential is found in example 1 of this document.
    [0066]
    [0067] In a particular embodiment, the metal has a valence equal to or greater than 2, or in other words, is in an oxidation state equal to or greater than 2. In a more particular embodiment, the oxidation state in which it is found the metal is 2 or 3. In another particular embodiment, the metal is selected from zinc, iron, calcium, magnesium, copper, chromium, manganese, nickel, ruthenium, barium, strontium, bismuth, platinum, zirconium, germanium, titanium, molybdenum, cobalt and tin.
    [0068]
    [0069] In a particular embodiment the polyamino acid is selected from a peptide, a protein and a monoclonal antibody.
    [0070]
    [0071] In a particular embodiment, the peptides of the invention comprise two or more amino acids.
    [0072] In a particular embodiment, the peptides and proteins of the invention are selected from hormones, growth factors, cytokines, hematopoietic factors, enzymes, receptors, antigenic proteins, coagulation factors, adhesion molecules and cellular receptors. In a more particular embodiment, the polyamino acids of the invention are selected from insulin, GLP-1 proteins, GLP-2 proteins, somatropin, anakinra, dornase alpha, serum proteins, SPARC or osteonectin proteins, protein C, keratin subfamily A, human growth hormone or somatotropin, gonadotropin, angiopoietin, colony stimulating factors, epidermal growth factor, erythropoietin, fibroblast growth factor, GDNF family of ligands, growth differentiation factor 9, hepatocyte growth factor, hepatoma-derived growth, insulin-like growth factors, keratinocyte growth factor, migration stimulating factor, macrophage stimulating protein, neurotrophins, placental growth factor, platelet-derived growth factor, thrombopoietin, transforming growth factors, vascular endothelial growth, chemokines, interfer ones, interleukins, lymphokines, tumor necrosis factors, Fc fusion proteins, peptides and derivatives of contulaquine-G, antifylamine, opioid peptides, lipopeptides and antigens.
    [0073]
    [0074] In a more particular embodiment, the GLP-1 proteins comprise exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, taspoglutide and semaglutide. In a more particular embodiment, the GLP-2 proteins comprise teduglutide. In a more particular embodiment, colony stimulating factors comprise macrophage colony stimulating factor, granulocyte colony stimulating factor and granulocyte macrophage colony stimulating factor. In a more particular embodiment, the interferons comprise interferon alfa IIb, interferon alfacon-1 and interferon alfa-n3. In a more particular embodiment, tumor necrosis factors comprise tumor necrosis factor alpha. In a more particular embodiment, Lipopeptides comprise surotomycin. In a more particular embodiment, the antigens comprise tetanus toxoids, diphtheria toxoids, hepatitis B.
    [0075]
    [0076] In a particular embodiment, the monoclonal antibodies of the invention are selected from elotuzumab, panitumumab, trastuzumab, bevacizumab, adalimumab, ritusimumab and anti-TNF antibodies.
    [0077]
    [0078] For the complex to form, it is necessary that the polyamino acid has a net charge opposite to the metal charge, that is, a negative charge. For this, the polyamino acid can be ionized and the resulting net charge is negative, or the polyamino acid has a functional group with a negative charge. The net charge of the polyamino acid may be positive or negative depending on the pH of the medium. When the polyamino acid, and in particular the peptide, the protein or the monoclonal antibody, is in a medium with a pH higher than the isoelectric point then it will be negatively ionized.
    [0079]
    [0080] In a particular embodiment, the polynucleotides are selected from transferring RNA, interfering RNA, silencing RNA, DNA, antisense oligonucleotides, micro-RNA and double stranded RNA.
    [0081]
    [0082] For the complex to form between the metal and the polynucleotide, the polynucleotide must have a net negative charge. The net charge of a polynucleotide depends on the nucleotides that form it and a person skilled in the art knows that the net charge of the polynucleotide is negative.
    [0083] In a particular embodiment, the complex comprises insulin and zinc. In another particular embodiment, the complex comprises iron and tRNA.
    [0084]
    [0085] The cover of the nanosystems of the invention comprises at least one charged molecule.
    [0086] In a particular embodiment, the shell-loaded molecule is selected from molecules that have at least one functional group selected from guanidino, primary amine, secondary amine, tertiary amine, quaternary amine and thiol,
    [0087]
    [0088] In a particular embodiment, the shell-loaded molecule is selected from molecules that have at least one functional group selected from carboxylate, phosphate, nitrate, carbonate and sulfate.
    [0089] In a particular embodiment, the charged charge molecule is selected from among ionizable surfactants or with a charged functional group, ionizable polymers or with a charged functional group, ionizable polysaccharides, polyamino acids, homo and co-polymers containing ionizable residues, quaternary amines, and combinations thereof. In a more particular embodiment, the charged loading molecule is selected from ethyl lauroyl arginate, cetyltrimethylammonium bromide, hexadecyltrimethyl ammonium bromide, benzetonium chloride, hyaluronic acid, chitosan, chitosan-polyethylene glycol, polyarginine, polyarginine polyethenglycol glycine-polydylene glycol polyethylene glycol, protamine, chondroitin sulfate, polyglutamic acid, puliglutamic acid-polyethylene glycol, polyaspartic acid, polyaspartic acid-polyethylene glycol, polyisolic acid, heparan sulfate, keratan sulfate, and combinations thereof.
    [0090]
    [0091] In a particular embodiment, the shell may further comprise non-ionizable and uncharged molecules, that is, neutral molecules. In a more particular embodiment, the non-ionizable and non-chargeable molecules are selected from non-ionic surfactants, non-charged polymers, polyalcohols, and combinations thereof. In a more particular embodiment, the neutral shell molecule is selected from the group consisting of fatty acid esters, polyethylene glycol esters, sucrose monoesters, sorbitanos, polysorbates, poloxamers, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, and combinations thereof. In a more particular embodiment, the neutral molecule is polyethylene glycol stearate 40.
    [0092]
    [0093] The interaction between the complex and the shell is based on an electrostatic interaction, so that the molecule present in the shell can be ionizable and thus have an ion with a charge opposite to the charge of the complex. Alternatively, the molecule present in the shell may have a functional group with a charge opposite to the charge of the complex. And the net charge of the cover molecule will be the opposite of that of the complex.
    [0094]
    [0095] A person skilled in the art can measure the charge of the complex as explained above, and then select the cover molecule. In addition, you can incorporate neutral or uncharged molecules since they do not vary the total net charge of the roof. The incorporation of neutral molecules to the shell allows to increase the stability of the nanosystems.
    [0096] For example, when the complex has a negative net charge, the person skilled in the art will select a molecule with a positive charge for the cover such as ethyl lauroyl arginate, cetyltrimethylammonium bromide or hexadecyltrimethylammonium bromide. And you can also incorporate neutral molecules such as polyethylene glycol stearate. For example, when the complex has a positive net charge, the person skilled in the art will select a molecule with a negative charge for the shell, such as, for example, hyaluronic acid that can be ionized in a basic medium as is evident to one skilled in the art.
    [0097]
    [0098] An additional advantage of the cover is that it confers stability to the nanosystem, even when the nanosystem is found in biological fluids in vitro. Thus, it is demonstrated in example 5 of the present invention that the coated nanosystem is stable in Simulated Intestinal Fluid, a property that the uncovered system does not have (example 5).
    [0099]
    [0100] Another additional advantage of the nanosystems of the invention over uncoated complexes, is that they allow very controlled release of polyamino acids or polynucleotides, preventing an initial burst and maintaining release over time, for at least 4 hours. These characteristics make them especially suitable for the administration of polyamino acids and polynucleotides orally, preferably insulin.
    [0101] The nanosystems of the present invention have an average diameter of less than 1. ^ m, that is, they have a size between 1 and 999 nm, preferably less than 200 nm, more preferably less than 170 nm, even more preferably less than 120 nm. The size of the nanosystems is mainly influenced by the composition and preparation conditions and can be measured by dynamic light scattering (DLS), as shown in the examples. The size of the nanosystems does not vary significantly when they are lyophilized or when they are lyophilized over time, or when they are reconstituted after being lyophilized, as shown in example 6, obtaining in all cases systems of size nanometric
    [0102]
    [0103] The nanosystems of the invention have a low polydispersion index (PDI) that can be measured by dynamic light scattering (DLS). In a particular embodiment, the polydispersion index of the nanosystems is preferably less than 0.5, more preferably less than 0.4. This indicates that they form a homogeneous population of nanoparticles in which a similar and controlled behavior of each of the nanosystems that make it up is expected.
    [0104] The charge of the nanosystems of the invention can be neutral, positive or negative. Preferably the charge of the nanosystems of the invention is neutral. In a particular embodiment the nanosystems of the invention have an approximately neutral charge. In a particular embodiment, the nanosystems of the invention have a charge between -5.0 mV and 5.0 mV. In a preferred embodiment the nanosystems of the invention have a load between -1.0 mV and 1.0 mV.
    [0105]
    [0106] In another particular embodiment, the nanosystems of the invention have a positive charge of less than 50mV, preferably less than 30mV. In a particular embodiment, the nanosystems of the invention have a load between 0.1 mV and 30 mV.
    [0107]
    [0108] A person skilled in the art knows methods to be able to measure the burden of the nanosystem. According to the present invention the charge of the nanosystem is the Zeta potential value measured by Doppler Laser anemometry. A particular description of how to measure said Zeta potential is found in the examples.
    [0109]
    [0110] In a particular embodiment, the nanosystems of the invention have a positive charge of less than 30mV and a polydispersion index of less than 0.5.
    [0111]
    [0112] In a particular embodiment, a nanosystem of the invention consists of insulin, zinc, polyethylene glycol stearate and ethyl lauroyl arginate and has an average size of less than 120 nm. In a particular embodiment, a nanosystem of the invention consists of insulin, zinc, polyethylene glycol stearate and ethyl lauroyl arginate and has an average size of less than 120 nm and a polydispersion index of less than 0.5.
    [0113]
    [0114] In another particular embodiment, a nanosystem of the invention consists of insulin, zinc, polyethylene glycol stearate and cetyltrimethylammonium bromide and has an average size of less than 120 nm.
    [0115]
    [0116] In addition, it is noteworthy that both the complexes and the nanosystems of the invention have a cubic or orthopedic shape with perfectly defined corners or vertices as seen in the Transmission Electron Microscopy (TEM) images (see Figure 3.
    [0117]
    [0118] The morphology of the nanosystems of the invention could have an advantageous influence when administering polyamino acids or polynucleotides orally to mucous membranes, improving the capacity for internalization, retention in the gastrointestinal tract and in vivo transport orally.
    [0119] Thus, in a particular embodiment, the nanosystems of the invention are further characterized by having a cubic or orthopedic shape.
    [0120]
    [0121] In another aspect the invention is directed to a process for obtaining a nanosystem as described above, which comprises adding a solution of at least one ionizable or charged molecule, to a complex comprising a) a polyamino acid or a polynucleotide characterized for having a negative charge, and b) a metal,
    [0122]
    [0123] with the proviso that the net charge of the complex is opposite to the charge of the ionizable or charged molecule.
    [0124]
    [0125] The ionizable or charged molecule will be part of the shell. For example, the ionizable molecule can have a guanidino or amino group that are protonable groups and thus have a positive charge. For example, the ionizable molecule can have a carboxylic acid group that can be deprotonated and thus have a negative charge. For example, the ionizable or charged molecule can have a quaternary amino group that is positively charged.
    [0126]
    [0127] In a particular embodiment, the ionizable or charged molecule is selected from molecules that have at least one functional group selected from guanidino, primary amine, secondary amine, tertiary amine, quaternary amine and thiol,
    [0128]
    [0129] In a particular embodiment, the ionizable or charged charge molecule is selected from molecules that have at least one functional group selected from carboxylate, phosphate, nitrate, carbonate and sulfate.
    [0130]
    [0131] In a particular embodiment, the ionizable or charged charge molecule is selected from ionizable surfactants or with a charged functional group, ionizable polymers or with a charged functional group, ionizable polysaccharides, polyamino acids, homo and co-polymers containing residues ionizable, quaternary amines, and combinations thereof. In a more particular embodiment, the charged loading molecule is selected from ethyl lauroyl arginate, cetyltrimethylammonium bromide, hexadecyltrimethyl ammonium bromide, benzetonium chloride, hyaluronic acid, chitosan, chitosan-polyethylene glycol, polyarginine, polyarginine polyethenglycol glycine-polydylene glycol polyethylene glycol, protamine, chondroitin sulfate, polyglutamic acid, puliglutamic acid-polyethylene glycol, polyaspartic acid, acid polyaspartic-polyethylene glycol, polysalic acid, heparan sulfate, keratan sulfate, and combinations thereof.
    [0132]
    [0133] In a particular embodiment, the process may further comprise adding non-ionizable and uncharged molecules, that is, neutral molecules. In a more particular embodiment, non-ionizable and non-chargeable molecules are selected from non-ionic surfactants, non-charged polymers, polyols, and combinations thereof. In a more particular embodiment, the neutral shell molecule is selected from the group consisting of fatty acid esters, polyethylene glycol esters, sucrose monoesters, sorbitanos, polysorbates, poloxamers, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, and combinations thereof. In a more particular embodiment, the neutral molecule is polyethylene glycol stearate 40.
    [0134]
    [0135] In a particular embodiment, the invention is directed to a process for the preparation of the complex described above, which comprises
    [0136]
    [0137] a) mixing a polyamino acid or a polynucleotide, and a metal,
    [0138] b) when in step a) a polyamino acid is mixed, then modify the pH of the mixture to reach a pH equal to or greater than the isoelectric point of the polyamino acid,
    [0139] c) incubate the mixture at a temperature between 0 and 8 ° C,
    [0140] d) disperse the mixture in a buffered medium.
    [0141]
    [0142] Thus, one skilled in the art may modify the pH of the medium in which the complex is prepared to reach or exceed the isoelectric point of the polyamino acid of interest or add the already ionized polyamino acid to the medium. In a particular embodiment, the pH is adjusted between 4.0 and 6.0.
    [0143]
    [0144] The complexes can be prepared in an alternative manner comprising gradually mixing a solution of a polyamino acid or a polynucleotide, and a metal, under stirring.
    [0145]
    [0146] The following examples illustrate the invention, although they are not limiting thereof.
    [0147]
    [0148] Examples
    [0149]
    [0150] Example 1. Preparation of Insulin-Zn nanocomplexes.
    [0151] The nanocomplexes were obtained by a two-step procedure, consisting of the induction of the controlled interaction between insulin and zinc by means of pH adjustment, followed by incubation of the mixture at 4 ° C and subsequent dispersion of the complex in buffer:
    [0152] A 1.5 mg / mL insulin solution in 0.005N HCl was prepared, to which 20 mg / mL zinc acetate in ultrapure water was added according to an insulin molar ratio: zinc 1: 6. The pH of the mixture was then adjusted to 5.13 by the addition of NaOH under magnetic stirring, after which the mixture was incubated at 4 ° C. After incubation, the mixture was frightened at room temperature and then the sediment obtained in the previous stages was resuspended by vortexing. Then, the resuspended sediment on 10 mM PBS in 1: 1 v / v ratio was added under magnetic stirring. Finally, the suspension was allowed to equilibrate at room temperature before characterizing.
    [0153]
    [0154] Physicochemical properties of nanocomplexes
    [0155] The particle size distribution and the POI were determined by dynamic light scattering (DLS) and the zeta potential values (ZP) were determined from the electrophoretic mobility values by Doppler anemometry (LDA) in a Malvern Zetasizer device ( NanoZS ZEN 3600, Malvern Instruments, Worcestershire, UK), equipped with a beam of red laser light (X = 632.8 nm). The formulations were measured directly undiluted at 25 ° C, at least in three batches and with triplicate analysis of each batch. The efficiency of association (EA%) of insulin in the nanocomplexes was analyzed after separating the nanocomplexes from the suspension medium and its direct and indirect analysis. For this, 1 mL of the formulation was ultracentrifuged (Beckman Coulter, Optima L-90K, Brea, USA, 70.1Ti rotor) at 70,000 rpm (average RCF 336,140 g) for 3h at 15 ° C, after which a sediment is obtained of the nanocomplexes separated from the suspension medium. After separating the supernatant, the pellet was dissolved in 0.1% TFA to induce dissociation of the insulin-Zn complex. Aliquots of both the supernatant (indirect quantification) and the pellet dissolved in TFA (direct quantification) were analyzed by high performance liquid chromatography (HPLC) using an isocratic reverse method in an Agilent equipment (1100 LC series, with diode network detector set at 214 nm, Santa Clara, USA), with a C18 column (Superspher® RP-18 endcapped) as a stationary phase. The EA% was calculated according to the following equation:
    [0156] Amount of insulin in isolated nanocomplexes EA ( %) = xlüü Total amount of insulin
    [0157]
    [0158] The load capacity or association (loading capacity, % w / w) was calculated by dividing the amount of associated insulin (AE x Total insulin in the formulation) by the total weight of the nanocomplexes.
    [0159] Table 1. Properties of the Insulin-Zn nanocomplexes
    [0160]
    [0161]
    [0162]
    [0163]
    [0164] The morphological evaluation was carried out in a transmission electron microscope (TEM, CM12, Philips, Holland), for which the samples were deposited on copper grids and stained with phosphotungstic acid (2% w / v in acetate buffer 500 mM pH 5.5, to avoid dissociation of the complex at acidic pH), for 2 min and allowed to dry overnight in a desiccator. The morphology of the complex is shown in Figure 1.
    [0165]
    [0166] Example 2. Insulin-Zn nanocomplex coating.
    [0167]
    [0168] The nanocomplexes were optimized with a coating of their surface in order to provide them with additional properties, mainly aimed at controlling colloidal stability in biological media, the release of the associated protein and improving mucodifusion. Several different coatings were successfully tested:
    [0169] - combination of the neutral surfactant polyethylene glycol stearate 40 (PEG st) and the cationic ethyl lauroyl arginate (LAE) surfactant.
    [0170] - combination of the neutral surfactant polyethylene glycol stearate 40 (PEG st) and the cationic surfactant cetyltrimethylammonium bromide (CTAB).
    [0171]
    [0172] Example 2.1 Coating with LAE and PEG st
    [0173] The coating with PEG st and LAE is carried out by adding solutions of these excipients on the suspension of the nanocomplexes prepared according to example 1, under stirring. The structure of the coating is understood as a surfactant layer: the positively charged LAE groups would interact with the negatively charged surface of the nanocomplexes, so that the hydrophobic LAE glues would be oriented outward. These tails would in turn interact with the hydrophobic tails of PEG st, which would consequently leave the PEG polar groups oriented towards the outside environment. This conformation is reflected in the change in zeta potential values (ZP) of the nanocomplexes from negative values to neutrality as the concentration of surfactants increases and in NMR studies.
    [0174] Physicochemical properties
    [0175] The zeta potential values (ZP) were determined from the electrophoretic mobility values by Doppler anemometry (LDA) in a Malvern Zetasizer device (NanoZS ZEN 3600, Malvern Instruments, Worcestershire, UK), equipped with a laser red light beam (X = 632.8 nm). The formulations were measured directly undiluted at 25 ° C, at least in three batches and with triplicate analysis of each batch. See results in figure 2A.
    [0176]
    [0177] The particle size distribution and the PDI, zeta potential (ZP) and association efficiency (EA%) of insulin in the nanocomplexes were determined according to the methodology already described in example 1.
    [0178] Table 2. Properties of the Insulin-Zn nanocomplexes before and after stabilization by coating with the selected LAE-PEG st.
    [0179]
    [0180]
    [0181] The morphological evaluation was carried out in a transmission electron microscope was performed in the manner described in example 1, and the morphology of these coated nanocomplexes are shown in Figure 3.
    [0182]
    [0183] Example 2.2 Coating with CTAB and PEG st.
    [0184]
    [0185] Coating with CTAB and PEG st was performed following the method described in example 2.1. Zeta potential measurements were obtained as a function of the amount of coating material as shown in Figure 2B.
    [0186]
    [0187] Example 2.3 Fluorescent marking
    [0188]
    [0189] The formulation prepared in example 2.1. It has been fluorescently labeled to be tested in additional biodistribution studies.
    [0190]
    [0191] To label insulin, a solution of 10 mg / mL of insulin in 0.1M bicarbonate buffer pH 8.58 was prepared, on which a) 100 ^ L of a 10 mg / mL solution of FITC in ethanol was added, or either b) 50 ^ L of a 10 mg / mL Cy5-NHS solution in DMSO, drop by drop under magnetic stirring (300 rpm). The mixture was kept under magnetic stirring (300 rpm) for 1h and in the dark, after which the labeled insulin was purified from the fluorophore moieties by gel filtration columns (PD10). The precipitation of the labeled insulin was then induced by adjusting the pH to its pl (5.49), centrifuged (10 min, 4 ° C, 15,000g) and the supernatant was discarded to remove the salts from the buffer. Finally, the labeled insulin pellet was resuspended in ultrapure water and lyophilized for conversion to powder.
    [0192]
    [0193] Example 2.4. Cover Study
    [0194]
    [0195] The structure of the LAE-PEG st shell of the nanosystem prepared according to example 2.1 was eluted using NMR techniques. The schematic approach to the roof structure described in Example 2.1 is schematically represented in Figure 4A. To confirm the roof layout and rule out that they are aggregates or that there is no real interaction between the roof and the complex, waterLOGSY ( Water-Ligand Obserbed via Gradient SpectroscopY) experiments were carried out , for which they had to be obtained previously the proton nuclear magnetic resonance (NMR) spectra of the Nanosystem and its components as well as Diffusion Order Spectroscopy (DOSY NMR)
    [0196]
    [0197] For this, samples of the suspension of coated nanocomplexes were analyzed as well as solutions of each of the components of the system separately at the same concentration and in the same dispersion medium as the nanosystems, measured in conventional standard tubes from 5 mm to 25 °. The NMR spectra were obtained with a Varian Inova 17.6T spectrometer (Agilent) operating at a proton frequency of 750 MHz, and processed with the MestreNova v10.0.1 software using the TMS peak as a reference (5 = 0 ppm). Diffusion filters were applied to suppress the solvent (WATERGATE, 1d! H Dfilter) in all samples to attenuate the peak of water and potential low molecular weight impurities in the samples. The waterLOGSY experiments were carried out by applying a 180 ° inversion pulse on the water signal at 4.7 ppm by means of a synchronized selective pulse of 1, 2 and 3 ms and mixing time of 0.5s, to assess the environment chemical groups of relevance and to elucidate their position in the nanostructure.
    [0198]
    [0199] The NMR spectra of the nanosystem and its components are shown in Figure 4B to 4E). The characteristic signals of the protons -OCH 2 -CH 2 - of the PEG chain were clearly located in the PEG st spectrum at 3.64-3.67 ppm (Figure 4C). On the other hand, the proton signals corresponding to the aliphatic chains were easily located in the spectra of PEG st (1.26 ppm) and LAE (1.27 ppm) (Figure 4C and 4D). The presence of these signals was also located in the spectrum of the coated nanosystem (protons of the carbon -OCH 2 -CH 2 - of PEG groups at 3.71 ppm and carbons of aliphatic chains at 1.27 ppm) (Figure 4E).
    [0200]
    [0201] The DOSY experiments (Figure 4F) showed a similar diffusion coefficient for all the signals obtained in the spectrum of the coated nanosystem, suggesting that they came from the same structure (nanosystem) and therefore a minimal or zero contribution of possible LAE surfactant impurities and PEG st not associated to the system.
    [0202]
    [0203] Finally, the waterLOGSY experiment (Figure 4G) of the coated nanosystems clearly showed an inverted peak at 3.71 ppm corresponding to the PEG regions, confirming its external arrangement on the surface of the nanocomplex, in contact with the aqueous medium. By On the other hand, the signal corresponding to the aliphatic chains of the surfactants (1.27 ppm) remained uninverted, indicating its internal arrangement in the structure. Finally, the changes in the PEG inverted signal were proportional to the pulse length applied (from 3 to 1 ms, figure 4G), confirming that the observed effect was due to the waterLOGSY effect. In conclusion, the results of this characterization reinforced the hypothesis of the structure adopted by the surfactants in the nanosystem shell.
    [0204]
    [0205] Example 3. Study of the stability of nanocomplexes coated in Simulated Intestinal Fluid (SIF).
    [0206]
    [0207] The stability of the coated nanosystems prepared in examples 2.1 and 2.2. in SIF it was evaluated based on the evaluation of size and count rate. For this, the nanosystem suspension was diluted in the medium of interest at the highest dilution at which the equipment can obtain a measurement within the acceptable concentration range. The samples were incubated at 37 ° C and with horizontal agitation (300 rpm, Heidolph Instruments GmbH & Co. KG, Schwabach, Germany). At defined intervals (0; 0.5; 1; 2; and 4h), the evolution of particle size and distribution and mean count rate (MCR) (when working with a fixed attenuator) or derived count rate (DCR) were analyzed (when working with automatic attenuator), both indicative of the number of particles that remain in suspension, in a Malvern Zetasizer device (NanoZS ZEN 3600, Malvern Instruments, Worcestershire, UK). The results are shown in Figures 5A, 5B and 5C. The results show that the uncoated nanocomplexes give rise to micrometric sizes, possibly due to the aggregation of the nanosystems, from the first measurement times (Figure 5A). On the contrary, nanosystems coated with both LAE and PEG st (Figure 5B) and CTAB and PEG st (Figure 5C) are able to maintain their size in SIF for at least 4h while the count rate, indicative of the number of particles which remain in suspension, remained unchanged, thus confirming the colloidal stability of the coated nanosystems in this medium.
    [0208]
    [0209] Example 4. Release study
    [0210]
    [0211] To study the release of insulin from the uncoated nanocomplexes prepared in example 1 and coated nanocomplexes prepared in example 2.1. and suspended in SIF, the suspension thereof was diluted in SIF (1: 5) and incubated at 37 ° C with horizontal agitation (300 rpm, Heidolph Instruments GmbH & Co. KG, Schwabach, Germany). At intervals defined (0; 0.5; 1; 2; and 4h), 1 mL aliquots were ultracentrifuged (Beckman Coulter, Optima L-90K, Brea, USA, 70.1Ti rotor) at 70,000 rpm (average RCF 336,140 g) for 3h at 15 ° C, after which a sediment of the coated nanocomplexes is obtained, containing the non-released insulin, separated from the suspension medium. After separating the supernatant, the pellet was dissolved in 0.1% TFA to induce dissociation of the insulin-Zn complex. Uncoated nanocomplexes, where no pellets are observed, were processed in the same way. Both the supernatant (indirect quantification) and the pellet dissolved in TFA (direct quantification) were analyzed by high performance liquid chromatography (HPLC) using an isocratic reverse method, in an Agilent device (1100 LC series, with diode network detector set at 214 nm, Santa Clara, USA), with a C18 column (Superspher® RP-18 endcapped) as a stationary phase.
    [0212] The results obtained are shown in Figure 6A and 6B, confirming that uncoated nanosystems release virtually all of the insulin at 30 min (Figure 6A), while coated nanosystems according to Example 2.1 are able to control the release of insulin in SIF for at least 4h (Figure 6B), a period especially suitable for oral administration since this is the estimated time of intestinal transit along the small intestine, an area in which greater absorption is estimated. On the other hand, it should be mentioned that most of the protein and peptide nanotransporters described in the literature hardly present this quality, since they frequently present problems of premature release or burst release (Müller, Mader and Gohla, 2000; Santalices et al., 2017).
    [0213]
    [0214] Example 5. Evaluation of protection against proteolysis of coated nanocomplexes.
    [0215]
    [0216] This test was carried out by quantifying the amount of insulin that remained without degrading after incubating the formulation with 1% supplemented SIF w / v with pancreatin (4 USP) (SIF-p). For this and prior to the study, serial dilutions of SIF-p and an insulin solution were prepared in the nanocomplex suspension medium and incubated for 20 min (37 ° C, horizontal agitation at 300 rpm), after which the degradation reaction was stopped by adding 0.1N HCl and placing the mixture on ice. Then insulin undegraded analyzed by HPLC (method described in earlier sections) and the ratio of pancreatin - insulin which the half life of insulin (t1 / 2) was ~ 20 min was selected for further study. For proteolysis evaluation, 250 ^ L of 1) the nanocomplex suspension was mixed coated described in example 2.1; 2) an insulin solution at the same concentration and in the same dispersion medium as the formulation (5 mM PBS), and 3) only dispersion medium (negative pancreatin control), with SIF-p according to the ratio of Pancreatin-insulin previously selected and incubated at 37 ° C with horizontal agitation (300 rpm, Heidolph Instruments GmbH & Co. KG, Schwabach, Germany). At defined intervals (0; 0.5; 1 and 2h), the enzymatic activity was stopped by adding 300 ^ L of 0.1N HCl to each aliquot, which also caused the disruption of the nanocomplexes by dissociation at acidic pH, and placed The resulting mixture on ice. The remaining amount of insulin was analyzed by HPLC following the method described in previous sections.
    [0217] The results obtained (see Figure 7) showed a clear protection effect of the formulation against enzymatic degradation, where free insulin degraded by 76.2% with respect to its initial concentration in just 30 min, while the insulin associated with The nanocomplexes did not drop to these levels until 2h after the start of the study.
    [0218]
    [0219] Example 6. Lyophilization of the coated Insulin-Zn complex and stability study during storage.
    [0220]
    [0221] The coated nanocomplexes obtained in example 2.1. they were lyophilized in the presence of different concentrations of cryoprotectant (trehalose). For this, a suspension of coated nanocomplexes was mixed together with concentrated solutions of trehalose (1: 1) under magnetic stirring (300 rpm), after which the mixture is immediately frozen at -80 ° C and then lyophilized (Genesis VirTis 25EL pilot lyophilizer) The product (lyophilized powder) obtained was stored at room temperature in a desiccator. To reconstitute it, ultrapure water was added in the volume corresponding to the initial concentration of nanocomplexes prior to mixing with the cryoprotectant, and resuspension in a vortex (30s) was favored. The physicochemical characterization of the resulting suspension was carried out according to the methodology previously described in example 1, and concentrations of cryoprotectants were selected at which physicochemical parameters similar to the original formulation were obtained (see Figure 8).
    [0222] The coated nanocomplexes, lyophilized with trehalose and reconstituted to their original form with ultrapure water, proved to be able to be concentrated by ultrafiltration up to 4.69 and maintain their initial size up to 24 hours at room temperature.
    [0223] This study was carried out by loading ultrafiltration systems previously filtered with ultrapure water (Centrifugal Filter Units Amicon Ultra - 15 (ref. UFC 903024) with 14 mL of reconstituted lyophilized formulation. Then, the systems were centrifuged in consecutive periods of 3 min (Eppendorf Centrifuge) , model 5430 R, 4,000 rcf, 15 ° C), among which the concentrate was homogenized and aliquots were taken for insulin concentration analysis by HPLC (corresponding method described in previous sections) and particle size maintenance (size measurement and PDI, corresponding method described in previous sections).
    [0224] Table 3. Estimated insulin concentration and analyzed insulin concentration (HPLC) in the lyophilized and reconstituted formulation based on centrifugation time.
    [0225]
    [0226]
    [0227] The colloidal stability at room temperature of the concentrated formulations was evaluated four (x4) and eight (x8) times from their initial volume, by size measurement, PDI and count rate (Malvern Zetasizer equipment, method described in example 1). The x8 concentrated formulations maintained their initial size for 48 hours in colloidal form and at room temperature, while the x4 concentrates did so for 24 hours. See figure 9.
    [0228]
    [0229] Example 7. Interaction with intestinal tissues in vivo.
    [0230]
    [0231] The in vivo biodistribution of the coated nanocomplexes obtained in Example 2.1 was studied by radioactively marking the formulation and then administering it orally to rats that were viewed by monophotonic emission computed tomography (SPECT / CT). The animals (Wistar rats, 250 g) were housed and cared for under standard conditions with free access to water and food ad libitum. All procedures were carried out following protocols previously approved by the guides of the Ethics and Biosafety Committee of the University of Navarra.
    [0232] For nanosystem labeling, previously lyophilized formulations with trehalose were concentrated from 0.64 mg / mL to 1.71 mg / mL of final insulin concentration to ensure administration of a volume suitable for oral administration. This was carried out by ultrafiltration systems (Amicon Ultra 30KDa, with Ultracel® regenerated low adhesion cellulose membrane) centrifuged at 5,000 g and RT in 2 min periods (Mikro® 20, Hettich®, Hettich Lab Technology, Tuttlingen, Germany ), preventing the formation of aggregates and / or adsorption to membranes by gentle pipetting, and controlling the maintenance of particle size (NanoS90 equipment, Malvern Instruments, Worcestershire, UK). The nanosystems were radiolabeled adapting a previously described protocol (Areses et al., 2011): 750 ^ L of concentrated formulation was mixed with 40 ^ L of an acidified solution of 0.5 mg / mL SnCh (2mM HCl in Romil-Sps water) to the consequent reduction of technetium species in a closed vial, which was purged with helium for 5 min to minimize oxygen content and prevent oxidation of pre-reduced tin. Next, 210 ^ L of the 99mTcO f generator eluent in 0.9% w / v NaCl was added to the vial and the mixture was allowed to react for 30 min at RT. Next, the vial was opened and the quality control of the marking was carried out by thin layer radiochromatography (ITCL). For this, 2 ^ L of the sample obtained was deposited on a Whatmann 3MM strip (1x15cm) and was developed with 0.9% NaCl w / v up to 14 cm, and the radiochemical purity was determined with a MiniGita radiochromatographic system (Raytest GmbH, Dortmund, Germany). Prior to administration, each individual dose of the radiolabelled formulation (260 ^ L) was mixed with 70 ^ L of 22.5 mM PBS to ensure stability during passage through the stomach. Male Wistar rats (n = 4) were fasted for 12 hours before the experiments and were given an oral dose of 330 ^ L of the final radiolabelled formulation (equivalent to 50 IU / Kg insulin dose) or technetium free as a control, both dosages equivalent to 250 p, Cis per rat (evaluated with ATOMLAB 500, Biodex®, Shirley NY, USA). The animals were anesthetized with 2% isoflurane gas (0.2 L / min oxygen flow) and viewed in a SPECT-CT device (Symbia, Siemens Medical System, USA) at 1, 2, 4, 6, 8 and 24 h. See figure 10.
    [0233] Radiolabelled nanosystems remained mainly in the small intestine (up to 2h) and in the cecum for the duration of the study, while the control of free technetium remained in the stomach (see Figure 10A and 10B). This intestinal transit profile shows the retention of the nanosystems in the blind section at the times of 4h, 8h and 11h agrees with the estimated transit times in the rat animal model and in humans, where the speed of intestinal contents transport It is much smaller in the distal segments than in the proximal. It should also be noted that anesthesia with isoflurane is related to the reduction of motility in the GI tract and transit times due to muscle relaxation.
    [0234]
    [0235] Example 8. Preservation of the bioactivity of the associated protein in the formulation after the formulation and also lyophilization processes.
    [0236]
    [0237] The bioactivity of the protein in the nanosystems described in Example 8 before and after lyophilization was carried out by subcutaneous administration (sc) of a dilution of the formulation in 5 mM PBS at a dose corresponding to 1 IU / Kg to Sprague rats -Dawley, and comparison of the glycemic profile obtained against the administration of a 1 IU / Kg dose of the same unprocessed insulin. Sprague-Dawley rats (250-300g) were obtained from the Central Animalario, University of Santiago de Compostela (Spain). They were kept under 12h light / 12h dark cycles and fed a standard rodent diet (Panlab A04, Panlab laboratories). All experiments were reviewed and approved by the Committee Ethics of the University of Santiago de Compostela (ref. 1500AE / 12 / FUN01 / FIS02 / CDG3), in accordance with the European and Spanish regulations for the use of animals in animal studies, and therefore carried out in compliance with the Council Directive 2010/63 / EU and European Parliament. The results (Figure 11) showed the same glycemic profile in all cases, indicating that the bioactivity of insulin remained intact after the production processes of the nanosystems and their lyophilization.
    [0238]
    [0239] Example 9. Pharmacological effect in healthy rats and diabetic rats
    [0240]
    [0241] Sprague-Dawley rats (250-300g) were obtained from the Central Animalario, University of Santiago de Compostela (Spain). They were kept under 12h light / 12h dark cycles and fed a standard rodent diet (Panlab A04, Panlab laboratories). All experiments were reviewed and approved by the Ethical Committee of the University of Santiago de Compostela (ref. 1500AE / 12 / FUN01 / FIS02 / CDG3), in accordance with European and Spanish regulations for the use of animals in animal studies, and therefore carried out in compliance with Council Directive 2010/63 / EU and European Parliament.
    [0242]
    [0243] In the case of healthy rats, one week before the experiment, a surgical implantation of an intestinal catheter was applied with the proximal end of the tunneled cannula subcutaneously to exit from the back of the neck, and they were sutured. The animals were allowed to recover for 6 days and were monitored based on their general condition and weight daily. Prior to administration, they were fasted with free access to water for 4h, and blood samples (time 0h) were taken from the caudal vein immediately before administration to take baseline glucose levels with a hand held glucometer (Glucocard ™ G + meter, Arkray Factory, Japan). Only animals with basal levels equal to or greater than 70 mg / dL were used for the study. Next, intestinal injections of either the coated systems described in Example 2.1 or an insulin solution in the same dispersion medium (negative control) at a dose of 50 IU / Kg (n = 6) were applied. Blood glucose levels were monitored every hour until the end of the study.
    [0244] In the case of diabetic rats (T1DM), the rats were diabetized 7 days before the experiment. For this, they were fasting overnight with free access to water, after which they were given an intraperitoneal (ip) injection of STZ 60 mg / kg in sodium citrate buffer (50 mM, pH 4.5), prepared at the time and used immediately. The diabetization procedure was applied a second time in animals that had normoglycemic values (<150 mg / dL) after 48 hours. The rats were maintained on a standard diet for 7 days with daily monitoring of the general condition, weight and blood glucose values by means of a caudal vein sample. Those rats with glucose values above 500 mg / dL were given an sc injection of insulin 1 IU / Kg to prevent the death of the animals due to hyperglycemia, except the day before the experiment. Before the experiment, the rats were fasted overnight (12h) and baseline glucose levels were analyzed by sampling the caudal vein and measuring in a glucometer. Those animals with initial values greater than 250 mg / dL were considered diabetic and used in the study, while those with values below 250 mg / dL or above 599 mg / dL (maximum glucometer limit) were discarded. Next, the animals were anesthetized with 2% isoflurane gas (oxygen flow of 0.2 L / min), an incision was opened in the abdominal cavity to locate the intestine and intra-intestinal injection of the nanocomplexes described in example 2.1 or a solution of free insulin in the same dispersion medium (negative control) at a dose of 50 IU / Kg (n = 8). Next, the animals were sutured and allowed to wake up, after which they were monitored for blood glucose values every hour and until the end of the study.
    [0245]
    [0246] The results obtained in the normoglycemic model (see figure 12) show a modest but significant reduction in glucose levels of up to 42% based on the initial values, maintained up to 6h. In this way it was confirmed that the nanosystem-associated insulin was internalized and reached the blood circulation in sufficient quantity to exert a prolonged decrease in glucose levels.
    [0247]
    [0248] In the case of the diabetic rat model (see figure 13), a significant decrease in glycemia of up to 80% was obtained with respect to the initial values, with clear differences compared to the negative control, and reaching at least 8 hours of effect. In conclusion, the results offered a much clearer effect compared to the normoglycemic rat model, confirming in both models the absorption of insulin from the coated nanocomplexes after intestinal administration.
    [0249] Example 10. Scaling study
    [0250]
    [0251] The formulation prepared in Example 2.1 has been successfully scaled from 2 mL production volumes to 20 and 200 mL volumes showing similar properties after characterization.
    [0252]
    [0253] The properties of the lyophilized formulation were maintained for 2 months of storage.
    [0254]
    [0255] Example 11. Preparation of nanocomplexes with tRNA-Zn.
    [0256]
    [0257] To prepare tRNA-zinc nanocomplexes, 500 ^ L of a 0.01N tRNA solution at 0.1 ^ g / mL concentration were prepared, to which a solution of zinc acetate 20 mg / mL in corresponding volume was added at a 5: 1 or 10: 1 load ratio (moles of zinc: nucleotide). The mixture was kept under magnetic stirring (300 rpm) and 0.1N Na0H was added until neutral pH was reached, after which it was incubated for 3h at 4 ° C. The mixture was then kept at room temperature for 4 min to allow it to temper, vortexed for 10s, and its size and zeta potential (ZP) was measured.
    [0258]
    [0259]
    [0260]
    [0261]
    [0262] Example 12. Preparation of nanocomplexes with negative charge tRNA-Fe coated with different positively charged molecules.
    [0263]
    [0264] A solution of tRNA in water (free of RNases) was prepared at a concentration of 0.1 mg / mL and a solution of ferric chloride hexahydrate in 100 mM acetate buffer (pH 6) at a concentration of 0.246 mg / mL. Next, the ferric chloride solution was dripped onto the tRNA solution under magnetic stirring (1: 1), and kept under stirring. The resulting nanocomplexes are characterized by measuring their size and zeta potential (ZP) and the association of RNA by agarose gel electrophoresis (1% w / v, 100V, 20 min).
    [0265]
    [0266] To coat the nanocomplexes, the formulation was dripped onto a positively charged polymer coating solution (0.1 mg / mL polyarginine (pArg) or 1 mg / mL chitosan) under stirring (1: 1), and kept under stirring magnetic
    [0267]
    [0268] The resulting coated nanocomplexes were characterized by measuring their size and zeta potential (ZP) and the association of RNA by agarose gel electrophoresis (1% w / v, 100V, 20 min).
    [0269]
    [0270]
    [0271]
    [0272]
    [0273]
    [0274]
    [0275]
    [0276] Example 13. Preparation of nanocomplexes with positively charged tRNA-Fe coated with different negatively charged molecules.
    [0277]
    [0278] A solution of tRNA in water (free of RNases) was prepared at a concentration of 0.1 mg / mL and a solution of ferric chloride hexahydrate in a 100 mM acetate buffer (pH 6) at a concentration of 2,216 mg / mL. The tRNA solution was then dripped onto the ferric chloride solution under magnetic stirring (1: 1), and kept under stirring. The systems were characterized measuring its size and zeta potential (ZP) and the association of RNA by agarose gel electrophoresis (1% w / v, 100V, 20 min)
    [0279]
    [0280] To coat the nanocomplexes, the formulation was dripped onto a polymer solution with a negatively charged coating (2 mg / mL hyaluronic acid (HA), chondroitin sulfate (CS), polyethylene glycol copolymer and polyglutamic acid (PEG (5k) -PGA (10) or polysalic acid (PSA)) under stirring (1: 1), and kept under magnetic stirring.The resulting systems were characterized by measuring their size and zeta potential (ZP) and the association of RNA by agarose gel electrophoresis (1% w / v, 100V, 20 min).
    [0281]
    [0282]
    [0283]
    [0284]
    [0285]
    权利要求:
    Claims (27)
    [1]
    1. Nanosystem characterized by being constituted by:
    (i) a complex comprising a) a polyamino acid or a polynucleotide characterized by having a negative charge and b) a metal, and
    (ii) a cover comprising at least one charged molecule,
    with the proviso that the net charge of the complex (i) is opposite to the charge of the shell molecule.
    [2]
    2. Nanosystem according to claim 1 wherein the polyamino acid is selected from a peptide, a protein and a monoclonal antibody.
    [3]
    3. Nanosystem according to claims 1-2, wherein the metal has a valence equal to or greater than 2.
    [4]
    4. Nanosystem according to any of claims 1-3, wherein the metal is selected from zinc, iron, calcium, magnesium, copper, chromium, manganese, nickel, ruthenium, barium, strontium, bismuth, platinum, zirconium, germanium, titanium , molybdenum, cobalt and tin.
    [5]
    5. Nanosystem according to any of the preceding claims, further characterized by having a cubic or orthopedic shape.
    [6]
    6. Nanosystem according to any of claims 2-5, wherein the protein is selected from hormones, growth factors, cytokines, hematopoietic factors, enzymes, receptors, antigenic peptides, antigenic proteins, coagulation factors, adhesion molecules and cellular receptors .
    [7]
    7. Nanosystem according to any of claims 2-5, wherein the monoclonal antibody is selected from elotuzumab, panitumumab, trastuzumab, bevacizumab, adalimumab, ritusimumab and anti-TNF antibodies.
    [8]
    8. Nanosystem according to any one of the preceding claims, wherein the polynucleotide is selected from transferring RNA, interfering RNA, silencing RNA, DNA, antisense oligonucleotides, micro-RNA and double stranded RNA.
    [9]
    9. Nanosystem according to any of the preceding claims, wherein the molecule with charge of the shell is selected from among ionizable surfactants or with a charged functional group, ionizable polymers or with a charged functional group, ionizable polysaccharides, ionizable polyamino acids, homo and co-polymers containing ionizable residues, and combinations thereof.
    [10]
    10. Nanosystem according to any of the preceding claims, wherein the shell may further comprise non-ionizable and uncharged molecules.
    [11]
    11. Nanosystem according to any of the preceding claims, wherein the non-ionizable and non-chargeable molecules are selected from non-ionic surfactants, non-charged polymers, polyols, and combinations thereof.
    [12]
    12. Nanosystem according to any of the preceding claims, which has an average size of less than 200 nm, preferably less than 170 nm, more preferably less than 120 nm.
    [13]
    13. Nanosystem according to any of claims 2-12, wherein the protein is insulin and the metal is zinc.
    [14]
    14. Nanosystem according to any of claims 2-12, wherein the polynucleotide is a tRNA and the metal is iron.
    [15]
    15. Nanosystem according to claim 1, consisting of insulin, zinc, polyethylene glycol stearate and ethyl lauroyl arginate and having an average diameter of less than 120 nm.
    [16]
    16. Nanosystem according to claim 1, consisting of insulin, zinc, polyethylene glycol stearate and cetyltrimethylammonium bromide and having an average diameter of less than 120 nm.
    [17]
    17. Nanosystem according to any of the preceding claims, characterized in that it is in lyophilized form.
    [18]
    18. Method for obtaining a nanosystem according to claim 1, comprising adding a solution of at least one ionizable or charged molecule, to a complex comprising a) a polyamino acid or a polynucleotide characterized by having a negative charge, and b) a metal,
    with the proviso that the net charge of the complex is opposite to the charge of the ionizable or charged molecule.
    [19]
    19. Method for preparing the complex described in claim 18, which comprises
    a) mixing a polyamino acid or a polynucleotide, and a metal,
    b) adjust the pH of the mixture to a pH equal to or greater than the isoelectric point of the polyamino acid,
    c) incubate the mixture at a temperature between 0 and 8 ° C,
    d) disperse the mixture in a buffered medium.
    [20]
    20. Complex comprising a protein and a transition metal and has an average diameter of less than 200 nm, preferably less than 170 nm, more preferably less than 120 nm.
    [21]
    21. Complex according to claim 20, consisting of insulin and zinc and having an average diameter of less than 120 nm.
    [22]
    22. Complex obtainable by the method described in claim 19.
    [23]
    23. Pharmaceutical composition comprising a nanosystem according to any of claims 1-17 or a complex according to any of claims 20-22, and pharmaceutically acceptable excipients.
    [24]
    24. Nanosystem according to any of claims 1-17, or pharmaceutical composition according to claim 23, for use in medicine.
    [25]
    25. Nanosystem or pharmaceutical composition according to claim 24, for use in the treatment and / or prevention of diseases associated with hormonal, metabolic or immunological disorders, inflammatory processes, degenerative diseases and tumors.
    [26]
    26. Nanosystem or pharmaceutical composition according to claim 24 or 25, for use in the treatment of diabetes.
    [27]
    27. Nanosystem or pharmaceutical composition according to any of claims 24 26, for oral administration.
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    ES2738173B2|2020-12-03|
    引用文献:
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    IN2012DE00305A|2012-02-03|2015-04-10|Jamia Hamdard|
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