WO2022136393A1

WO2022136393A1 - Zein nanoparticles for use in hyperglycemic conditions

Info

Publication number: WO2022136393A1
Application number: PCT/EP2021/087031
Authority: WO
Inventors: Carlos Javier GONZÁLEZ NAVARRO; Juan Manuel Irache Garreta; Ana Luisa MARTÍNEZ-LÓPEZ; Cristian REBOREDO FUENTES; José Luis VIZMANOS PÉREZ
Original assignee: Universidad De Navarra
Priority date: 2020-12-23
Filing date: 2021-12-21
Publication date: 2022-06-30
Also published as: EP4267114A1

Abstract

The invention relates to the use of nanoparticles comprising a zein matrix and a basic amino acid, and compositions comprising said nanoparticles, in the treatment and/or prevention of conditions characterized by increased glucose levels and/or fat accumulation, in particular to the medical use in the treatment and/or prevention of diabetes and to the non-therapeutic use in extending the lifespan of an organism.

Description

ZEIN NANOPARTICLES FOR USE IN HYPERGLYCEMIC CONDITIONS

FIELD OF THE INVENTION

The invention relates to the use of zein nanoparticles in the treatment and/or prevention of conditions characterized by increased glucose levels and/or fat accumulation, in particular to the medical use in the treatment and/or prevention of diabetes, obesity or metabolic syndrome, and to the non-therapeutic use in extending the lifespan of an organism.

BACKGROUND OF THE INVENTION

Increased glucose levels and/or fat accumulation are involved in a variety of therapeutic and non-therapeutic conditions, such as diabetes and lifespan.

Aging is a biological process that curses with physiological and molecular deterioration [V. Piskovatska et al., “Health benefits of anti-aging drugs” in Subcellular Biochemistry, vol. 91, Springer New York, 2019, pp. 339-392; and E. J. E. Kim and S. J. V. Lee, “Recent progresses on anti-aging compounds and their targets in Caenorhabditis elegans” Translational Medicine of Aging, vol. 3. KeAi Communications Co., pp. 121-124, Jan. 2019], Slowing down or delaying this time-associated degeneration would mean an extension of the lifespan and/or an improvement of the quality of life of people [B. Klimova et al., Curr. Med. Chem., 2018, 25(17), 1946-1953], This context generates a market niche that has been increasing during the last decades and, that is expected to keep growing. By 2019, the global anti-ageing market reached a value of 54.2 billion USS. Moreover, this market is predicted to reach values of 85.1 billion USS worldwide by 2025.

Several anti-ageing compounds, such as spermidine, rapamycin, metformin, or resveratrol, have demonstrated the capability of delaying the consequences of ageing [T. W. Snell et al., Biogerontology, 2016, 77(5-6), 907-920], Among the different mechanisms involved in anti-ageing drugs, those that are most well-known are the antioxidants, calorie restriction mimetics, inductors of autophagy, senolytics, immunomodulators, telomerase activators and epigenetic drugs [A. M. Vaiserman et al., Ageing Res. Rev., 2016, 31, 9-35],

Calorie restriction (CR), also known as a dietary restriction (DR), is the most reliable antiageing treatment so far [A. M. Vaiserman et al., Ageing Res. Rev., 2016, 31, 9-35; and S. H. Lee and K. J. Min, BMB Reports, 2013, 46(A), 181-187], It refers to a state at which animals receive a reduced intake of calories but with all the essential nutrients being provided. So, compared to a healthy diet, a 30 to 70% calorie reduction is achieved without malnutrition. It has been reported in several studies that a 30-60% reduction in the food intake, compared to ad libitum, led to an increase in the lifespan of several animal species [A. M. Vaiserman et al., Ageing Res. Rev., 2016, 31, 9-35; and F. Madeo et al., Nat. Rev. Drug Discov., 2014, 73(10), 727-740], Since calorie restriction mimetics comprise a wide variety of different interventions, they also target different pathways. Among these pathways, they can act by reducing the circulating levels of glucose and insulin, which are closely related to the “glycation theory of ageing” [A. M. Vaiserman et al., Ageing Res. Rev., 2016, 31, 9-35; R. Weindruch et al., J. Gerontol. A. Biol. Sci. Med. Sci., 2001, 56(spec 1), 20-33; and M. A. Lane, G. S. Roth, and D. K. Ingram, Methods Mol. Biol., 2007, 371, 143-149],

Thus, new strategies for extending the lifespan could be aimed at providing agents which reduce glucose levels or fat accumulation.

Increased glucose levels and fat accumulation are also involved in diabetes.

Diabetes is a global epidemic which is expected to grow up to 642 million patients by 2030.

Diabetes is a chronic, metabolic disease characterized by elevated levels of blood glucose, which leads over time to serious damage to the heart, blood vessels, eyes, kidneys and nerves. Type 1 diabetes results from the pancreas’ failure to produce enough insulin. The most common is type 2 diabetes, usually in adults, which occurs when the body becomes resistant to insulin or there is not enough insulin produced. This type of diabetes is largely the result of excess body weight and physical inactivity. Gestational diabetes occurs when pregnant women without a previous history of diabetes develop high blood glucose levels.

Blood glucose levels are used for the diagnosis of diabetes, according to the following criteria: fasting plasma glucose level > 7.0 mmol/L (126 mg/dL) or plasma glucose > 11.1 mmol/L (200 mg/dL) two hours after a 75 g oral glucose load as in a glucose tolerance test. People with fasting glucose levels from 6.1 to 6.9 mmol/L (110 to 125 mg/dL) are considered to have impaired fasting glucose. People with plasma glucose at or above 7.8 mmol/L (140 mg/dL), but not over 11.1 mmol/L (200 mg/dL), two hours after a 75 gram oral glucose load are considered to have impaired glucose tolerance. These are considered prediabetic states.

Diabetes management concentrates on keeping blood sugar levels as close to normal, without causing low blood sugar. This can usually be accomplished with dietary changes, exercise, weight loss, and use of appropriate medications.

Most medications used to treat diabetes act by lowering blood sugar levels through different mechanisms. These medications are known as hypoglycemic agents, antidiabetic agents, or antihyperglycemic agents. Among these, bisguanides, thiazolidinediones, and LYN kinease activators are insulin sensitizers, which address the problem of insulin resistance. Biguanides, such as metformin, phenformin and buformin, reduce hepatic glucose output and increase uptake of glucose by the periphery. Thiazolidinediones, such as rosiglitazone, pioglitazone and troglitazone, bind to PPARy which is involved in transcription of genes regulating glucose and fat metabolism. LYN kinase activators, such as tolimidone, potentiate insulin signaling. Another group of medicaments are the secretagogues, such as sulfonylureas (tolbutamide, acetohexamide, tolazamide, chlorpropamide, glipizide, glibenclamide, glimepiride, gliclazide, glycopyramide, gliquidone) and meglitinides (repaglinide and nateglinide) which increase insulin output from the pancreas. Another group of antidiabetic drugs are the alpha-clucosidease inhibitors, such as acarbose, miglitol and voglibose, which slow the digestion of starch in the small intestine, so that glucose from the starch of a meal enters the bloodstream more slowly, and can be matched more effectively by an impaired insulin response or sensitivity.

Obesity is a complex multifactorial disease defined by excessive adiposity that can impair health and it is also one of the key risk factors for many diseases such as coronary heart disease, hypertension and stroke, certain types of cancer, type 2 diabetes, etc. In fact, obesity is the most important modifiable risk factor for type 2 diabetes. Body mass index (BMI) is a surrogate marker of adiposity calculated as weight (kg)/height² (m²). The BMI categories for defining obesity vary by age and sex in infants, children and adolescents. For adults, obesity is defined by a BMI greater than or equal to 30 kg/m² A BMI ranging from 25 to 29.99 kg/m² is also associated with increase disease risk and is referred to as pre-obesity or overweight. In the case of children aged between 5-19 years, overweight is BMI-for-age greater than 1 standard deviation above the WHO Growth Reference median and obesity is greater than 2 standard deviations above the WHO Growth Reference median. Finally, for children under 5 years of age, overweight is weight-for- height greater than 2 standard deviations above WHO Child Growth Standards median whereas obesity is weight-for-height greater than 3 standard deviations above the WHO Child Growth Standards median. The main treatment for obesity consists of weight loss via calorie restricted dieting and physical exercise. The most effective treatment for obesity is bariatric surgery. Five medications have evidence for long-term use orlistat, lorcaserin, liraglutide, phentermine-topiramate, and naltrexone-bupropion. However, these drugs may have undesired side effects.

Numerous studies have implicated abdominal obesity as a major risk factor for insulin resistance, type-2 diabetes mellitus, cardiovascular disease, stroke, metabolic syndrome and death [Huffman et. al, 2009, Biochimica et biophysica acta, 1790(10), 1117-1123; Liu et al., J Med Internet Res. 2021, 23(8):e24017; Wood et al., PLoS ONE, 2021, 16(11): e0258545].

According to the new IDF definition, a person is defined as having metabolic syndrome if having central obesity (generally defined as waist circumference with ethnicity specific values but, if BMI is >30kg/m², central obesity can be assumed and waist circumference does not need to be measured) plus any two of the following four factors: raised triglycerides > 150 mg/dL (1.7 mmol/L) or specific treatment for this lipid abnormality; reduced HDL cholesterol < 40 mg/dL (1.03 mmol/L) in males < 50 mg/dL (1.29 mmol/L) in females or specific treatment for this lipid abnormality; raised blood pressure systolic BP > 130 or diastolic BP > 85 mm Hg or treatment of previously diagnosed hypertension; raised fasting plasma glucose (FPG) > 100 mg/dL (5.6 mmol/L), or previously diagnosed type 2 diabetes (if FPG is above 5.6 mmol/L or 100 mg/dL, OGTT is strongly recommended but is not necessary to define presence of the syndrome). Metabolic syndrome is associated with the risk of developing cardiovascular disease and type-2 diabetes. Generally, the individual disorders that compose the metabolic syndrome are treated separately. Diuretics and ACE inhibitors may be used to treat hypertension. Various cholesterol medications may be useful if LDL cholesterol, triglycerides, and/or HDL cholesterol is abnormal. Dietary carbohydrate restriction reduces blood glucose levels, contributes to weight loss, and reduces the use of several medications that may be prescribed for metabolic syndrome.

Nanoparticles are extensively used as carriers for oral drug delivery purposes. In particular, nanoparticles based on proteins show advantages for pharmaceutical applications due to their inherent biodegradabilidy, in vivo biocompatibility and, in general, lower cost than other materials such as polymers and lipids. Zein nanoparticles have demonstrated an important capability to extend the plasma levels of different drugs and other bioactive compounds by facilitating its absorption and improving their oral biovailability. WO 2012/007628 Al discloses the use of zein nanoparticles to encapsulate biologically active compounds. Furthermore, zein nanoparticles have been disclosed [D. Lucio et al., Eur. J. Pharm. Biopharm., 2017, 121, 104-112] as suitable oral carriers for glibenclamide, which is one of the drugs most commonly used for the treatment of diabetes.

Despite the above mentioned therapeutic strategies for the treatment of diabetes, metabolic syndrome, obesity, and the non-therapeutic approaches to extend the lifespan of an organism, there remains a need for further agents to regulate increased glucose levels and/or fat accumulation that can be used for these purposes. BRIEF DESCRIPTION OF THE INVENTION

The inventors have surprisingly found that nanoparticles comprising a zein matrix and a basic amino acid, but which do not comprise any antidiabetic drug, such as glibenclamide, i.e. unloaded or empty nanoparticles, induced a significant reduction in blood glucose levels and fat accumulation in vivo. These nanoparticles were also capable of delaying ageing and of increasing the lifespan in animal models. These nanoparticles also decreased significantly the accumulation of fat in obese animals, including visceral fat. This effect was accompanied of a significant decrease of both serum and hepatic triacylglycerides (TAG) level, and marked decrease levels of MCP-1 (Monocyte Chemoattractant Protein-1), a key mediator of low-grade inflammation widely associated with insulin resistance and recruitment of macrophages in adipose tissue. Thus, these nanoparticles are suitable for the treatment and/or prevention of conditions related to high blood glucose levels and fat accumulation, such as diabetes, obesity and metabolic syndrome, and in extending the lifespan. The results obtained for the unloaded zein nanoparticles are particularly unexpected since free zein, i.e. where the protein does not form part of a nanoparticle, was not capable of producing the observed effects.

Thus, in a first aspect, the invention relates to a nanoparticle comprising a zein matrix and a basic amino acid for use in the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation (such as diabetes, obesity and metabolic syndrome), wherein the nanoparticles does not comprise any biologically active ingredient.

In a second aspect, the invention relates to a composition comprising at least one nanoparticle as defined in the first aspect and a pharmaceutically acceptable excipient for use in the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation (such as diabetes, obesity and metabolic syndrome).

In a third aspect, the invention relates to the use of a nanoparticles as defined in the first aspect in the manufacture of a medicament for the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation (such as diabetes, obesity and metabolic syndrome).

In a fourth aspect, the invention relates to the use of a composition comprising at least one nanoparticles as defined in the first aspect and a pharmaceutically acceptable excipient in the manufacture of a medicament for the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation (such as diabetes, obesity and metabolic syndrome).

In a fifth aspect, the invention relates to a method of treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation (such as diabetes, obesity and metabolic syndrome) comprising administering to a subject in need thereof a nanoparticle as defined in the first aspect.

In a sixth aspect, the invention relates to a method of treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation (such as diabetes, obesity and metabolic syndrome) comprising administering to a subject in need thereof a composition comprising at least one nanoparticle as defined in the first aspect and a pharmaceutically acceptable excipient

In a seventh aspect, the invention relates to the non-therapeutic use of a nanoparticle as defined in the first aspect for extending the lifespan of an organism.

In an eight aspect, the invention relates to the non-therapeutic use of a composition comprising at least a nanoparticle as defined in the first aspect and a carrier acceptable in food or nutraceutics, for extending the lifespan of an organism.

In a ninth aspect, the invention relates to a nanoparticle comprising a zein matrix, a basic amino acid and polyethylene glycol or a derivative thereof for use in the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation (such as diabetes, obesity and metabolic syndrome). In a further aspect, the invention relates to a composition comprising at least one nanoparticle as defined in the ninth aspect and a pharmaceutically acceptable excipient for use in the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation (such as diabetes, obesity and metabolic syndrome).

In a further aspect, the invention relates to the use of a nanoparticles as defined in the ninth aspect in the manufacture of a medicament for the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation (such as diabetes, obesity and metabolic syndrome).

In a further aspect, the invention relates to the use of a composition comprising at least one nanoparticles as defined in the ninth aspect and a pharmaceutically acceptable excipient in the manufacture of a medicament for the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation (such as diabetes, obesity and metabolic syndrome).

In a further aspect, the invention relates to a method of treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation (such as diabetes, obesity and metabolic syndrome) comprising administering to a subject in need thereof a nanoparticle as defined in the ninth aspect.

In a further aspect, the invention relates to a method of treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation (such as diabetes, obesity and metabolic syndrome) comprising administering to a subject in need thereof a composition comprising at least one nanoparticle as defined in the ninth aspect and a pharmaceutically acceptable excipient.

In a further aspect, the invention relates to the non-therapeutic use of a nanoparticle as defined in the ninth aspect for extending the lifespan of an organism. In a further aspect, the invention relates to the non-therapeutic use of a composition comprising at least a nanoparticle as defined in the ninth aspect and a carrier acceptable in food or nutraceutics, for extending the lifespan of an organism.

DESCRIPTION OF THE FIGURES

Figure 1 shows the Scanning Electron Microscopy (SEM) images of spray-dried nanoparticles. A: naked nanoparticles (NP); B: PEG-coated nanoparticles at a PEG-to- zein ratio of 0.5 (NP50). In the white square, it is shown a magnification of a section of each picture.

Figure 2 shows the surface hydrophobicity of the different formulations. Values are normalized to the hydrophobicity of naked nanoparticles (NP). Data expressed as mean ± SD (n=3). *: p < 0.05; **: p < 0.01.

Figure 3 is a comparison of the capability of nanoparticles (bare and coated with PEG at different ratios) to diffuse through pig intestinal mucus. Values are normalized to the diffusion of bare nanoparticles (NP diffusion in intestinal mucus = 1). Data expressed as mean ± SD (n=3). **: p < 0.01.

Figure 4 shows the effects of zein nanoparticle (NP) supplementation on glucose content of C. elegans. Data represents the mean ± SD (n = 250 worms). *** p < 0.001 compared to control.

Figure 5 shows the effect of the different formulations over the fat accumulation of C. elegans. NGM: nematode growth medium (negative control); Orl: orlistat (positive control); Free zein: zein in purified water; NP: naked empty zein nanoparticles; NP50: empty zein nanoparticles coated with PEG at a PEG-to-zein ratio of 0.5. Data expressed as mean ± SD (n=3 wells with at least 25 worms/well). *: p < 0.05; **: p < 0.01 compared to control. #: p < 0.05; ##: p < 0.01 compared to free zein. Figure 6 shows the effects of zein nanoparticles (NP) treatment on intracellular ROS levels in wild-type N2 strain C. elegans. Data represents three independent experiments, values are mean ± SD (n = 100). ***p < 0.001 compared to control. Control: untreated worms; NP (LI worms treated from LI larvae stage until 18^th-day adulthood, NP (L4 ->): worms treated from 1-day adult until 18^th-day adulthood.

Figure 7 shows the effects of zein nanoparticles (NP) treatment on the expression of genes in wild-type N2 strain C. elegans. The mRNA expression levels of daf-16 and skn-1 as determined by qRT-PCR and normalized to expression of pmp-1. Data represents three independent experiments, values are mean ±S.D. *p<0.05, ***p<0.001 versus respective controls. Control: untreated worms; NP: worms treated from LI larvae stage until 9^th-day adulthood, NP after: worms treated from 1-day adult until 9^th-day adulthood.

Figure 8 shows the effect of the different treatments over the blood glucose levels of healthy male Wistar rats. Control: receiving water; Free zein: suspension of pure zein in water; NP: naked zein nanoparticles; NP50: zein nanoparticles coated with PEG at a PEG- to-zein ratio of 0.5. All the treatments administered at a dose of 50 mg/kg. Data expressed as mean ± SD (n = 6). *: p < 0.05; **: p < 0.01 compared to control. #: p < 0.05; ##: p < 0.01 compared to free zein, f : p < 0.05; f f : p < 0.01 compared to NP.

Figure 9 shows the effect of the oral administration of zein nanoparticles on the blood levels of insulin (A) and GLP-1 (B) in healthy male Wistar rats. Control: animals receiving water. NP: naked zein nanoparticles; NP50: zein nanoparticles coated with PEG at a PEG-to-zein ratio of 0.5. All the treatments administered at a dose of 50 mg/kg. Data expressed as mean ± SD (n = 6). *: p < 0.05; **: p < 0.01 compared to control. #: p < 0.05.

Figure 10 shows the effect of the different treatments over the blood glucose levels of healthy male Wistar rats after an intraperitoneal injection of glucose (2 g/kg). All the treatments were administered orally at a dose of 50 mg/kg. Control: animals receiving water; Free zein: zein in purified water; NP50: empty zein nanoparticles coated with PEG at a PEG-to-zein ratio of 0.5. All the treatments administered at a dose of 50 mg/kg. Data expressed as mean ± SD (n = 6). *: p < 0.05; **: p < 0.01 compared to control. #: p < 0.05 compared to NP.

Figure 11 shows the Kaplan-Meier survival curves representing the percentage of the SAMP8 mice alive. Control mice received water. NP mice received a suspension of zein nanoparticles. Both groups started receiving the treatment at 7 months of age (n = 10 mice per group).

Figure 12 represents the analysis of the body composition of the obese rats after 6 weeks of administration. (A) Proportion of fat mass relative to the body weight of each rat; (B) Ratio between the amount of fat mass and lean mass (fat mass/lean mass of each individual rat) at the end of the experiment. SC: animals fed with standard control diet (2014 Teklad global 14% protein rodent maintenance diets); HFS: animals fed with high fat/high sucrose (HFS) diet (D12451 - rodent diet); HFS-NP: animals fed with HFS supplemented with zein nanoparticles. Individual values are shown as dark symbols, the thick horizontal lines represent the mean value of each group, and the thin and shorter horizontal lines represent the standard deviation (SD) of the mean of each group, n = 12 per group. ** p < 0.01; ***, p < 0.001 for the indicated comparison.

Figure 13 represents the effect of supplementation of HFS-fed animals with zein nanoparticles on the retroperitoneal (A), epididymal (B), visceral (C) and subcutaneous (D) fat weight. SC: animals fed with standard control diet (2014 Teklad global 14% protein rodent maintenance diets); HFS: animals fed with high fat/high sucrose (HFS) diet (D12451 - rodent diet); HFS-NP: animals fed with HFS supplemented with zein nanoparticles. Visceral fat corresponds to the sum of mesenteric, epididymal and retroperitoneal fat depots. Data are presented as mean ± S.D., n = 12 per group. * p < 0.05; **, p < 0.01; ***, p < 0.001 for the indicated comparison.

Figure 14 represents the effect of supplementation of HFS-fed animals with zein nanoparticles during 6 weeks on their serum lipid profile. (A) Triglycerides (TAG), (B) HDL-Cholesterol, (C) Total cholesterol and (D) Atherogenic index. SC: animals fed with standard control diet (2014 Teklad global 14% protein rodent maintenance diets); HFS: animals fed with high fat/high sucrose (HFS) diet (D12451 - rodent diet); HFS-NP: animals fed with HFS supplemented with zein nanoparticles. Data are presented as mean± S.D., n = 12 per group. * p < 0.05; **, p < 0.01; ***, p < 0.001 for the indicated comparison.

Figure 15 represents the effect of supplementation of HFS-fed animals with zein nanoparticles on the plasma levels of the cytokine MCP-1 (Monocyte Chemoattractant Protein-1). SC: animals fed with standard control diet (2014 Teklad global 14% protein rodent maintenance diets); HFS: animals fed with high fat/high sucrose (HFS) diet (D12451 - rodent diet); HFS-NP: animals fed with HFS supplemented with zein nanoparticles. Data are presented as mean± SD, n = 12 per group. ** p < 0.01 for the indicated comparison.

Figure 16 shows the effect of supplementation of HFS-fed animals with zein nanoparticles on lipid steatosis. SC: animals fed with standard control diet (2014 Teklad global 14% protein rodent maintenance diets); HFS: animals fed with high fat/high sucrose (HFS) diet (D12451 - rodent diet); HFS-NP: animals fed with HFS supplemented with zein nanoparticles. Data are presented as mean± SD, n = 12 per group. ** p < 0.01; *** p < 0.001 for the indicated comparison.

DETAILED DESCRIPTION OF THE INVENTION

Use of nanoparticles comprising a zein matrix and a basic amino acid and which do not comprise any biologically active ingredient

The first aspect of the present invention is directed to a nanoparticle comprising a zein matrix and a basic amino acid for use in the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation, wherein the nanoparticle does not comprise any biologically active ingredient.

This aspect may also be worded as the use of a nanoparticle comprising a zein matrix and a basic amino acid in the manufacture of a medicament for the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation, wherein the nanoparticle does not comprise any biologically active ingredient.

It may also be worded as a method of treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation comprising the administration of a nanoparticle comprising a zein matrix and a basic amino acid, wherein the nanoparticle does not comprise any biologically active ingredient.

As used herein, “nanoparticle” refers to colloidal systems of the type of spheres or similar shapes with an average size less than 1 micrometer (pm).

The nanoparticles are characterized by having an average particle size less than 1 pm, typically comprised between 1 and 999 nm, preferably between 10 and 900 nm, more preferably between 50 and 500 nm, more preferably between 100 and 450 nm, even more preferably between 140 and 400 nm, even more preferably between 150 and 300 nm, still more preferably from 150 to 250 nm.

As used herein, “average size” refers to the average diameter of the population of nanoparticles which move together in an aqueous medium. The average size of these systems can be measured by standard processes known by the person skilled in the art, and which are described, for example, in the experimental part (see below).

As used herein, the term “zein” includes any globular protein belonging to the group of prolamines; said protein is generally synthesized during the development of the endosperm (nutritive tissue formed in the embryo sac of seed plants and usually forms a food deposit for the embryo of the seeds of various angiosperm plants). Zein can be obtained from any suitable source, although it is preferably obtained from corn. Various methods and techniques for extracting zein from com endosperm are known; commercial zein is generally extracted from corn gluten meal (US 2009/0258050).

The study of zein reveals an extreme variability at the genetic level and, therefore, a complex situation among the different proteins forming part of the group of proteins known as zeins. Native zein is actually a large and heterogeneous family of several groups of proteins which differ in their molecular size, solubility, and charge. More than twenty different zein have been estimated to exist. The analysis of zein extracts by means of high-performance liquid chromatography (HPLC), ion-exchange chromatography, gel exclusion chromatography, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing (IEF), amino acid analysis, and DNA cloning techniques have led to a better understanding of zein proteins.

The analysis of the composition of the amino acids of zein reveals a large amount of leucine, alanine, glutamine, and phenylalanine; however, lysine and tryptophan are absent or, alternatively, are present in very small amounts. The high proportion of non-polar amino acid residues and the exceptional lack of ionic groups are responsible for the hydrophobic nature thereof and for the particular solubility thereof.

The protein bodies of zein are formed by three types of structurally different proteins: alpha-zein (a-zein), gamma-zein (y-zein) [which includes beta zein (0-zein)], and deltazein (6-zein). Said proteins can be classified into four classes (a-zein, 0-zein, y-zein and 6-zein) based on the differences in solubility and sequence.

Zein extracted without reducing agents forms a large multigene family of polypeptides referred to as a-zein. a-zeins, generally the most abundant fraction of native zein, contain about 40 amino acids in the amino terminus which precede a series of 9 or 10 repeated peptides of 20 amino acids. These repeats are believed to be a-helices and wind the protein into a rod-shaped molecule.

The other fractions of zein (0-, y-, and 6-zein) must be extracted using alcohols solutions of alcohols containing reducing agents to break the disulfide bonds. By way of illustration, mercaptoethanol is used for laboratory extraction. 0-, y-, and 6-zeins show no sequence homology with a-zein. y-Zein is soluble in both aqueous and alcoholic solvents in reducing conditions. Each of the y-zeins has a unique N-terminus sequence. By way of example, in the 50 kDa y-zein, this region is 136 amino acids long and it is very rich in histidine. The 27 kDa y-zein has a series of eight tandem repeats of a hexapeptide which produce 11 amino acids after the amino terminus. The first eight amino acids of the 16 kDa y-zein protein are identical to those of the 27 kDa y-zein, but the 16 kDa y-zein has three degenerate versions of prolinerich repeats. y-Zein normally represents between 10 and 15% of the total of the zeins.

P-Zein, which is related to y-zein, includes a methionine-rich 17 kDa polypeptide and constitutes up to 10% of the total zein. Approximately the last 140 amino acids of P- and y-zeins are 85% identical. P-Zein has no repetitive peptides and seems to mostly consist of P-sheets and turn conformation.

6-zein is a 10 kDa protein and is a minor fraction of zein. 6-zeins are the most hydrophobic of the group, contain no repetitive peptides, and are exceptionally methionine- and cysteine-rich.

Zein has been considered as a “Generally Recognized as Safe” (GRAS) product by the Food and Drug Administration (United States) since 1985 [CAS (Chemical Abstract Service) number: 9010-66-6],

In the present invention, the source or the grade of zein is not limited to a single zein and, in fact, any zein can be used to put the present invention into practice. By way of illustration, the commercial zeins which can be used in the present invention include, but are not limited to, the zein supplied by Sigma-Aldrich (product number Z 3625); Wako Puras Chemical Industries (product numbers 261-00015, 264-01281 and 260-01283); Spectrum Chemical (product numbers Z1131 and ZE105); ScienceLab units SLZ1150; SJZ Chem-Pharma Company (product name ZEIN (GLIDZIN); Arco Organics (catalog numbers 17931-0000, 17931-1000, and 17931-5000); and Freeman Industries, zein regular grade F4000, zein regular grade F4400, zein special grade F6000, etc. In a particular embodiment, the commercial zein supplied by Sigma- Aldrich (product number Z 3625), obtained from corn, is used. As used herein, the term “zein” includes both native zein and modified zein. The term “modified zein” includes any zein having an amino acid sequence which is normally not naturally-occurring, but which behave similarly to authentic zeins and which are soluble in alcohol. Amino acid substitutions, especially those which do not substantially modify the hydrophobicity, may be introduced. By way of illustration, amino acid substitutions can be performed within the repeated sections, or a single amino acid can be substituted, and substitutions can also be performed in the segments connecting the domains of repeated sequences. Insertions and substitutions can also be introduced in the carboxyl terminus and the amino terminus of the zein molecule. Additionally, deletions can be performed in the amino acid sequence provided that the resulting protein is functionally equivalent to zein, i.e., that it maintains its properties.

Virtually any zein can form the matrix of the nanoparticle; nevertheless, in a particular embodiment, said zein is a zein from corn, such as the zein supplied by Sigma-Aldrich (product number Z 3625).

In “matrix” nanoparticle systems, also known as nanospheres, the polymer or macromolecule, i.e. zein, adopts a solid structure (matricial conformation).

The nanoparticles comprise a basic amino acid. As used herein, a “basic amino acid” refers to an organic molecule containing an amino group (-NH2) and a carboxyl group (- COOH) and positive charge. The basic amino acid of the nanoparticles for use of the invention refers to a free amino acid that is not covalently bonded to any other atom, i.e. it does not form part of any oligomeric, peptidic or other molecular structures, but refers to the amino acid as being an independent molecule. Said basic amino acid is preferably a basic alpha-amino acid such as lysine, arginine, histidine or mixtures thereof. Preferably, the basic amino acid present in the nanoparticles of the invention is lysine.

In the nanoparticles, the weight ratio of zein to basic amino acid is in the range of from 0.01 : 1 to 50: 1, preferably from 0.5: 1 to 25: 1, more preferably from 1 : 1 to 20: 1, more preferably from 5: 1 to 15: 1, even more preferably from 6: 1 to 7: 1, still more preferably about 6.7: 1. As shown in the examples, the nanoparticles are able to diffuse through the mucus layer, improving the absorption through the oral mucosa.

Surprisingly, as shown in the examples, the biological activity of the nanoparticles described herein is achieved in the absence of any pharmaceutically active ingredients used for the treatment of diabetes. Thus, the nanoparticles do not comprise any biologically active ingredient.

The term “biologically active ingredient” or “drug” refers to any compound having therapeutic activity. Non-limiting illustrative examples of biologically active ingredients include antidiabetic drugs, statins, ACE inhibitors, angiotensin receptor blockers, B- receptor blockers, a blockers, calcium channel blockers, diuretics, aldosterone inhibitors, anticoagulants, heparin, antiplatelet drugs, fibrinolytics, anti-hemophilic factors, haemostatic drugs, antimicrobial agents, antipyretics, analgesics, antimalarial drugs, antibiotics, antiseptics, hormone replacements, antacids, reflux suppressants, antiflatulents, antidopaminergics, proton pump inhibitors, FL-receptor antagonists, cytoprotectants, prostaglandin analoges, laxatives, antispasmodics, antidiarrheals, bile acid sequestrants, opioids, cardiac glycosides, antiarrhythmics, nitrate, antianginals, vasoconstrictors, vasodilators, psychedelics, hypnotics, anaesthetics, antipsychotics, eugeroics, antidepressants, antiemetics, anticonvulsants/antiepileptics, anxiolytics, barbiturates, movement disorder drugs, stimulants, benzodiazepines, cyclopyrrolones, dopamine antagonists, antihistamines, cholinergics, anticholinergics, emetics, cannabinoids, 5-HT (serotonin) antagonists, antifungals, alkalinizing agents, quinolones, cholinergics, anticholinergics, antispasmodics, 5-alpha reductase inhibitor, vaccines, immunoglobulins, immunosuppressants, interferons, monoclonal antibodies, etc.

Examples of antidiabetic drugs are sulfonylureas (such as, tolbutamide, acetohexamide, tolazamide, chlorpropamide, glipizide, glimepiride, gliclazide, glycopyramide, gliquidone), bisguanides (such as metformin, phenformin and buformin), thiazolidinediones (such as rosiglitazone, pioglitazone and troglitazone), LYN kinease activators LYN (such as tolimidone), meglitinides (such as repaglinide and nateglinide), alpha-glucosidease inhibitors (such as acarbose, miglitol and voglibose), GLP-1 agonists (such as exenatide, dulaglutide, semaglutide, liraglutide and lixisenatide), DPP-4 inhibitors (such as saxagliptin, sitagliptin, vildagliptin, linagliptin and alogliptin), dual PPAR agonists (such as sarolitazar), amylin analogues (such as pramlintide), SGLT-2 inhibitors (such as canagliflozin, dapagliflozin, empagliflozin, ertugliglozin, ipragliflozin, luseogliflozin, remogliflozin etabonate, sergliflozin etabonate, sotagliflozin, tofogliflozin), and insulin and analogues.

Examples of antidiabetic drugs of phytochemical origin which are commonly used for the treatment and/or prevention of diabetes, such as quercetin, berberina, rutin, fisetin, kaempferol, isorhamnetin, morin, hesperidin, naringenin, eriodictyol, apigenin, luteolin, tangerein, chrysin, wogonin, diosmin, baicalein, genistein, daidzein, myrecelin, curcumin, resveratrol, cyaniding, delphinidin, pelargonidin, glycan (Gymnema sylvestre), 1-DOPA (Mucana Prune ns). vasicine, vasicinone and moran A.

In a particular embodiment, the nanoparticles do not comprise PVM/MA. As it is used herein, “poly (methyl vinyl ether-co-maleic anhydride)” (PVM/MA) refers to the copolymer of methyl vinyl ether and maleic anhydride (commercialized by International Specialty Products, ISP, under trademark Gantrez®AN). Therefore expressions, “PVM/MA”, poly(anhydride), or Gantrez® AN are synonyms, and are indistinctly used in this description. Gantrez® AN copolymer contains alternating units of methylvinylether and maleic anhydride, having the formula:

The fundamental character of this polymerization requires that a maleic anhydride unit must be adjacent to a methylvinylether unit and vice versa, resulting in a true alternating copolymer. The anhydride groups in Gantrez® AN structure allows the chemical interaction of this polymer with hydroxyl containing molecules through a nucleophilic substitution reaction mechanism. In another particular embodiment, the nanoparticles used in this aspect of the invention do not comprise a polyethylene glycol polymer or derivative thereof. Polyethylene glycol polymers and derivatives thereof are defined below.

In another particular embodiment, the nanoparticles consist of a zein matrix and a basic amino acid as previously defined.

The nanoparticles defined herein can be formulated in a composition. Thus, another aspect of the invention relates to a composition comprising at least one nanoparticle as defined in the first aspect and a pharmaceutically acceptable carrier for use in the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation.

This aspect may also be worded as the use of composition comprising at least one nanoparticle as defined in the first aspect and a pharmaceutically acceptable excipient in the manufacture of a medicament for the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation.

It may also be worded as a method of treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation comprising the administration of a composition comprising at least one nanoparticle as defined in the first aspect and a pharmaceutically acceptable excipient.

Said compositions comprises a plurality of said nanoparticles.

Said nanoparticles can be presented in the form of a suspension, preferably in an aqueous medium, or, alternatively, they can be presented in the form of a dry powder, for example as a lyophilizate together with a cryoprotective agent

Pharmaceutically acceptable excipient are well known in the pharmaceutical art, and are described, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams and Wilkins (22^nd Ed., 2012). The choice of pharmaceutical excipient can be selected with regard to the intended route of administration and standard pharmaceutical practice.

The pharmaceutical composition of the invention may be formulated in a solid (for example, tablets, capsules, coated tablets, granulates, suppositories, sterile crystalline or amorphous solids which can be reconstituted to provide liquid forms, etc.), liquid (for example suspension or dispersion of the nanoparticles, etc.) or semisolid (gels, pomades, creams and the like) pharmaceutical dosage form. The described compositions will comprise suitable carriers or vehicles for each formulation. Furthermore, the pharmaceutical composition can contain, as appropriate, stabilizers, suspensions, preservatives, surfactants and the like. These excipients will be chosen according to the selected pharmaceutical dosage form.

In a specific embodiment, said pharmaceutical composition is formulated as a pharmaceutical dosage form suitable for its administration by a route of access to mucosae. In a preferred embodiment, the pharmaceutical composition provided by this invention is orally administered; therefore the carrier or vehicle comprises one or more pharmaceutical excipients acceptable for the administration by oral route. Oral formulations are conventionally prepared by methods known by persons skilled in the art. A review of the different forms of administration of active ingredients, of the excipients to be used, and of the processes for manufacturing them can be found in the book “Tratado de Tecnologia Farmaceutica”, Volume III, by Ramon Martinez Pacheco, (2017), Ed. Sintesis, Madrid; and “Pharmaceutics: The Science of Dosage Form Design”, by Michael E. Aulton, 2^nd ed. 2001, Elsevier, ISBN-13: 978-0443055171.

The nanoparticles and compositions described above are for the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation, such as diabetes, obesity and metabolic syndrome.

In a particular embodiment, nanoparticles and compositions described above are for the treatment and/or prevention of diabetes, preferably for the treatment and/or prevention of prediabetes or type-2 diabetes, more preferably for the treatment and/or prevention of prediabetes.

As previously explained, type-2 diabetes occurs when the body becomes resistant to insulin or there is not enough insulin produced. This type of diabetes is largely the result of excess body weight and physical inactivity. It is characterized by at least one of the following blood glucose levels: fasting plasma glucose level > 7.0 mmol/L (126 mg/dL) or plasma glucose > 11.1 mmol/L (200 mg/dL) two hours after a 75 g oral glucose load as in a glucose tolerance test.

A prediabetic state or prediabetes is characterized by impaired fasting glucose levels and/or impaired glucose tolerance. Impaired fasting glucose levels are from 6.1 to 6.9 mmol/L (110 to 125 mg/dL). Impaired glucose tolerance is defined by a plasma glucose at or above 7.8 mmol/L (140 mg/dL), but not over 11.1 mmol/L (200 mg/dL), two hours after a 75 gram oral glucose load.

In a particular embodiment, the nanoparticles and compositions described above are for the treatment and/or prevention of obesity.

As previously explained, obesity is a obesity is a complex multifactorial disease defined by excessive adiposity that can impair health and it is also one of the key risk factors for many diseases such as coronary heart disease, hypertension and stroke, certain types of cancer, type 2 diabetes. Body mass index (BMI) is a surrogate marker of adiposity calculated as weight (kg)/height² (m²). The BMI categories for defining obesity vary by age and sex in infants, children and adolescents. For adults, obesity is defined by a BMI greater than or equal to 30 kg/m² A BMI ranging from 25 to 29.99 kg/m² is also associated with increase disease risk and is referred to as pre-obesity or overweight. In the case of children aged between 5-19 years, overweight is BMLfor-age greater than 1 standard deviation above the WHO Growth Reference median and obesity is greater than 2 standard deviations above the WHO Growth Reference median. Finally, for children under 5 years of age, overweight is weight-for-height greater than 2 standard deviations above WHO Child Growth Standards median whereas obesity is weight-for-height greater than 3 standard deviations above the WHO Child Growth Standards median.

In another particular embodiment, the nanoparticles and compositions described above are for the treatment and/or prevention of metabolic syndrome.

As previously explained, according to the new IDF definition, a person is defined as having metabolic syndrome if having central obesity (generally defined as waist circumference with ethnicity specific values but, if BMI is >30kg/m², central obesity can be assumed and waist circumference does not need to be measured) plus any two of the following four factors: raised triglycerides > 150 mg/dL (1.7 mmol/L) or specific treatment for this lipid abnormality; reduced HDL cholesterol < 40 mg/dL (1.03 mmol/L) in males < 50 mg/dL (1.29 mmol/L) in females or specific treatment for this lipid abnormality; raised blood pressure systolic BP > 130 or diastolic BP > 85 mm Hg or treatment of previously diagnosed hypertension; raised fasting plasma glucose (FPG) > 100 mg/dL (5.6 mmol/L), or previously diagnosed type 2 diabetes (if FPG is above 5.6 mmol/L or 100 mg/dL, OGTT is strongly recommended but is not necessary to define presence of the syndrome).

As used herein the terms “treat”, “treatment”, or “treatment of’ refers to reducing the potential for a certain disease or disorder, reducing the occurrence of a certain disease or disorder, and/or a reduction in the severity of a certain disease or disorder, preferably, to an extent that the subject no longer suffers discomfort and/or altered function due to it. It also refers to mitigating or decreasing at least one clinical symptom and/or inhibition or delay in the progression of the condition and/or prevention or delay of the onset of a disease or illness.

The term “prevention”, “preventing” or “prevent”, as used herein, refers to avoiding the appearance of a certain disease or disorder. The prevention can be complete (e.g. the total absence of a disease). The prevention can also be partial, such that for example the occurrence of a disease in a subject is less than that which would have occurred without the administration of the combination or composition of the present invention. Prevention also refers to reduced susceptibility to a clinical condition. The prevention also includes reducing the risk of suffering the disease.

The dose of nanoparticles of the invention to be administered to a subject in need of treatment and/or prevention with the nanoparticles should be sufficient to trigger a beneficial therapeutic response in the patient over time. Thus, nanoparticles are administered to a patient in an amount sufficient to prevent, alleviate, reduce, cure or at least partially arrest symptoms and/or complications from the disease. An amount adequate to accomplish this is defined as a “therapeutically effective dose”. The dose can vary within a broad range, and will depend, for example on the frequency of administration and on the potency and duration of action, on the nature and severity of the disease and symptoms, and on the sex, age, weight, co-medication and individual responsiveness of the subject to be treated and on whether the therapy is acute or prophylactic. Only by illustrative purposes, the dose of the nanoparticles to be administered to a subject may be comprised, for example, between approximately 0.1 and approximately 50 mg per kg of body weight, preferably, between 0.1 and 10 mg per kg of body weight.

The subject to which the nanoparticles are administered is a human or animal; preferably subjects are mammals, more preferably humans.

Aging is associated with a lot of metabolic changes that reduce the lifespan, including a decline in the modulation of metabolic function and insulin sensitivity [T. Finkel, Nat. Med., 2015, 27(12), 1416-1423], A high glucose diet has been associated with reduced lifespan in C. elegans [A. Schlotterer et al., Diabetes, 2009, 55(11), 2450-2456; and S. J. Lee, C. T. Murphy, and C. Kenyon, Cell Metab., 2009, 70(5), 379-391],

Therefore, the further aspect of the present invention is directed to the non-therapeutic use of a nanoparticle comprising a zein matrix and a basic amino acid and which does not comprise any biologically active ingredient, as defined above, for extending the lifespan of an organism. The nanoparticles used in this aspect of the invention are as those described above for the first aspect.

In particular, the nanoparticles used in this aspect of the invention are characterized by having an average particle size less than 1 pm, typically comprised between 1 and 999 nm, preferably between 10 and 900 nm, more preferably between 50 and 500 nm, more preferably between 100 and 450 nm, even more preferably between 140 and 400 nm, even more preferably between 150 and 300 nm, still more preferably from 150 to 250 nm.

As previously explained, virtually any zein can form the matrix of the nanoparticles used in this aspect of the invention; nevertheless, in a particular embodiment, said zein is a zein from corn, such as the zein supplied by Sigma-Aldrich (product number Z 3625).

The basic amino acid present in the nanoparticles is as previously described. Preferably, the basic amino acid of the nanoparticles used in this aspect of the invention is selected from the group consisting of lysine, arginine, histidine and mixtures thereof, preferably lysine.

In the nanoparticles used according to this aspect of the invention, the weight ratio of zein to basic amino acid is in the range of from 0.01 : 1 to 50: 1, preferably from 0.5: 1 to 25: 1, more preferably from 1: 1 to 20:1, more preferably from 5:1 to 15:1, even more preferably from 6: 1 to 7: 1, still more preferably about 6.7: 1.

Preferably, the nanoparticles used in this aspect of the invention do not comprise a polyethylene glycol polymer or derivative thereof.

The nanoparticles used in this aspect of the invention do not comprise any biologically active ingredient, as defined above. More particularly, the nanoparticles used in this aspect consist of the zein matrix and the basic amino acid.

In a particular embodiment, the nanoparticles do not comprise PVM/MA. “PVM/MA” is as previously defined. The nanoparticles used in this aspect of the invention can be formulated in a composition. Thus, another aspect of the invention relates to the non-therapeutic use of a composition comprising at least one nanoparticle as defined herein and an excipient acceptable in food or nutraceutics, for extending the lifespan of an organism. Said composition comprises a plurality of said nanoparticles.

As previously explained, said nanoparticles can be presented in the form of a suspension, preferably in an aqueous medium, or, alternatively, they can be presented in the form of a dry powder, for example as a lyophilizate together with a cryoprotective agent.

In particular the compositions for use according to this aspect are a food, or nutraceutical product.

In a particular embodiment, the product is a food, a feed or a food for special medical purposes comprising the nanoparticles described herein and a food acceptable vehicle or carrier.

As used herein, the term “food” is any substance or product of any nature, solid or liquid, natural or processed which due to its characteristics, applications, components, preparation and state of preservation, can usually or ideally be used for some of the following purposes: a) as normal nutrition for human beings or animals or as pleasurable foods; or b) as dietetic products, in special cases of human or animal food.

The term “feed” includes all the natural materials and finished products of any origin which, separately or conveniently mixed with one another, are suitable as animal food. Examples include cattle feed, chicken feed, horse feed, poultry feed.

The term “food for special medical purposes” means foods specially processed or formulated and intended for the dietary management of people with certain diseases, disorders or medical conditions. These special foods are intended for people whose nutritional requirements cannot be met by normal foods. In particular, they are intended for the exclusive or partial feeding of patients with a limited, impaired or disturbed capacity to take, digest, absorb, metabolise or excrete ordinary food or certain nutrients contained therein, or metabolites, or with other medically-determined nutrient requirements, whose dietary management cannot be achieved by modification of the normal diet alone, with other food intended for a particular diet, or both. Food for special medical purposes are to be used under the supervision of a medical practitioner and other appropriate health professionals.

A ready-to-eat food is that which does not need to be diluted by means of an aqueous solution suitable for consumption for example. In principle, the ingredients present in a ready-to-eat food are balanced and there is no need to add additional ingredients to the food to make it ready to eat, such considered by a person skilled in the art. A concentrated food is that in which one or more ingredients are present at a higher concentration than in a ready-to-eat food, therefore for use it is necessary to dilute it by means of an aqueous solution suitable for consumption for example. Non-limiting, illustrative examples of foods provided by this invention include both dairy products and derivatives, for example, fermented milks, yoghurt, kephir, curd, cheeses, butters, ice creams, milk-based desserts, etc., and non-dairy products, such as baked products, cakes and pastries, cereals, chocolates, jams, juices, other fruit derivatives, oils and margarines, prepared dishes, etc. In another particular embodiment, the product is a nutraceutical product comprising the nanoparticles described herein and a nutraceutical acceptable carrier.

In another particular embodiment, the product is nutraceutical product comprising the nanoparticles described herein and a nutraceutical acceptable vehicle or carrier.

As used herein, the term “nutraceutical product” refers to a product suitable for use in human beings or animals, comprising one or more natural products with therapeutic action which provide a health benefit or have been associated with disease prevention or reduction, for example, it includes food supplements or dietary supplements presented in a non-food matrix (e.g., capsules, powder, etc.) of a concentrated natural bioactive product usually present (or not) in the foods and which, when taken in a dose higher than that existing in those foods, exerts a favorable effect on health which is greater than effect which the normal food may have. Therefore, the term “nutraceutical product” includes isolated or purified food products as well as additives or food supplements which are generally presented in dosage forms normally used orally, for example, capsules, tablets, sachets, drinkable phials, etc.; such products provide a physiological benefit or protection against diseases, generally against chronic diseases. If desired, the nutraceutical product provided by the invention can contain one or more nutraceuticals (products or substances associated with disease prevention or reduction), for example, flavonoids, omega-3 fatty acids, etc., and/or one or more prebiotics (non-digestible food ingredients which stimulate probiotic activity and/or growth), for example, oligofructose, pectin, inulin, galactooligosaccharides, lactulose, human milk oligosaccharides, dietary fiber, etc.

Acceptable excipient in food and nutraceutics have been described above with respect to the food and nutraceutical products, but also include the pharmaceutically acceptable excipients previously described.

The choice of excipient can be selected with regard to the type of composition or product, the intended route of administration and standard practice.

The composition of the invention may be formulated as a liquid, semi-solid or solid form.

In a specific embodiment, said composition is formulated as a dosage form suitable for its administration by a route of access to mucosae. In a preferred embodiment, the composition provided by this invention is orally administered; therefore the carrier or vehicle comprises one or more excipients acceptable for the administration by oral route.

The organisms to which the nanoparticles are administered are preferably mammals, more preferably humans.

As used herein, the term “extending the lifespan of an organism” refers to increasing the lifespan of an organism relative to the lifespan in the absence of the nanoparticles (e.g., relative to the projected lifespan of an individual, a population, an individual with certain diseases or lifestyle choices, etc.). In particular, said increase in the lifespan is considered as being suitable, preferably said increase in the lifespan is of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or at least 80% with respect to the projected lifespan in the absence of the nanoparticles. In particular, the increase in the lifespan is from 10% to 50%.

In particular, said increase in the lifespan is characterized by a reduction of the glucose levels, fat accumulation and/or intracellular ROS levels in the organism to which the nanoparticles are administered.

Use of nanoparticles comprising a zein matrix, a basic amino acid and polyethylene glycol or a derivative thereof

The further aspect of the present invention is directed to a nanoparticle comprising a zein matrix, a basic amino acid and polyethylene glycol or a derivative thereof for use in the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation.

This aspect may also be worded as the use of a nanoparticle comprising a zein matrix, a basic amino acid and polyethylene glycol or a derivative thereof in the manufacture of a medicament for the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation.

It may also be worded as a method of treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation comprising the administration of a nanoparticle comprising a zein matrix, a basic amino acid and polyethylene glycol or a derivative thereof.

The term “nanoparticle” is as previously defined and are characterized by having an average particle size less than 1 pm, typically comprised between 1 and 999 nm, preferably between 10 and 900 nm, more preferably between 50 and 500 nm, more preferably between 100 and 450 nm, even more preferably between 140 and 400 nm, even more preferably between 150 and 300 nm, still more preferably from 150 to 250 nm. The term “zein” is as previously defined. Virtually any zein can form the matrix of the nanoparticle for use of the invention; nevertheless, in a particular embodiment, said zein is a zein from com, such as the zein supplied by Sigma- Aldrich (product number Z 3625).

The nanoparticles comprise a basic amino acid, as previously defined. Said basic amino acid is preferably a basic alpha-amino acid such as lysine, arginine, histidine or mixtures thereof. Preferably, the basic amino acid present in the nanoparticles of the invention is lysine.

In the nanoparticles defined herein, the weight ratio of zein to basic amino acid is in the range of from 0.01 : 1 to 50: 1, preferably from 0.5: 1 to 25: 1, more preferably from 1 : 1 to 20: 1, more preferably from 5: 1 to 15: 1, even more preferably from 6: 1 to 7: 1, still more preferably about 6.7: 1.

The nanoparticle further comprises a polyethylene glycol or derivative thereof. These particles are also named PEGylated nanoparticles.

In the present description, the term “polyethylene glycol” (PEG), is understood to be any hydrophilic polymer soluble in water containing ether groups linked by 2 carbon atoms, optionally branched ethylene groups. Therefore this definition includes branched or nonbranched polyethylene glycols.

PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE) (the three names are chemically synonymous and refers to an oligomer or polymer of ethylene oxide). The term also includes derivatives of one of the terminal hydroxyl groups, which can be modified (one of two ends). Thus, according to the present invention polyethylene glycol derivatives maintaining one primary hydroxy (-OH) group can be used. Polyethylene glycols are water-soluble polymers that have been approved (FDA) for the oral, parenteral and topical administration of drugs. Polyethylene glycols are produced by means of polymerization of ethylene oxide (EO) in the presence of water, monoethylene glycol or di ethylene glycol as reaction initiators in an alkaline medium (1,2-Epoxide Polymers: Ethylene Oxide Polymers and Copolymers” in Encyclopedia of Polymer Science and Engineering; Mark, H.F. (Ed.), John Wiley and Sons Inc., 1986, pp. 225- 273). When the desired molecular weight (generally controlled by means of in-process measurements of viscosity) is reached, the polymerization reaction ends by neutralizing the catalyst with an acid (lactic acid, acetic acid or the like). The result is a linear polymer having a very simple structure:

H-(O- CH₂-CH₂)n-OH where (n) is the number of EO monomers or units. This term polyethylene glycol is normally used to indicate the significant influence of hydroxyl terminal groups on the physicochemical properties of these molecules. The term PEG is normally used in combination with a numerical value. In the pharmaceutical industry the number indicates the average molecular weight, whereas in the cosmetic industry the number accompanying the letters PEG refers to the polymerized EO units forming the molecule (Handbook of Pharmaceutical Excipients, Rowev R.C., Sheskey P. J., Weller P. J. (Eds.), 4^th Edition, Pharmaceutical Press and American Pharmaceutical Association, London, UK, 2003). PEGs are included in various pharmacopeias, although the nomenclature differs (International Harmonisation: Polyethylene glycol (PEG): Pharmeuropa 1999, 11, 612-614). According to the Handbook of Pharmaceutical Excipients (Fourth Edition), 2003 Edited by R.C. Rowe, P.J. Sheskey and P.J. Weller Published by the Pharmaceutical Press (London, UK) and the American Pharmaceutical Association (Washington, USA), polyoxyethylene glycols are also referred to as polyethylene glycols, macrogols, or PEG. The British and European Pharmacopoeias use polyethylene glycols and macrogols, while the US pharmacopoeia (USP) uses polyethylene glycols. In the present invention, the number accompanying the letters PEG refers to the average molecular weight.

As it is used herein “molecular weight” is defined as the average molar mass of a molecule. Unlike small molecules, the molecular weight of a polymer is not one unique value. Rather, a given polymer will have a distribution of molecular weights depending for example on the way the polymer is produced. Therefore, as it is used herein, the term molecular weight for polymers refers to the distribution of molecular weight, or of the average molecular weight. The average molecular weight of PEG may be determined by conventional methods in the art, such as ¹ H-RMN, size exclusion chromatography (SEC), or based on the hydroxyl value, as described, by way of illustration, in Yoncheva K., et al. [Eur J Pharm Sci 2005;24(5),411-9], Gonzalez -Fernandez et al. [Polymer, 2020, 72, 1269] or Ling et al., [Nanoscale Research Letters, 2013, S(l), 538],

In one variant of the invention the polyethylene glycols used preferably have a molecular weight comprised in the range from 400 to 40 kDa. In one preferred variant of the invention the polyethylene glycol used has a molecular weight equal to or greater than 400 Da; values comprised from 10 to 40 kDa are especially preferred, more preferably from 20 to 40 kDa, even more preferably about 35 kDa.

From a toxicological point of view, PEGs are considered rather non-toxic and non- immunogenic (Hermansky S.J et al., Food Chem Toxic., 1995, 33, 139-140; Final Report on the Safety Assessment of PEGs: J.A. C. T., 1993, 12, 429-457; Polyethylene glycol, 21 CFR 172.820, FDA). The maximum daily intake defined by the WHO is 10 mg/kg (Polyethylene glycols; Twenty-third report of the Joint FAO/WHO Expert Committee on Food Additives; World Health Organisation, Geneva; Technical Report Series 1980, 648, 17-18).

Polyethylene glycol derivatives have advantages that are similar to traditional PEGs such as their aqueous solubility, physiological inactivity, low toxicity and stability under very different conditions. These derivatives include very different products and are characterized by the functional group substituting the hydroxyl, such as -NH2 (among the most reactive ones), phenol, aldehyde, isothiocyanate, -SH groups, etc. The following can be pointed out among the polyethylene glycol derivatives that can be used in the invention:

- Polyoxyethylene esters: PEG monomethyl ether monosuccinimidyl succinate ester; PEG monomethyl ether monocarboxymethyl ether; PEG adipate; PEG distearate; PEG monostearate; PEG hydroxystearate; PEG dilaurate; PEG dioleate, PEG monooleate, PEG monoricinoleate; PEG coconut oil esters.

- Polyoxyethylene alkyl ethers: PEG monomethyl ether or methoxy PEG (mPEG); PEG dimethyl ether.

- Others: Poly(ethylene glycol terephthalate); polyoxyethylene derivatives and sorbitan esters and fatty acids; ethylene oxide and propylene oxide copolymers; ethylene oxide with acrylamide copolymers.

- PEG derivatives: O,O'-Bis-(2-aminoethyl)polyethylene glycol (DAE-PEG 2000); O,O'-Bis-(2-aminopropyl)polypropylene glycol-polyethylene glycol-polypropylene glycol.

In a particular embodiment, the polyethylene glycol or derivative thereof is a polyethylene glycol.

Illustrative non-limiting examples of PEG which can be used in the present invention include polyethylene glycol 35000 (PEG35), polyethylene glycol 20000 (PEG20), polyethylene glycol 10000 (PEG10), and polyethylene glycol 6000 (PEG6). In a preferred embodiment of the invention, the polyethylene glycol is PEG35. PEGs are commercially available.

The PEG is present on the surface of the nanoparticles comprising a zein matrix and a basic amino acid. Thus, the PEG is present as a coating. The nanoparticles may be totally or partially coated with PEG, preferably totally coated with PEG. PEG chains bind to the hydrophobic surface of the zein through multiple van der Waals interactions forming a layer all around the nanoparticle, thus, leading preferably to a total coating of the nanoparticles.

In a preferred embodiment, the weight ratio of the polyethylene glycol or derivative thereof and zein is in the range of from 0.05: 1 to 1 :5, preferably from 0.05: 1 to 1 :1, preferably from 0.25: 1 to 1 : 1, more preferably from 0.4: 1 to 0.6: 1, even more preferably about 0.5: 1. As shown in the examples, the nanoparticles for use according to the invention are able to diffuse through the mucus layer, improving the absorption through the oral mucosa. This diffusion is particularly enhanced for PEGylated nanoparticles.

Surprisingly, as shown in the examples, the biological activity of the nanoparticles described herein is achieved in the absence of any pharmaceutically active ingredients used for the treatment of diabetes. Thus, in one embodiment, the nanoparticles do not comprise any biologically active ingredient as previously defined. In another embodiment, the nanoparticles do not comprise any antidiabetic drug. In another embodiment, the nanoparticles do not comprise glibenclamide. In a particular embodiment, the nanoparticles consist of the zein matrix, the basic amino acid and the polyethylene glycol or derivative thereof.

In an alternative embodiment, the nanoparticle further comprises a biologically active ingredient, in particular an antidiabetic drug, more preferably an antidiabetic drug other than glibenclamide.

The “biologically active ingredients” and in particular the “antidiabetic drugs” are as previously defined.

In a particular embodiment, the nanoparticles do not comprise PVM/MA. “PVM/MA” is as previously defined.

The nanoparticles defined herein can be formulated in a composition. Thus, another aspect of the invention relates to a composition comprising at least one nanoparticle comprising a zein matrix, a basic amino acid and polyethylene glycol or a derivative thereof, and a pharmaceutically acceptable carrier for use in the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation.

This aspect may also be worded as the use of composition comprising at least one nanoparticle comprising a zein matrix, a basic amino acid and polyethylene glycol or a derivative thereof, and a pharmaceutically acceptable excipient in the manufacture of a medicament for the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation.

It may also be worded as a method of treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation comprising the administration of a composition comprising at least one nanoparticle comprising a zein matrix, a basic amino acid and polyethylene glycol or a derivative thereof, and a pharmaceutically acceptable excipient.

Said compositions comprises a plurality of said nanoparticles.

Pharmaceutically acceptable excipient are well known in the pharmaceutical art and are as previously defined.

The pharmaceutical composition may be formulated in a solid (for example, tablets, capsules, coated tablets, granulates, suppositories, sterile crystalline or amorphous solids which can be reconstituted to provide liquid forms, etc.), liquid (for example suspension or dispersion of the nanoparticles, etc.) or semi solid (gels, pomades, creams and the like) pharmaceutical dosage form. The described compositions will comprise suitable carriers or vehicles for each formulation. Furthermore, the pharmaceutical composition can contain, as appropriate, stabilizers, suspensions, preservatives, surfactants and the like. These excipients will be chosen according to the selected pharmaceutical dosage form.

In a specific embodiment, said pharmaceutical composition is formulated as a pharmaceutical dosage form suitable for its administration by a route of access to mucosae. In a preferred embodiment, the pharmaceutical composition provided by this invention is orally administered; therefore the carrier or vehicle comprises one or more pharmaceutical excipients acceptable for the administration by oral route. Oral formulations are conventionally prepared by methods known by persons skilled in the art. A review of the different forms of administration of active ingredients, of the excipients to be used, and of the processes for manufacturing them can be found in the book “Tratado de Farmacia Galenica”, by C. Fault i Trillo, 10 Edition, 1993, Luzan 5, S.A. de Ediciones.

The nanoparticles and compositions described herein are for the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation, such as diabetes, obesity and metabolic syndrome

In a particular embodiment, the nanoparticles and compositions described above are for the treatment and/or prevention of diabetes, preferably for the treatment and/or prevention of type-2 diabetes or a prediabetes, more preferably for the treatment and/or prevention of type-2 diabetes.

In a particular embodiment, the nanoparticles and compositions described above are for the treatment and/or prevention of obesity. As previously explained, obesity is a complex multifactorial disease defined by excessive adiposity that can impair health and it is also one of the key risk factors for many diseases such as coronary heart disease, hypertension and stroke, certain types of cancer, type 2 diabetes, etc. Body mass index (BMI) is a surrogate marker of adiposity calculated as weight (kg)/height² (m²). The BMI categories for defining obesity vary by age and sex in infants, children and adolescents. For adults, obesity is defined by a BMI greater than or equal to 30 kg/m² A BMI ranging from 25 to 29.99 kg/m² is also associated with increase disease risk and is referred to as pre-obesity or overweight. In the case of children aged between 5-19 years, overweight is BMI-for-age greater than 1 standard deviation above the WHO Growth Reference median and obesity is greater than 2 standard deviations above the WHO Growth Reference median. Finally, for children under 5 years of age, overweight is weight-for-height greater than 2 standard deviations above WHO Child Growth Standards median whereas obesity is weight-for-height greater than 3 standard deviations above the WHO Child Growth Standards median.

As previously explained, according to the new IDF definition, a person is defined as having metabolic syndrome if having central obesity (generally defined as waist circumference with ethnicity specific values but, if BMI is >30kg/m², central obesity can be assumed and waist circumference does not need to be measured) plus any two of the following four factors: raised triglycerides > 150 mg/dL (1.7 mmol/L) or specific treatment for this lipid abnormality; reduced HDL cholesterol < 40 mg/dL (1.03 mmol/L) in males < 50 mg/dL (1.29 mmol/L) in females or specific treatment for this lipid abnormality; raised blood pressure systolic BP > 130 or diastolic BP > 85 mm Hg or treatment of previously diagnosed hypertension; raised fasting plasma glucose (FPG) > 100 mg/dL (5.6 mmol/L), or previously diagnosed type 2 diabetes (if FPG is above 5.6 mmol/L or 100 mg/dL, OGTT is strongly recommended but is not necessary to define presence of the syndrome). As used herein the terms “treat”, “treatment”, “treatment of’, “prevention”, “preventing” “prevent”, are as previously defined.

A further aspect of the present invention is directed to the non-therapeutic use of a nanoparticle comprising a zein matrix, a basic amino acid and polyethylene glycol or a derivative thereof for extending the lifespan of an organism.

The nanoparticles used in this aspect of the invention are as those described above.

In particular, the nanoparticles used in this aspect of the invention are characterized by having an average particle size less than 1 pm, typically comprised between 1 and 999 nm, preferably between 10 and 900 nm, more preferably between 50 and 500 nm, more preferably between 100 and 450 nm, even more preferably between 140 and 400 nm, even more preferably between 150 and 300 nm, still more preferably from 150 to 250 nm. As previously explained, virtually any zein can form the matrix of the nanoparticles used in this aspect of the invention; nevertheless, in a particular embodiment, said zein is a zein from corn, such as the zein supplied by Sigma-Aldrich (product number Z 3625).

The nanoparticles used in this aspect of the invention comprise a polyethylene glycol polymer or derivative thereof as described above.

Illustrative non-limiting examples of PEG which can be used in the present invention include polyethylene glycol 35000 (PEG35), polyethylene glycol 20000 (PEG20), polyethylene glycol 10000 (PEG10), and polyethylene glycol 6000 (PEG6). In a preferred embodiment of the invention, the polyethylene glycol is PEG35. PEGs are commercially available. The PEG is present on the surface of the nanoparticles comprising a zein matrix and a basic amino acid. Thus, the PEG is present as a coating. The nanoparticles may be totally or partially coated with PEG, preferably totally coated with PEG. PEG chains bind to the hydrophobic surface of the zein through multiple van der Waals interactions forming a layer all around the nanoparticle, thus, leading preferably to a total coating of the nanoparticles.

In a preferred embodiment, the weight ratio of the polyethylene glycol or derivative thereof and zein is in the range of from 0.05: 1 to 1 :5, preferably from 0.05: 1 to 1 :1, preferably from 0.25: 1 to 1 : 1, more preferably from 0.4: 1 to 0.6: 1, even more preferably about 0.5: 1.

In a particular embodiment, the nanoparticles used in this aspect of the invention do not comprise any biologically active ingredient, as defined above. More particularly, the nanoparticles used in this aspect consist of the zein matrix, the basic amino acid and the polyethylene glycol or derivative thereof.

In an alternative embodiment, the nanoparticles used in this aspect of the invention comprise a biologically active ingredient.

The nanoparticles used in this aspect of the invention can be formulated in a composition. Thus, another aspect of the invention relates to the non-therapeutic use of a composition comprising at least one nanoparticle as defined herein, i.e. comprising a zein matrix, a basic amino acid and polyethylene glycol or a derivative thereof, and an excipient acceptable in food or nutraceutics, for extending the lifespan of an organism. Said composition comprises a plurality of said nanoparticles. As previously explained, said nanoparticles can be presented in the form of a suspension, preferably in an aqueous medium, or, alternatively, they can be presented in the form of a dry powder, for example as a lyophilizate together with a cryoprotective agent.

In particular the compositions for use according to this aspect are a food, or nutraceutical product, as previously defined.

In another particular embodiment, the product is nutraceutical product as previolsy defined comprising the nanoparticles described herein and a nutraceutical acceptable vehicle or carrier.

Acceptable excipient in food and nutraceutics have been described above. The choice of excipient can be selected with regard to the type of composition or product, the intended route of administration and standard practice.

The term “extending the lifespan of an organism” is as previously defined. In particular, the increase in the lifespan is of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or at least 80% with respect to the projected lifespan in the absence of the nanoparticles. In particular, the increase in the lifespan is from 10% to 50%.

Preparation of the nanoparticles

The zein nanoparticles for use according to the present invention may be prepared using a desolvation method, such as that described by Penalva et al. [J. Agric. Food Chem., 2015, 63(23), 5603-5611] or as that described in WO 2012/007628 Al as “process [1]” (which is incorporated herein by reference). The detailed description of the preparation of the nanoparticles can be found in the examples. Said method comprises: a) preparing a hydroalcoholic solution containing a zein and a basic amino acid; and b) adding water to the solution of step a).

The hydroalcoholic solution used in step a) contains water and an alcohol, typically ethanol; in a particular embodiment, said hydroalcoholic solution comprises between 25% and 75% (w/v) of alcohol, preferably between 30% and 60%, more preferably about 55%.

The amount of zein present in the hydroalcoholic solution formed in step a) can vary within a wide range; nevertheless, in a particular embodiment, the amount of zein contained in said hydroalcoholic solution is comprised between 0.1% and 20% (w/v, expressed as weight in mg and volume in mL), preferably between 5% and 15%, more preferably about 10%.

The amount of basic amino acid present in the hydroalcoholic solution formed in step a) can vary within a wide range. Generally, said amount is usually expressed according to the amount of zein to be dissolved. Said weight ratio of zein to basic amino acid is preferably in the range of from 0.01 : 1 to 50: 1, preferably from 0.5: 1 to 25: 1, more preferably from 1: 1 to 20:1, more preferably from 5: 1 to 15: 1, even more preferably from 6: 1 to 7: 1, still more preferably about 6.7: 1.

In step b) of process, water is added in an amount sufficient for the formation of the nanoparticles for use according to the invention. Although the amount of water to be added can vary within a wide range, in a particular embodiment, water is added in an amount sufficient for the final proportion of alcohol in the medium to be comprised between 10% and 60% (w/v), preferably between 15% and 30%, more preferably between 20% and 25%.

The nanoparticles are isolated by removal of the alcohol by conventional means, such as evaporation at reduced pressure to provide a suspension of the nanoparticles. This suspension may be concentrated and purified by tangential filtration. The resulting suspension may be dried by conventional means such as spray-drying, in particular using the experimental conditions described in the examples.

Drug loaded nanoparticles may also be prepared as described above but further adding the antidiabetic drug to the solution of step a). These nanoparticles may also be prepared following the procedures described in WO 2012/007628 Al as “process [2]” and “process [3]” (which are incorporated herein by reference).

PEGylated nanoparticles may be prepared by simple incubation of the nanoparticles obtained as described above with the corresponding polyethylene glycol or derivative thereof. For this purpose, a solution of polyethylene glycol or a derivative thereof in water is prepared, preferably to a concentration of polyethylene glycol or derivative thereof in said solution between 50 and 200 mg/mL, more preferably between 50 and 150 mg/mL, still more preferably about 100 mg/mL. Said solution is contacted with a suspension of the nanoparticles in water previously obtained.

The amount of solution of polyethylene glycol or derivative thereof contacted with the suspension of nanoparticles can vary within a wide range. Said amount depends on the target weight ratio of polyethylene glycol or derivative thereof to zein. Said weight ratio of the polyethylene glycol or derivative thereof and zein is in the range of from 0.05: 1 to 1 :5, preferably from 0.05: 1 to 1 :1, preferably from 0.25: 1 to 1 :1, more preferably from 0.4:1 to 0.6: 1, even more preferably about 0.5: 1.

The contacting is preferably carried out by mixing at a temperature in the range of 15 to 30 °C, preferably of 20 to 25 °C, for a period of time of about 15 min to 1 h, preferably for about 30 min.

The resulting suspension of PEGylated nanoparticles may be concentrated and purified by tangential filtration. The resulting suspension may be dried by conventional means such as spray-drying, in particular using the experimental conditions described in the examples.

The compositions for use according to the invention may be prepared by mixing the ingredients contained in said compositions.

As used in the present document “about” refers to a range of values close to a specified value, such as ± 10% of a specified value, preferably ± 5% of a specified value. Furthermore, regardless of whether or not the term “about” is specified, the person skilled in the art understands that any numerical value expressed herein encompasses a close range of values. Such variations of a specified value can result from the experimental errors during the corresponding measurement.

The present invention also relates to the following embodiments:

Embodiment 1. A nanoparticle comprising a zein matrix and a basic amino acid for use in the treatment and/or prevention of diabetes, wherein the nanoparticle does not comprise glibenclamide.

Embodiment 2. The nanoparticle for use according to embodiment 1, wherein the basic amino acid is selected from the group consisting of lysine, arginine, histidine and mixtures thereof. Embodiment 3. The nanoparticle for use according to embodiment 2, wherein the basic amino acid is lysine.

Embodiment 4. The nanoparticle for use according to any one of the preceding embodiments, wherein the weight ratio of zein to basic amino acid is in the range of from 0.01 : 1 to 50: 1, preferably from 0.5: 1 to 25: 1, more preferably from 1 : 1 to 20: 1, more preferably from 5: 1 to 15: 1, even more preferably from 6: 1 to 7: 1.

Embodiment 5. The nanoparticle for use according to any one of the preceding embodiments, wherein its average size is in the range of from 100 to 450 nm, preferably from 150 to 250 nm.

Embodiment 6. The nanoparticle for use according to any one of the preceding embodiments, which further comprises a polyethylene glycol or derivative thereof.

Embodiment 7. The nanoparticle for use according to embodiments 6, wherein the polyethylene glycol or derivative thereof has a molecular weight in the range of from 400 Da to 40 kDa, preferably from 10 to 40 kDa, more preferably from 20 to 40 kDa, even more preferably about 35 kDa.

Embodiment 8. The nanoparticle for use according to embodiment 6 or 7, wherein the polyethylene glycol is polyethylene glycol 35000 (PEG35).

Embodiment 9. The nanoparticle for use according to any one of embodiments 6 to 8, wherein the weight ratio of the polyethylene glycol or derivative thereof and zein is in the range of from 0.05: 1 to 1 :5, preferably from 0.05: 1 to 1 : 1, preferably from 0.25: 1 to 1 : 1, more preferably from 0.4: 1 to 0.6: 1, even more preferably about 0.5: 1.

Embodiment 10. The nanoparticle for use according to any one of the preceding embodiments, wherein the nanoparticle does not comprise any antidiabetic drug. Embodiment 11. The nanoparticle for use according to any one of embodiments 1 to 9, wherein the nanoparticle further comprises an antidiabetic drug other than glibenclamide.

Embodiment 12. A composition comprising at least one nanoparticle as defined in any one of the preceding embodiments and a pharmaceutically acceptable excipient for use in the treatment and/or prevention of diabetes.

Embodiment 13. The nanoparticle for use according to any one of embodiments 1 to 11 or the composition for use according to embodiments 12, wherein diabetes is type-2 diabetes or prediabetes.

Embodiment 14. Non-therapeutic use of a nanoparticle as defined in any one of embodiments 1 to 11 for extending the lifespan of an organism.

Embodiment 15. Non-therapeutic use of a composition comprising at least a nanoparticle as defined in any one of embodiments 1 to 11 and a carrier acceptable in food or nutraceutics, for extending the lifespan of an organism.

The following examples represent specific embodiments of the present invention. They do not intend to limit in any way the scope of the invention defined in the present description.

EXAMPLES

Materials and methods

1. Material

Zein, lysine, poly(ethylene glycol) 35.000 Da (PEG35), Nile red, Orlistat, glucose, mannitol, agarose, sodium hydroxide, aprotinin, Rose Bengal sodium salt, sodium phosphate monobasic, 5 -fluoro-2’ -deoxyuridine (FUdR), 2’,7’-dichlorodihydro fluorescein diacetate (H2DCF-DA) and hypochlorite sodium were purchased from Sigma- Aldrich (Steinheim, Germany and St Louis, MO). Ethanol (HPLC grade), DPPIV inhibitor and sodium chloride were obtained from Merck (Darmstadt, Germany). Lumogen® Red was provided by BASF (Germany). O C T ™ Compound Tissue-Tek was purchased from Sakura Finetek Europe (Alphen aan Der Rijn, The Netherlands). Deionized water (18.2 MQ-cm resistivity) was prepared by a water purification system (Wasserlab, Pamplona, Spain).

2. Preparation of nanoparticles

2.1. Preparation of bare nanoparticles (NP)

Empty nanoparticles

Zein nanoparticles were prepared by a desolvation method as described previously [R. Penalva et al., J. Agric. Food Chem., 2015, 63(23), 5603-5611], with minor modifications, followed by an ultrafiltration purification step and subsequent spraydrying. Briefly, 200 mg zein and 30 mg lysine were dissolved in 20 mL ethanol 55% with magnetic stirring for 10 minutes at room temperature. Nanoparticles were formed by the addition of 20 mL purified water. The ethanol was removed using a rotatory evaporator under reduced pressure (Buchi Rotavapor R-144; Buchi, Postfach, Switzerland) and the resulting suspension was concentrated and purified by tangential filtration. Finally, the suspension was dried in a Buchi Mini Spray Dryer B-290 apparatus (Buchi Labortechnik AG, Flawil, Switzerland) under the following experimental conditions: (i) inlet temperature, 90 °C; (ii) outlet temperature, 45-50 °C; (iii) air pressure, 4-6 bar; (iv) pumping rate, 5 mL/min; (v) aspirator, 80%; and (vi) airflow, 400- 500 L/h.

Lumogen® red-loaded nanoparticles

For Lumogen® red-loaded nanoparticles, 2.6 mL of a Lumogen® red solution at 0.4 mg/mL was added to the solution of zein and lysine, prior to the formation of the nanoparticles. After incubation, the nanoparticles were formed, purified and dried as described above.

2.2. Preparation of PEG-coated nanoparticles

The coating of nanoparticles with PEG35 was performed by simple incubation between the just formed nanoparticles (before the purification step) and PEG 35,000 at different PEG-to-zein ratios. For this purpose, a stock solution of PEG 35,000 was prepared by dissolving the polymer in water to a final concentration of 100 mg/mL. Then, different volumes of this stock solution were added to the suspension of freshly nanoparticles. The mixture was maintained under magnetic agitation for 30 minutes at room temperature. After this time, nanoparticles were concentrated and purified by tangential filtration and drying as descried above.

Fluorescently-labelled nanoparticles were prepared by encapsulating Lumogen® red as described in section 2.1.

3. Physico-chemical characterization of nanoparticles

3.1. Size, polydispersity index, zeta potential and yield

Particle size and polydispersity index (PDI) were measured by photon correlation spectroscopy (PCS) after dispersion in ultrapure water (1/10), at 25 °C, by dynamic light scattering (angle of 90°). Zeta-potential (Q was determined by electrophoretic laser Doppler anemometry after the dispersion of nanoparticles in purified water as follows: 200 pL of the samples were diluted in 2 mL of a 0.1 rnM KC1 solution adjusted to pH 7.4. All the already mentioned measurements were performed using a Zetasizer analyzer system (Brookhaven Instruments Corporation, New York, USA). The shape and surface morphology of the nanoparticles was studied by scanning electron microscopy (SEM) after resuspension in water.

3.2. Yield of zein forming nanoparticles

The amount of zein forming nanoparticles was estimated by two different techniques. One technique consisted on the quantification of the zein present in the elute obtained from the purification/concentration step. The amount of zein present in the elute was quantified spectrophotometrically by measuring the absorbance at 300 nm wavelength. Two standard curves were added to the procedure, one containing increasing concentrations of pure zein dissolved in ethanol 70%, and, another one containing the same dilutions but in the presence of lysine at the same proportion as the one used to formulate the nanoparticles (15%, w/w). The other technique consisted on a capillary electrophoresis quantification of the nanoparticles after centrifugation, as purification step. For that purpose, 5 mg of the nanoparticle formulation was dispersed in water and centrifuged at 15,000 x g for 20 min. Supernatants were discarded and the pellets were digested with ethanol 75%. Then, the amount of protein was quantified by capillary electrophoresis-Experion system (Bio-Rad Laboratories, Hercules, CA). Samples were analyzed under reducing and non-reducing conditions according to the manufacturer’s instructions (Experion Pro260 Analysis kit; Bio-Rad Laboratories). A solution of molecular mass protein standards (ladder) included in the kit was used to determinate the content of protein in a sample. For data analysis, Experion software version 3.10 (BioRad Laboratories) was used. The amount of protein forming nanoparticles in the formulation was estimated as the ratio between the amount of the protein quantified in the pellet of the centrifuged samples and the total amount of protein used for the preparation of nanoparticles and expressed in percentage.

3.3. Surface hydrophobicity evaluation

The surface hydrophobicity of the formulations was evaluated using the Rose Bengal test [S. Doktorovova et al., Eur. J. Pharm. Sci., 2012, 45(5), 606-612] with some minor modifications. Briefly, 500 pL of nanoparticle suspensions (from 0.03 to 3 mg/mL) was mixed with 1 mL of a Rose Bengal aqueous solution (100 pg/mL). All samples were incubated under constant shaking at 1500 rpm, for 30 min at 25 °C (Labnet VorTemp 56 EVC, Labnet International, Inc.). Afterwards, the samples were centrifuged at 13,500 x g for 30 min (centrifuge MIKRO 220, Hettich, Germany). The amount of Rose Bengal in the supernatants was calculated by measuring the absorbance at 548 nm, using a microplate reader (BioTek PowerWave XS, USA). Further, the total surface area (TSA) of each sample was plotted against the partitioning quotient (PQ) calculated in accordance with the following equation:

Rose Bengal Bound PQ = - r; - , „ . - ; (equation 3]

Rose Bengal Unbound

The slope of the line of the chart represents the hydrophobicity of the formulation. The higher the slope, the higher the hydrophobicity.

4. Ex vivo mucus diffusion studies in porcine intestinal mucus

4.1. Collection and preparation of porcine mucus The native porcine mucus was harvested from small intestine. The intestines were collected from the slaughterhouse and kept in ice-cold PBS (for a maximum period of 2 h) prior to the mucus collection. Intestine was cut into small portions that were opened to expose the lumen. Then, the exposed lumen was cleaned with PBS and the mucus collected using a spatula. The scraping was very gently in order not to drag epithelial tissue. A single pool of mucus was obtained, which was then distributed in 0.5 g-aliquots in microtubes that were frozen at -80 °C.

4.2. Evaluation of the diffusion of nanoparticles in mucus by Multiple Particle tracking (MPT)

The diffusion of the nanoparticles through pig intestinal mucus, as an in vitro measurement of their mucus-permeating properties, was assessed by the Multiple Particle Tracking (MPT) technique [M. Abdulkarim et al., Eur. J. Pharm. Biopharm., 2015, 97, 230-238; and J. Rohrer et al., Eur. J. Pharm. Biopharm., 2016, 98, 90-97], MPT involves video capturing and post-acquisition analysis for the individual movement of hundreds of fluorescently labelled particles within a mucus matrix [J. GrieBinger et al., Eur. J. Pharm. Biopharm., 2015, 96, 464-476], 25 pL of a suspension of fluorescently labelled nanoparticles (0.002%) were inoculated into approximately 0.5 g of mucus aliquots. Then, each sample was incubated in gently agitation for 2 h at 37 °C in order to ensure effective particle distribution before the video recording. 2-dimensional videos were captured in a Leica DM IRB wide-field epifluorescence microscope (*63 magnification oil immersion lens) using a high-speed camera (Allied Vision Technologies, UK) capturing 30 frames/second; 10 s videos (i.e. complete video comprised 300 frames). At least 100 individual trajectories were tracked and analyzed from each mucus inoculated with fluorescent nanoparticles. The MPT of each formulation was carried out in triplicates, leading to a minimum of 300 individual trajectories assessed.

Videos were analyzed using Fiji (Image J). Only trajectories longer than 30 frames were considered, in order to ensure a continuous presence of the individual nanoparticles in the X-Y plane. The trajectory of each nanoparticle was then converted into numeric pixel data and, finally, into metric distances (based on the recording settings). The displacement of each nanoparticle overtime was expressed as the squared displacement (SD), and the mean square displacement (MSD) was calculated as the geometric mean of that nanoparticle’s squared displacement along its entire trajectory. MSD was determined as follows:

MSD = (XAt)² + (KAt)² [equation 4]

For each nanoparticle formulation studied, the “ensemble mean square displacement” (<MSD>) was determined for each of the replicates by calculating the geometric mean of the 100 individual trajectories. Then, the effective diffusion coefficient (Deff) of each formulation was calculated by:

Where 4 is a constant relating to the 2-dimensional mode of video capturing and At is the selected time interval.

In parallel, the diffusion of the nanoparticles in water (D^Q) was calculated by the Stokes- Einstein equation at 37°C:

D^Q = kT6nr\r [equation 6]

In which K is the Boltzmann constant, T is absolute temperature, q is water viscosity and r is the mean radius of nanoparticles.

Finally, the diffusion of all the formulations was expressed as the ratio (%) between their Deff and their D^Q (diffusions in mucus and in water, respectively). This ratio provides a measure of the relative diffusion of the nanoparticles in mucus when considering their Brownian motion in water.

5. In vivo evaluation of nanoparticles using C. elegans

5.1. Strains and culture conditions

Caernoharbditis elegans (C. elegans) was maintained and cultured as described previously [S. Brenner, Genetics, 1974, 77, 71-94], Wild-type N2 Bristol strain was obtained from the Caenorhabditis Genetics Center (CGC, University of Minnesota, MN), and were cultured at 20 °C on NGM (Nematode Growth Medium) agar with Escherichia coll OP50 as normal nematode diet. For all experiments, age-synchronized worms were obtained by hypochlorite treatment, a condition in which only eggs can survive, and eggs were let hatch overnight in M9 buffer solution [J. GrieBinger et al., Eur. J. Pharm. Biopharm., 2015, 96, 464-476],

5.2. Nanoparticles intake

To evaluate the intake of nanoparticles by the worms, Lumogen® red-loaded nanoparticles were supplemented to the growth medium of C. elegans. After the drying of the supplemented NGM plates, 50 pL of a culture of OP50 were added over the solid- NGM-containing plates and let dry in darkness. Finally, about 500 adult worms were added to each well and incubated at 20 °C for 2 hours. After incubation time, worms were collected and fixed in glass slides with 2% agarose supplemented with sodium azide (1%) to kill the worms. Then, the already fixed worms were observed with a Nikon eclipse 80i epi-fluorescent microscope using the rhodamine filter to see the Lumogen®-loaded nanoparticles. The contrast image of the whole worms was obtained with the DAPI filter since C. elegans shows autofluorescence in that range.

5.3. In vivo efficacy of the nanoparticles in C. elegans

Treatment with nanoparticles

The efficacy evaluation was performed as described previously [D. Lucio et al., Eur. J. Pharm. Biopharm., 2017, 121, 104-112], with minor modifications. All assays were performed by triplicate in 6-well cell culture plates containing 4 mL of glucose- supplemented (0.5%; w/v) NGM, with or without treatments. Orlistat (6 pg/mL) was used as a positive control of fat reduction in C. elegans [P. Martorell et al., J. Agric. Food Chem., 2012, 60(44), 11071-11079], The effect of the formulations was evaluated by adding a suspension of empty nanoparticles: 1.6 mg/mL of NP or 2.5 mg/mL of NP50. Moreover, a negative control of NGM without glucose was added to ensure the increased fattening associated to the high glucose conditions. All the treatments were added to the plates after dilution/resuspension in purified water. After the addition of the treatments to the liquid-NGM-containing wells, they were mixed and allowed to solidify and dry in a dark environment. Then, 50 pL of a culture of OP50 were added over the solid-NGM- containing plates and let dry in darkness. Finally, about 500 LI synchronized worms were added to each well and incubated at 20 °C for 46 hours.

Fat content quantification

The fat content quantification in the nematodes was performed by the fixative-based the Nile red method [E. C. Pino et al., J. Vis. Exp., 2013, 73, e50180; and D. Navarro-Herrera et al., Food Fund., 2018, 9(3), 1621-1637], Briefly, L4 worms grown under the different conditions were harvested and washed with 0.01% of Triton X-100 in phosphate-buffered saline (PBST) and fixed in 40% isopropanol. Staining was performed by adding Nile Red solution (3 pg/mL) and incubating in the dark. After that, the worms were washed in PBST and mounted on a 2% agarose pad for microscopy visualization. Fluorescent images of Nile Red stained worms were captured at 80* magnification on a Nikon SMZ18 stereomicroscope (Nikon Instruments Inc., Tokyo, Japan) equipped with an epifluorescence system and a DS-FI1C refrigerated color digital camera. The images were taken under a GFP filter (Ex 480-500, DM 505; BA 535-550). Image analysis was performed using ImageJ program.

Lifespan assays

The lifespan assay was monitored at 20 °C under high glucose conditions (HG) [J. Apfeld and C. Kenyon, Nature, 1999, 402(6763), 804-809], NGM plates containing 40 mM of FUdR were used to prevent the growth of new worms. The pre-fertile period of adulthood was used as time zero (t = 0). Nematodes were regarded as dead if they do not move after repeated mechanical stimulus. All experiments were repeated at least three times and -100 worms were used for each experiment.

Measurement of intracellular ROS

Intracellular ROS in C. elegans was quantified using the molecular probe H2DCF-DA as described previously [Z. Wang et al., Exp. Gerontol., 2016, 82, 139-149], For ROS detection, 60 worms at certain ages were harvested for each treatment and washed with PBST. The worms were resuspended in M9 buffer. Then fresh H2DCF-DA was added to a final concentration of 50 pM. Samples were analyzed in the black flat bottomed 96-well plate every 30 min for a total period of 180 min in a fluorescence spectrophotometer (TECAN, Grbdig, Austria) with an automatic microplate reader at excitation/emission wavelengths of 485 and 525 nm. At least three independent replicates were performed.

Lipofuscin accumulation

Age-synchronous L4 larvae were cultured in the presence of FUdR (100 pM) and treated with or without 5 mg/L NP for 10 days for intestinal lipofuscin accumulation test. Ten- day old wild-type nematodes were collected on a microscope slide with 2% agar pad and anesthetized with sodium azide (1%). The autofluorescence of lipofuscin was captured by a Nikon eclipse 80i epifluorescent microscope. The experiment was performed in triplicate. The relative fluorescence intensity was quantified using ImageJ software to determine the lipofuscin levels.

Glucose concentration

Glucose levels in C. elegans were quantified as described [M. Mendler et al., Diabetologia, 2014, 55(2), 393-401], with some modifications. Approximately 1000 worms for each treatment were harvested and washed using PBST buffer. Pelleted worms were then lysed by beat beating in homogenization buffer (PBST, 10 mmol/L NP40) containing a protease inhibitor cocktail (Complete; Roche Diagnostics, Risch-Rotkreutz, Switzerland). Nematodes were homogenised using a mini-Beadb eater (Biospec Products, Bartlesville, OK) at speed 10 for 2 x 2 min. Then, worm homogenates were centrifuged at 16,000 x g for 5 min, and the supernatant fractions were assays for protein concentration (BCA Protein Assay Kit; Thermo Fisher Scientific, Waltham, MA) and glucose concentration (Accu-Check Aviva; Roche Diagnostics, Risch-Rotkreutz, Switzerland).

RNA extraction and quantitative PCR analysis

Total RNA was extracted from L4 worms with TRIzol™ isolation reagent (Invitrogen Life Technologies Inc., Paisley, UK). The purity and concentration of RNA were determined at 260/280 nm using a NanoDrop spectrophotometer (Thermo-Fisher Scientific, Wilmington, DE). RNA purification and reverse transcription were performed as described [68], Quantitative RT -PCR was carried out in a CFX348 Touch™ Real-Time PCR detection system (BioRad Laboratories, Hercules, CA) using the TaqMan ™ Universal PCR master mix and the corresponding specific probes from Life Technologies (TaqMan ™ Gene Expression Assays, Applied Biosystems, Foster City, CA). Gene expression levels were normalized compared to the expression of peroxisomal membrane protein related gene (pmp-S) as housekeeping control. Gene expression difference between NP treated and untreated samples were estimated using the relative quantification 2'^AACt.

6. In vivo evaluation of nanoparticles in healthy rats

6.1. Strain and housing conditions

Male Wistar rats weighing 180-220 g were purchased from Envigo (Indianapolis, USA). Animals were housed under controlled temperature (23 ± 2 °C) with 12-hour light/dark cycles and with free access to normal chow and water. All experiments were performed after a minimum acclimation period or 7 days. Prior to any procedure, animals were fasted overnight. During the procedures, animals were kept fasted but with free access to water. All the procedures were performed following a protocol previously approved by the “Ethical and Biosafety Committee for Research on Animals” at the University of Navarra in line with the European legislation on animal experiments.

6.2. In vivo evaluation of the mucus-permeating properties of nanoparticles in healthy rats

These studies were carried out using a protocol previously approved (045-18). The biodistribution of the nanoparticles in the gastrointestinal tract of healthy rats was evaluated as previously described [H. H. Salman et al., Vaccine, 2007, 25(48), 8123— 8132], with minor modifications. Briefly, male Wistar rats were fasted overnight but with water ad libitum and then orally administered with a suspension of fluorescently labelled nanoparticles. Distribution of the nanoparticles in the gastrointestinal mucosa was visualized by fluorescence microscopy. For that purpose, 25 mg Lumogen® F red- labelled nanoparticles were orally administered to rats after resuspension in 1 mL of purified water. Two hours post-administration, animals were sacrificed by cervical dislocation and the guts were removed. Duodenum and caecum portions of 1 cm were collected, washed with PBS, and frozen at -80 °C after inclusion in the tissue proceeding medium O C T.™ Compound. Five pm sections were cut out from each sample using a cryostat and attached to glass slides. Finally, the slices were fixed with formaldehyde and stained with the contrasting agent 4',6-diamidino-2-phenylindole (DAPI) for 15 min before the cover assembly. The presence of fluorescently loaded zein nanoparticles in the intestinal mucosa and the cell nuclei of intestinal cells, dyed with DAPI, were visualized in a fluorescence microscope (Axioimager Ml, Zeiss) with a coupled camera (Axiocam ICc3, Zeiss) and fluorescent source (HBO 100, Zeiss). The images were captured with the software ZEN (Zeiss). The post-acquisition processing of the images was carried out with the software Fiji (Image J).

6.3. Evaluation of the hypoglycemic effect of nanoparticles

For this experiment, healthy rats were divided in different groups of 6 animals each. Each group of animals received orally one of the following treatments: (i) dispersion of free zein in water (50 mg/kg), (ii) zein nanoparticles dispersed in water (NP; 50 mg/kg), (iii) PEG-coated zein nanoparticles prepared at a polymer-to-zein ratio of 0.5 (PEG-NP; 50 mg/kg). All these formulations were dispersed in 1 mL water prior administration to animals through oral gavage, using a stainless-steel cannula. As controls, one group of animals received orally 1 mL water. At different times, 300 pL of blood samples were collected, from the tail vein, into K3EDTA tubes at To (just before oral administration of the treatments). Protease inhibitors (DPP-4 inhibitor, 50 pM; aprotinin 500 KIU) were added to the tubes just after the collection of the blood. At each extraction, glucose levels in blood were quantified using a glucometer (Accu-Check® Aviva glucometer; Roche Diagnostics, Basel, Switzerland). The blood-containing tubes were centrifuged at 2500 g for 10 minutes at 4 °C. The plasma was withdrawn, placed into new microtubes and stored at -80 °C until the quantifications of GLP-1, and insulin were performed.

Rat insulin was quantified using ELISA kit (EZRML13K; Merck; Darmstadt, Germany) and GLP-1 was quantified using an ELISA kit (YK160 GLP-1 EIA Kit; Yanaihara Institute Inc; Awakura, Japan).

6.4. Intraperitoneal Glucose Tolerance Test (ipGTT) in healthy rats

The ipGTT was performed to evaluate the change in the glycemic control of rats receiving different treatments when facing an intraperitoneal injection of glucose (2 g/kg). For this purpose, rats were divided into 4 different groups (n = 6): (i) untreated control, receiving 1 mL of ultrapure water; (ii) free zein dispersed in 1 mL water (50 mg/kg); NP or uncoated zein nanoparticles (1 mL; 50 mg/kg); and PEG-NP or PEG-coated nanoparticles (at PEG- to-zein ratio of 0.5; 1 mL; 50 mg/kg). All the animals were fasted overnight prior to any procedure.

Treatments were administered by oral gavage 2 hours before the intraperitoneal injection of glucose. Blood glucose levels were measured at several times, before and after the intraperitoneal injection of glucose: -120 minutes (just before oral administration of nanoparticles), To (time of the intraperitoneal injection of glucose) and 15, 30, 60 and 120 minutes after the glucose overload. Blood samples (50 pL) were collected from the tail vein and the glycemia was measured using a glucometer (Accu-Check® Aviva glucometer; Roche Diagnostics, Basel, Switzerland).

7. In vivo evaluation of nanoparticles in SAMP8 mice

Male SAMP8 mice with 7 months of age were randomly divided into two groups (n = 10): untreated controls (receiving water) and treated with nanoparticles at a dose of 200 mg/kg every two days. SAMP8 mice were used due to their reduced lifespan, associated with an accelerated age-related degeneration [V. Karuppagounder et al., Ageing Res. Rev., 2017, 35, 291-296; E. M. Rhea and W. A. Banks, Exp. Gerontol., 2017, 94, 64-68; J. F. Flood and J. E. Morley, Neurosci. Biobehav. Rev., 1997, 22(1), 1-20; and T. Tsuduki et al., Nutrition, 2011, 27(3), 334-337] 7-month-old mice were chosen because this strain develops a normal growth until the age of around 6 months, when they start developing the typical age-related characteristics and pathologies [K. Yamamoto et al., Nutrition, 2016, 32(1), 122-128], Moreover, only males were used because it has been reported that female mice show less cognitive degeneration [J. E. Morley et al., Biochim. Biophys. Acta - Mol. Basis Dis., 2012, 7522(5), 650-656], The formulation was prepared as described previously and resuspended in water before administration. All the animals were administered through oral gavage, using a stainless-steel cannula. Animals were obtained from Harlan (Harlan Iberica, Barcelona, Spain) and housed under controlled temperature conditions (22 ± 1 °C), with 12-hour light/dark cycles. Food and water were available ad libitum. All the procedures were performed following a protocol previously approved by the “Ethical and Biosafety Committee for Research on Animals” at the University of Navarra in line with the European legislation on animal experiments (protocol 026-18).

To gather maximum life span data, animals were allowed to get older and die naturally. Moribund animals were immediately euthanized by cervical dislocation. Animals were considered as moribund if they fail to eat or drink, did not respond to touch stimuli, became completely blind (due to the degeneration of typical periophthalmic lesions [Y. Nomura and Y. Okuma, Neurobiol. Aging, 1999, 20(2), 111-115]) or developed tumors. The weight of the mice was followed up during the experiment.

8. In vivo evaluation of nanoparticles in a high-fat diet-induced obesity rat model

8.1. Strain and housing conditions

Wistar male rats were purchased from Envigo (Envigo Research Models and Services, Indianapolis, IN). The experiments began after acclimating the animals for at least 2 weeks under constant conditions of temperature (22 ± 1 °C), humidity (50% ± 10%), and artificial dark/light cycles (12 h each) with free access to standard control diet (2014, Teklad, Global 14% Protein Rodent Maintenance Diet) and water. All the procedures were performed following a protocol previously approved by the “Ethical and Biosafety Committee for Research on Animals” at the University of Navarra in line with the European legislation on animal experiments (protocol 016-21).

8.2. Experimental design

For the study, 36 rats with an initial average weight of 100 g ± 24 were randomly divided into three groups (n=12 rats per group). One group (control-diet group, SC) was fed with standard control diet (2014, Teklad, Global 14% Protein Rodent Maintenance Diet), whereas the two other groups received a high fat/high sucrose (HFS) diet (DI 2451, Research Diets) for 112 days (16 weeks). Water was allowed ad libitum to all groups. Body weight was measured weekly.

After 16 weeks and with average weight of 550 g ± 35, one of the HFS groups, named HFS-NP, received daily a single dose of zein nanoparticles (112 mg/kg; approx. 62 mg/day for animal) for 6 weeks. For this purpose, the formulation was prepared as described previously and dispersed in 1 mL water prior administration to animals through oral gavage, using a stainless-steel cannula. As controls, the animals belonging to both SC and HFS groups received orally 1 mL water. Prior to procedure, animals were fasted for 8 hours and, after administration, the water and food were supplied ad libitum. Body weight was measured weekly.

At the end of the 6 weeks treatment period, food was removed 8 h before sacrifice and the animals were euthanized by decapitation, trunk blood was collected, and plasma and serum samples were obtained for the biochemical analysis. Tissue samples from liver, white adipose tissue (WAT) depots (retroperitoneal, epididymal, and subcutaneous) were isolated, weighed and immediately stored at -80 °C.

8.3. Body composition analysis

Total body fat and lean mass were determined for each animal at week 5 of the treatment period by quantitative magnetic resonance spectroscopy using the EchoMRI™ system (Echo Medical Systems, Houston, TX). The scans were achieved by placing live animals into a thin-walled plastic cylinder (3 mm thick, 6.8 cm internal diameter), and a cylindrical plastic insert to limit the rat movement. While in the cylinder, the animals were briefly subjected to a low-intensity electromagnetic field (0.05 Tesla) for 2 min (Nixon et al., Obesity (Silver Spring). 2010; 18(8): 1652-9). Measures of body fat and lean mass proportions were performed in triplicate and were calculated relative to total body weight, together with the relation between fat and lean mass proportions at the end of the study.

8.4. Biochemical analysis

Triacylglycerides (TAG), total cholesterol, and high-density lipoprotein (HDL) cholesterol were measured in serum using a Pentra C200 clinical chemistry Analyser (HORIBA ABX, Montpellier, France) with the corresponding commercial kits (ABX Pentra, Montpellier, France). The concentrations of monocyte chemotactic protein- 1 (MCP-1) in plasma were quantified using a specific ELISA kit (Thermo Scientific and Life Technologies). Atherogenic index of plasma (AIP) was calculated as the quotient between TAG and HDL, and expressed as log (Bahadoran et al., Int J Food Sci Nutr. 2012; 63(7):767-71).

8.5. Hepatic determination of the triglyceride content

An aliquot of 100 mg of liver from each animal was mixed with 1 mL homogenization buffer (PBS) and then lysed by beat beating. The tissues were homogenized using a miniBeadbeater (Biospec Products, Bartlesville, OK) at speed 50 x 100 rpm for 1 min. The homogenates were transferred to a glass tube and mixed with 5 mL of 2: 1 chloroform/methanol mixture (v/v), vortexed for 10 min, and incubated overnight at 4 °C. After incubation, samples were vortexed and centrifuged at 1650 xg for 10 min at 4 °C. The bottom phase (chloroform) was collected in a clean tube. In the first tube, 1.5 ml of chloroform and 600 pL of 4 rnM MgCh were added to the upper phase; samples were incubated 30 min on ice and centrifuged (1650 xg, 10 min, 4 °C). Bottom phase (chloroform) was collected and added to the chloroform phase collected from first centrifugation. For all samples, 4 mL of this chloroform phase were mixed with 600 pL of Triton X-100 (1% (v/v) in chloroform) and evaporated at 40 °C using a Vacuum Evaporation System (Labconco Corp., MO). Finally, the dried extract was dissolved in 600 pl of water. The triglyceride concentration was measured using a triglyceride quantification Kit (Merck, Darmstadt, Germany) according to the manufacturer's instructions

8.6. Histopathological studies

A small portion of liver tissues was collected from all animals at the end of the study. Liver tissues were extracted and fixed in 3.7-4% formaldehyde buffered to pH = 7 and stabilized with methanol for histological analysis (Panreac Quimica S.L.U., Barcelona, Spain). The liver pads were then embedded in paraffin, sectioned (5-pm thick) and stained with haematoxylin-eosin dye for microscopic observations. Digital images were acquired with a Vectra/Polaris 3.0.3 scanner system (Akoya Biosciences) at a magnification of 40*.

8.7. Statistical analysis The means and standard errors were calculated for every data set. All the group comparisons and statistical analyses were performed using a one-way ANOVA test followed by a Tukey-Kremer multicomparison test. In all cases, p < 0.05 was considered as a statistically significant difference. All calculations were performed using Graphpad Prism v6 (California, USA).

9. Statistical analysis

The means and standard errors were calculated for every data set. All the group comparisons and statistical analyses were performed using a one-way ANOVA test followed by a Tukey-Kremer multicomparison test. In all cases, p < 0.05 was considered as a statistically significant difference. All calculations were performed using Graphpad Prism v6 (California, USA) and the curves were plotted with the Origin 8 software from Origin Lab (Massachusetts, USA).

Results

1. Nanoparticles characterization

The main physico-chemical characteristics of empty nanoparticles coated at different PEG-to-zein ratios are shown in Table 1. The coating with PEG did not significantly modify the size and zeta potential of the formulations, compared to naked nanoparticles (NP). All the formulations showed an average size of about 210 nm with poly dispersity index (PDI) values always lower than 0.1. The zeta potential of the formulations was always negative, with values around -50 mV. Regarding the yield of the process, expressed as the amount of zein transformed into nanoparticles, this parameter was close to 80%, when quantified spectrophotometrically or by capillary electrophoresis.

Table 1. Physico-chemical characteristics of empty zein nanoparticles without coating (NP) or coated with PEG at different PEG-to-zein ratios (expressed as percentages: 5%, 25%, 50% and 75%). Data expressed as mean ± SD, n > 3.

The morphology and surface analysis by scanning electron microscopy (SEM) showed that the formulations consisted of a homogeneous population of spherical-shaped nanoparticles. Size values obtained by this technique were similar to those obtained by photon correlation spectroscopy. Both coated and uncoated nanoparticles showed a smooth surface (Figure 1 A and IB).

2. Hydrophobicity evaluation

The results of the Rose Bengal test showed a direct relationship between the PEG-to-zein ratio and the surface hydrophobicity of the nanoparticles. The higher PEG-to-zein ratio, the higher reduction in the surface hydrophobicity (Figure 2).

3. Diffusion of nanoparticles in mucus

Figure 3 shows a comparison of the normalized values of diffusion in mucus (taking the capability of NP to diffuse in mucus as 1). Overall, significant differences were found for the different formulations tested. As expected, there is a direct relationship between the PEG-to-zein ratio of the coating of the nanoparticles and their capability to diffuse through the mucus. The higher PEG-to-zein ratio, the higher mucus diffusion. Thus, for PEG-coated nanoparticles at a ratio of 0.75 (NP75), the diffusion coefficient in mucus was 9 times higher than for uncoated nanoparticles (NP). However, no significant differences were found between nanoparticles coated at ratios of 0.25, 0.5 and 0.75.

4. In vivo evaluation of nanoparticles using C. elegans

4.1. Nanoparticles intake

Fluorescence micrographs of worms cultured in normal conditions or cultured in medium supplemented with Lumogen-loaded nanoparticles evidence the intake of nanoparticles by C. elegans. Fluorescently labelled nanoparticles can be observed along the gastrointestinal tract of the worm 2 hours after culture.

4.2. In vivo efficacy of the nanoparticles in C. elegans Glucose metabolism

In order to evaluate a potential effect of zein nanoparticles on the metabolism of glucose, the concentration of glucose in C. elegans was quantified (Figure 4). Under our experimental conditions, the glucose concentration on control worms was about 160 mmol/mg protein, whereas for worms treated with zein nanoparticles, the levels of glucose were significantly lower than in the control (about 35%; p < 0.001).

Fat accumulation

The effect of free zein and zein-based nanoparticles on the accumulation of fat by nematodes are presented in Figure 5. Values are normalized to the fat content quantified in control worms (NGM). Orlistat (Orl), used as positive control, reduced by 42% the fat content of worms while the treatment with free zein did not show any significant difference when compared to controls. On the contrary, both naked and PEG-coated nanoparticles showed significant differences in the fat accumulation by worms (10.82 and 14.18% reduction, respectively).

Lifespan

A high glucose diet has been associated with reduced lifespan in C. elegans [A. Schlotterer et al., Diabetes, 2009, 55(11), 2450-2456; and S. J. Lee, C. T. Murphy, and C. Kenyon, Cell Metab., 2009, 10(5), 379-391], For this reason the effect of zein nanoparticles on the lifespan of C. elegans under this condition was evaluated. The lifespan of untreated (control) and zein-NP (NP) treated wild-type nematodes was compared. NP treatment caused a significant increased mean lifespan (24.5 ± 0.4 days, p < 0.001), while in the untreated worms this value was 17.11 ± 0.28 days. In addition, the maximum lifespan was also lengthened by NP from 24.98 to 34.87 days. For comparisons, the mean and median lifespan of C. elegans treated with NP was 43% and 38% higher than for control worms. In a similar way, the maximum lifespan value for the animals treated with nanoparticles was 39.6% higher than for the control nematodes.

Lipofiicsin

In order to check the capability of zein nanoparticles in delaying aging, lipofuscin (as a model physiological marker of aging) was measured. Lipofuscin is an endogenous autofluore scent marker of cellular damage during aging in many organisms, including C. elegans [Z. Pincus and F. J. Slack, Dev. Dyn., 2010, 239(5), 1306-1314; and N. Papaevgeniou et al., Free Radic. Biol. Med., 2017, 108, S48], Interestingly, the presence of this biomarker in the nematodes treated with zein nanoparticles was two times lower than for control ones (3470 relative fluorescence units (rfu) vs 1703 rfu, p<0.001).

Oxidative stress

In order to gain insight into the effect of zein nanoparticles on the lifespan of worms, the levels of oxidative stress in the worms, ROS formation and expression of antioxidant stress genes were determined. The effect of NP on the intracellular ROS levels of NP- treated C. elegans was quantified during aging using the ROS indicator dichlodilhydro- fluorescein diacetate (DUE) [Z. Wang et al., Exp. Gerontol., 2016, 82, 139-149], Control nematodes showed an increase in ROS until 9^th day, thereafter a light decline in ROS levels was detected (Figure 6). The treatment with NP reduced ROS formation all along the period of aging evaluated; these ROS levels were similar to that found in standard conditions. In all the experiments, the NP were administered in LI larvae until 18^th-day of adulthood. Additionally, NP were administered in worms on the first day of adults until 18^th-day adulthood, skipping treatment during the early stages of development (LI to L4 larvae stages). The results showed that NP reduced ROS formations by 1.8-fold when compared with control nematodes (Figure 6).

In C. elegans, Daf-16 and SKN-1 are two classical transcription factors that are closely related to oxidative stress and longevity [X. Sun, W.-D. Chen, and Y.-D. Wang, Front. Pharmacol., 2017, 8, 548; and T. K. Blackwell et al., Free Radic. Biol. Med., 2015, SS(Pt B), 290-301], To examine this transcription factors, synchronized wild-type LI worms were treated with or without NP for 6 days. The qRT-PCR results showed a NP upregulation of transcriptional expression levels of daf-16 and skn-1, by 1.8 and 2.1-fold respectively, compared with their expression levels in control worms (Figure 7). The NP supplementation in worms after first stages of life (LI to L4 larvae stages) until 9^th adulthood showed an increased by 1.2 and 0.6 fold of daf-16 and skn-1 expression levels, respectively. 5. Distribution of nanoparticles in healthy rats

Fluorescence micrographs of duodenum slices obtained 2 hours post-administration of different Lumogen-loaded formulations to animals showed that both bare and PEG- coated nanoparticles at a PEG-to-zein ratio of 0.05 displayed a localization that seemed to be restricted to the mucus layer that covers the epithelium, without presence between intestinal villi. Nevertheless, PEG-coated nanoparticles at higher PEG-to-zein ratios (0.25 and 0.5) were clearly seen in close contact with the intestinal epithelium, occupying the inter-villi spaces and even reaching the intestinal crypts. Regarding the biodistribution of the same formulations in the caecum of rats 2 hours post-administration, no red strokes could be seen in caecum of rats treated with naked nanoparticles nor nanoparticles coated with PEG at PEG-to-zein ratios of 0.05 and 0.25. Only nanoparticles coated with PEG at a PEG-to-zein ratio of 0.5 could be seen in the caecum slices.

6. In vivo evaluation of the hypoglycemic effect of nanoparticles in healthy rats

Figure 8 shows the blood glucose levels of healthy male Wistar rats treated with the different treatments. The glycemia of rats treated with free zein at a dose of 50 mg/kg showed no differences compared to the control rats, which received water. On the contrary, when animals were treated with zein nanoparticles at the same dose, the blood glucose levels decreased significantly during the whole experiment. NP induced a 25% reduction in the glycemia 3 hours after the oral administration, reduction that was maintained for at least 6 hours. In the case of rats treated with NP50, the glycemic decrease was found to be 28% 3 hours after the oral administration, being slightly greater than those treated with NP. Moreover, in this case, the decrease increased to a 32% 6 hours after the administration, being significantly greater than the reduction induced by NP.

At the same extraction points at which the glycemia was evaluated, different hormones related to the glucose homeostasis were quantified. Figure 9 shows the concentration in blood of the different hormones after the administration of the treatments with the nanoparticles of the invention and the control group, normalizing the values to the initial concentrations. Figure 9A displays the changes in the insulin concentration of the rats treated with the different treatments. A reduction in the insulinemic values can be seen in the control animals, which received only water. 1.5 hours after the administration, control animals showed a 52% reduction in the insulinemia when compared to the initial values. This decrease reached the value of 96% 3 hours after the beginning of the experiment and 91% for 6 hours after. This reduction in the insulin levels, almost below the detection limit, can be associated with the use of isoflurane as anesthetic agent, which has demonstrated this effect [H. Zardooz et al., Physiol. Res., 2010, 59(6), 973-8], Regarding the groups treated with NP and NP50, both showed the same pattern: a slight decrease in the blood levels of insulin during the first 3 hours of experiment, partially countering the effect of isoflurane; and, a great increase 6 hours after the administration, achieving the values obtained from the animals treated with free zein. NP -treated animals showed a 23% decrease in the insulin levels in blood 1.5 hours after the administration. This decrease was reduced to 3% 3 hours after the administration. Finally, 6 hours postadministration of the naked nanoparticles, a 64% increase in the insulinemia was found, compared to the initial values. NP50 showed lesser effect than NP in stimulating the insulin release in rats, although no statistical differences were found. The changes in the insulinemia of NP50-treated animals was a 32% and 28% decrease 1.5 and 3 hours after the administration, followed by a 67% increase 6 hours post-administration.

The changes in the GLP-1 levels in blood are shown in Figure 9B. Control animals maintain values similar to the initial ones during the whole experiment, what is in concordance with results previously reported, in which the oral administration of water to rats did not modify the GLP-1 blood levels [M. Hayakawa et al., Biochem. Biophys. Res. Commun., 2018, 496(3), 898-903], In contrast, animals treated with naked nanoparticles show an 8% increase in the GLP-1 levels 1.5 hours after the administration, compared to the initial values. This increase raised along time, displaying a 26% increase after 3 hours and 39% after 6 hours. The NP50 treated group showed the highest increase in GLP-1 blood levels. A 20% increment was observed 1.5 hours after the oral administration of PEG-coated nanoparticles, increment that raised to 29% and 59% 3- and 6-hours post-administration, respectively.

7. Intraperitoneal Glucose Tolerance Test (ipGTT) in healthy rats The response to a glucose overload in rats was evaluated through an intraperitoneal glucose tolerance test. Two hours after the oral administration of treatments, rats were injected with a glucose solution equal to 2 g/kg. Figure 10A shows the changes in the glycemia of rats during the 2 hours after the challenge with glucose injection. All the treatments significantly reduced the glycemic increase 15 minutes after the challenge, compared to the controls. Only the group treated with NP50 showed a significant reduction in the glucose rise 30 minutes after the glucose injection, leading to a very significant decrease compared to control and free zein groups. From this point on, no differences were found among all treatments. Regarding the areas under the curve (AUC), which represent the total exposure to the glucose rise in rats, only the group treated with NP50 showed a significant decrease (Figure 10B). Only a 4% reduction in the AUC was found for both free zein treated group, compared to the control group while this reduction reached 11% for the NP50 treated group.

8. In vivo evaluation of nanoparticles in SAMP8 mice

For the study of the effect of zein nanoparticles (NP) dispersed in water on the survival of SAMP8 mice, the animals were treated for 13 months, receiving an oral dose of 200 mg/kg every 48 hours. The survival curves are represented in Figure 11. The NP -treated group displays a right shift of the curve, which represents an increment in the lifespan of the mice (p<0.05). This group (NP) shows also a delay in the age of the first death, median (with a 32% increase in the median lifespan expectancy) and maximum lifespan (the maximum lifespan of mice increased by 40% when treated with NP) (Table 1). When the area under the curve (AUC) of both survival curves was calculated, a 26% increase was observed for NP -treated mice.

Table 1. Summary of the effect of bare zein nanoparticles (NP) administration on the median, mean and maximum survival for SAMP8 mice.

9. In vivo evaluation of nanoparticles in a high-fat diet-induced obesity rat model Figure 12 represents the modifications in the body composition of obese rats (expressed as the fat content relative to the body weight and as the fat mass-to-lean mass ratio) when fed daily with zein nanoparticles during 6 weeks. Interestingly, after 6 weeks, supplementation of HFS-fed animals with zein nanoparticles induced a significant decrease of the fat content (about 24%; p<0.01), compared with control animals (HFS). The weight of adipose tissues is presented in Figure 13. Supplementation of HFS-fed animals clearly decreased the mass of retroperioneal (20%; p<0.01), epididymal (19%; p<0.01), subcutaneous (19%; p<0.001) and visceral fat (18%; p<0.05), when compared with control animals (HFS group).

Figure 14 compiles the biochemical parameters of lipid profile in animals at the end of the study. Serum HDL-Cholesterol and total cholesterol were similar for animals included in the HFS and the HFS-NP groups. On the contrary, the triglycerides (TAG) concentration were significantly lower in HFS-fed animals receiving a daily dose of zein nanoparticles (42% reduction) with respect with control HFS animals (p<0.001). Interestingly, these serum levels of TAG in the HFS-NP animals were similar to that quantified in the SC group. In addition, supplementation of HFS-fed animals with zein nanoparticles induced a significant reduction of their atherogenic index (38% reduction; p<0.001), reaching values similar to those observed in animals fed with the standard diet (SC) and indicating that NP consumption ameliorated the harmful cardiovascular effects of the HFS feedings.

Furthermore, the supplementation of HFS-fed animals with zein nanoparticles abolished the increased secretion of the pro-inflammatory cytokine MCP-1 observed in HFS-fed animals (Figure 15); reaching similar values that those observed in animals fed with the standard diet (SC group).

Figure 16 shows the levels of triglycerides in the liver of animals. For this purpose, a portion of the liver was analyzed. Again, supplementation of HFS-fed animals with zein nanoparticles significantly attenuated the typical lipid steatosis of these animals (about 30% reduction in the levels of triglycerides; p<0.01). Finally, histopathological studies (data not shown) revealed that the HFS diet led to an enlargement of the hepatocytes and an increase in the number of lipid droplets in the liver compared to the SD group. Likely, photomicrographs of hematoxylin-and-eosin-stained sections from HFS-fed animals supplemented with zein nanoparticles (revealed liver structures similar to those of animals fed with the standard diet (SC group).

Discussion

One of the objectives of the present study was to investigate whether oral administration of zein nanoparticles improves glucose tolerance in healthy rats. The results show that in a fasted state, zein nanoparticles induced an important decrease in the glucose blood levels (between 25-30%, compared with basal levels, 3 hours post-administration), while control animals did not show any change in the glycemia. This reduction in the blood glucose levels induced by zein nanoparticles, but not by control, seems to be due to their GLP-1 stimulating effect.

Another of the objectives of the present study was to investigate the effect induced by zein nanoparticles on lifespan and longevity of a C. elegans model and a mouse model with accelerated aging (SAMP8). The results showed that the treatment with zein nanoparticles prolonged of lifespan in both nematodes (about 35%) and aged mice (about 30%) compared to their respective controls. In C. elegans, NP treatment mitigated the negative effect of high-glucose conditions lead to an increase of the mean and maximum lifespan of nematodes. This lifespan extension may be related to a protective effect against the damage induced by the ROS derived from high-glucose conditions. An important factor for aging is the accumulation of oxidative stress caused by an increase in intracellular ROS levels. In this study, the administration NP led to a significant decrease of intracellular ROS levels in worms, even when NP exposure is started at adulthood. Another biomarker of oxidative damage is the accumulation of lipofuscin in the intestinal cells of the worms. The treatment with NP reduced lipofuscin accumulation, demonstrating that NP not only increases the lifespan but also retards aging in C. elegans. It is well known that genetic pathways and biochemical processes that modulate aging and longevity are strongly conserved between C. elegans and mammals [S. K. Kim, J. Exp. Biol., 2007, 270(9), 1607-1612; and A. Bitto et al., Cold Spring Harb. Perspect. Med., 2015, 5(11), a025114]. Thus, the longevity-improving effects ofNP in mice may be attributed, at least in part, to the anti-aging mechanisms described above for C. elegans. However, the lifespan-extension in the mice might also be related to an improvement or restoration of some metabolic functions. Aging is associated with a lot of metabolic changes that reduce the lifespan, including a decline in the modulation of metabolic function and insulin sensitivity [T. Finkel, Nat. Med., 2015, 27(12), 1416-1423], Recently, SAMP8 mice have been proposed as a model to study age-related metabolic complications. The short-life of these mice is related to the development of adipocyte hypertrophy and ectopic lipid accumulation in the liver that increase insulin resistance and lead to hyperglycemia and impaired glucose tolerance at 40-week-old [H. W. Liu et al., Exp. Gerontol., 2017, 99(1), 61-68], We found that supplementation with zein nanoparticles delays the age of the first death and increases the median and the maximum lifespan of the mice. These results suggest that NP-treatment may be mitigating these age- related metabolic disorders and, consequently, improving lifespan.

Another of the objectives of the present study was to investigate the effect of zein nanoparticles in obesity and metabolic syndrome. Numerous studies have implicated abdominal obesity as a major risk factor for insulin resistance, Type-2 diabetes mellitus, cardiovascular disease, stroke, metabolic syndrome and death (Huffman et. al, Biochimica et biophysica acta, 2009, 1790(10), 1117-1123; Liu et al., J Med Internet Res., 2021; 23(8):e24017; Wood et al., PLoS ONE, 2021, 16(11): e0258545). Daily supplementation of zein nanoparticles, at a dose of 112 mg/kg (equivalent in humans to ca. 18-33 mg/kg), decreases significantly the accumulation of fat in obese animals, including visceral fat. This effect is accompanied of a significant decrease of both serum and hepatic TAG level, the former associated to increased cardiovascular risk and the latter to hepatic steatosis that can drive to non-alcoholic fatty liver disease (NAFLD), insulin resistance and type-2 diabetes. Moreover, this effect is accompanied of a marked decrease (to control normal levels) levels of MCP-1 (Monocyte Chemoattractant Protein- 1), a key mediator of low-grade inflammation widely associated with insulin resistance and recruitment of macrophages in adipose tissue (Varma et al, Am. J. Physiol. - Endocrinol. Metab., 2009, 296m el300-el310). All these results suggest that zein nanoparticle may be useful for the prevention and /or treatment of obesity and metabolic syndrome.

Claims

1. A nanoparticle comprising a zein matrix and a basic amino acid for use in the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation, wherein the nanoparticles does not comprise any biologically active ingredient.

2. The nanoparticle for use according to claim 1, wherein the basic amino acid is selected from the group consisting of lysine, arginine, histidine and mixtures thereof.

3. The nanoparticle for use according to claim 2, wherein the basic amino acid is lysine.

4. The nanoparticle for use according to any one of the preceding claims, wherein the weight ratio of zein to basic amino acid is in the range of from 0.01 : 1 to 50: 1, preferably from 0.5: 1 to 25: 1, more preferably from 1 : 1 to 20: 1, more preferably from 5: 1 to 15: 1, even more preferably from 6: 1 to 7: 1.

5. The nanoparticle for use according to any one of the preceding claims, wherein its average size is in the range of from 100 to 450 nm, preferably from 150 to 250 nm.

6. The nanoparticle for use according to any one of the preceding claims, wherein the condition characterized by increased glucose levels and/or fat accumulation is selected from the group consisting of diabetes, obesity and metabolic syndrome.

7. A composition comprising at least one nanoparticle as defined in any one of claims 1 to 5 and a pharmaceutically acceptable excipient for use in the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation.

8. The composition for use according to claim 7, wherein the condition characterized by increased glucose levels and/or fat accumulation is selected from the group consisting of diabetes, obesity and metabolic syndrome.

9. Use of a nanoparticle as defined in any one of claims 1 to 5 in the manufacture of a medicament for the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation.

10. Use according to claim 9, wherein the condition characterized by increased glucose levels and/or fat accumulation is selected from the group consisting of diabetes, obesity and metabolic syndrome.

11. Use of a composition comprising at least one nanoparticle as defined in any one of claims 1 to 5, and a pharmaceutically acceptable excipient in the manufacture of a medicament for the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation.

12. Use according to claim 11, wherein the condition characterized by increased glucose levels and/or fat accumulation is selected from the group consisting of diabetes, obesity and metabolic syndrome.

13. Method of treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation comprising administering to a subject in need thereof a nanoparticle as defined in any one of claims 1 to 5.

14. Method according to claim 13, wherein the condition characterized by increased glucose levels and/or fat accumulation is selected from the group consisting of diabetes, obesity and metabolic syndrome.

15. Method of treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation comprising administering to a subject in need thereof a composition comprising at least one nanoparticle as defined in any one of claims 1 to 5 and a pharmaceutically acceptable excipient.

16. Method according to claim 15, wherein the condition characterized by increased glucose levels and/or fat accumulation is selected from the group consisting of diabetes, obesity and metabolic syndrome.

17. Non-therapeutic use of a nanoparticle as defined in any one of claims 1 to 5 for extending the lifespan of an organism.

18. Non-therapeutic use of a composition comprising at least a nanoparticle as defined in any one of claim 1 to 5 and a carrier acceptable in food or nutraceutics, for extending the lifespan of an organism.

19. A nanoparticle comprising a zein matrix, a basic amino acid and polyethylene glycol or a derivative thereof for use in the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation.

20. The nanoparticle for use according to claim 19, wherein the basic amino acid is selected from the group consisting of lysine, arginine, histidine and mixtures thereof.

21. The nanoparticle for use according to claim 20, wherein the basic amino acid is lysine.

22. The nanoparticle for use according to any one of claims 19 to 21, wherein the weight ratio of zein to basic amino acid is in the range of from 0.01 : 1 to 50: 1, preferably from 0.5: 1 to 25: 1, more preferably from 1 : 1 to 20: 1, more preferably from 5: 1 to 15: 1, even more preferably from 6: 1 to 7: 1.

23. The nanoparticle for use according to any one of claims 19 to 22, wherein the polyethylene glycol or derivative thereof has a molecular weight in the range of from 400 Da to 40 kDa, preferably from 10 to 40 kDa, more preferably from 20 to 40 kDa, even more preferably about 35 kDa.

24. The nanoparticle for use according to any one of claims 19 to 23, wherein the polyethylene glycol is polyethylene glycol 35000 (PEG35). 25. The nanoparticle for use according to any one of claims 19 to 24, wherein the weight ratio of the polyethylene glycol or derivative thereof and zein is in the range of from 0.05: 1 to 1 :5, preferably from 0.05: 1 to 1 : 1, preferably from 0.

25: 1 to 1 : 1, more preferably from 0.4:1 to 0.6: 1, even more preferably about 0.5: 1.

26. The nanoparticle for use according to any one of claims 19 to 25, wherein its average size is in the range of from 100 to 450 nm, preferably from 150 to 250 nm.

27. The nanoparticle for use according to any one of claims 19 to 26, wherein the nanoparticle does not comprise any antidiabetic drug.

28. The nanoparticle for use according to any one of claims 19 to 26, wherein the nanoparticle further comprises an antidiabetic drug other than glibenclamide.

29. The nanoparticle for use according to any one of claims 19 to 28, wherein the condition characterized by increased glucose levels and/or fat accumulation is selected from the group consisting of diabetes, obesity and metabolic syndrome.

30. A composition comprising at least one nanoparticle as defined in any one of claims 19 to 28 and a pharmaceutically acceptable excipient for use in the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation.

31. The composition for use according to claim 30, wherein the condition characterized by increased glucose levels and/or fat accumulation is selected from the group consisting of diabetes, obesity and metabolic syndrome.

32. Use of a nanoparticle as defined in any one of claims 19 to 28 in the manufacture of a medicament for the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation.

33. Use according to claim 32, wherein the condition characterized by increased glucose levels and/or fat accumulation is selected from the group consisting of diabetes, obesity and metabolic syndrome.

34. Use of a composition comprising at least one nanoparticle as defined in any one of claims 19 to 28, and a pharmaceutically acceptable excipient in the manufacture of a medicament for the treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation.

35. Use according to claim 34, wherein the condition characterized by increased glucose levels and/or fat accumulation is selected from the group consisting of diabetes, obesity and metabolic syndrome.

36. Method of treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation comprising administering to a subject in need thereof a nanoparticle as defined in any one of claims 19 to 28.

37. Method according to claim 36, wherein the condition characterized by increased glucose levels and/or fat accumulation is selected from the group consisting of diabetes, obesity and metabolic syndrome.

38. Method of treatment and/or prevention of a condition characterized by increased glucose levels and/or fat accumulation comprising administering to a subject in need thereof a composition comprising at least one nanoparticle as defined in any one of claims 19 to 28 and a pharmaceutically acceptable excipient.

39. Method according to claim 38, wherein the condition characterized by increased glucose levels and/or fat accumulation is selected from the group consisting of diabetes, obesity and metabolic syndrome.

40. Non-therapeutic use of a nanoparticle as defined in any one of claims 19 to 28 for extending the lifespan of an organism.

41. Non-therapeutic use of a composition comprising at least a nanoparticle as defined in any one of claim 19 to 28 and a carrier acceptable in food or nutraceutics, for extending the lifespan of an organism.