ITRM20130727A1 - NANOCOINTURERS INCLUDING SOLID LIPID NANOPARTICLES COATED WITH A HYDROPHILIC SHELL. - Google Patents

Description

<Nanoconstructs comprising solid lipid nanoparticles coated with a hydrophilic shell>

DESCRIPTION

The present invention relates to nanoconstructs comprising at least one solid lipid nanoparticle containing at least one pharmacologically and / or nutraceutically active substance coated with a cross-linked polymer shell and their use for the administration of food supplements and drugs.

State of the art

Many drugs and dietary supplements demonstrate prominent in vitro biological activity but do not produce relevant effects when administered in vivo. This difference in activity is often attributable to a lack of bioavailability, due to poor absorption, rapid metabolism and / or low stability. Some compounds are very lipophilic and are not effectively administered in aqueous preparations. Other compounds are very hydrophilic and therefore cannot diffuse across cell membranes but require specific transport (eg glucose transporters). The problems listed so far can be solved, in whole or in part, by incorporating the active principle in colloidal dispersions of nanometric size. These so-called "nanosystems" keep the substances in aqueous suspension and in biological fluids, making it possible for the body to absorb them in different ways. In the field of nanotechnology there is a wide range of different formulations for the administration of drugs / supplements. Types include: polymeric nanoconstructs [US 2010/0303922; US 2007/0190160; US 2008/0248126; US 2008/0241257], solid lipid nanoconstructs (SLN) [US 2006/0222716; US 2008/0206341], nanoemulsions [US 2007/0148194; US 2011/0045050; US 5,152,923], vesicles [US 2009/0011004; US 2010/0267846], liposomes [US 4356167; US 6761901], nanocapsules with a liquid core [US 6713533] or hybrid compositions [US 2008/0102127]. Nanoemulsions, vesicles and liposomes are characterized by low stability. Polymer nanoconstructs and the like often exhibit biocompatibility problems due to allergic reactions or degradation products of polymers. Among all these preparations, the Solid Lipid Nanoparticles, SLN constitute a good compromise between biocompatibility and stability. Said SLNs can be prepared starting from food-grade lipids and therefore completely biocompatible and biodegradable, at the same time possessing greater stability compared to nanoemulsions, vesicles and liposomes. SLNs in the current state of the art are however administered parenterally as they are unable to withstand extreme conditions such as digestion. A further weak point of the SLN is given by the poor stability in storage at uncontrolled temperature due to aggregation phenomena.

A more refined approach than the simple SLN involves a lipid core with an adsorbed layer on the surface composed of linear polycaprolactone polymers [Frozza et al, J Biomed Nanotechnol, 2010, 6 (6): 694-703], polyelectrolytes (sodium polystyrenesulfonate, polyacrylic acid, polylysine) [US 2006/0083781], lectin [US 2007/0237826] or chitosan [M. Garcia-Fuentes, C. Prego, D. Torres, M.J. Alonso, Eur J Pharmaceutical Sci, Volume 25, Issue 1, May 2005, Pages 133-143]. In these examples the polymers are simply adsorbed on the SLN surface by means of weak hydrophobic interactions or electrostatic interactions with a charged surfactant present in the lipid core (in which case careful pH control is required to maintain an adequate charge in the cover layer. ).

Fessi's group has formulated an interesting example of oil drops enclosed in a polyurea or polyester shell generated by polycondensation at the oil / water interface [US 7,348,031]. This last type of composition guarantees good stability in storage; however the described procedure only allows to obtain a liquid type core. The procedure described in US 7,348,031 was not applicable to a solid lipid: in fact, by using it, monodisperse nanoconstructs are not obtained.

The problem was therefore felt in the state of the art of obtaining SLN capable of withstanding extreme conditions such as digestion and, therefore, not necessarily administrable by parenteral route, which possessed greater stability in storage at uncontrolled temperature, as mentioned limited in the particles. of the state of the art from aggregation phenomena, and that they could be produced without the limitations known by the procedures of the state of the art.

This problem has been solved by the inventors through the present invention which relates to the nanoconstruct of independent claims 1 and 2; further embodiments are reported in the dependent claims.

Brief description of the figures

Three figures are attached to this description which represent:

Figure 1 is a graph of the size distribution of the encapsulated solid lipid nanoconstructs of example 1;

Figure 2: the plasma concentration of resveratrol and resveratrol sulfate after the administration of resveratrol (circles and squares in the graph) and of the encapsulated solid lipid nanoconstructs (triangles in the graph) of example 7 and

Figure 3: plasma concentration of resveratrol and resveratrol sulfate after the administration of resveratrol (circles and squares in the graph) and of the encapsulated solid lipid nanoconstructs (triangles in the graph) of example 8.

Detailed description of the invention

The present invention relates to a nanoconstruct comprising a solid lipid nanoparticle containing at least one pharmacologically active and / or nutrient substance (active ingredients) enclosed in a shell composed of cross-linked polymer. The core is composed of one or more solid lipids at room temperature and immiscible with water. The active ingredients are dispersed within the lipid core. The shell that surrounds and encloses the core, which cannot be obtained with SLN according to the methods known from the state of the art, derives from the reaction of monomers, oligomers, and / or polymers at the surface of the core. The shell formation reaction occurs at the interface between the core and the aqueous solution without chemical bonds being made between the core and the shell. In this way a cross-linked polymer is obtained which confers resistance to aggregation, degradation, decomposition and / or digestion. The presence of the shell prevents contact between active ingredients and enzymes making it possible to use bioreversible derivatives of the active ingredients, in particular derivatives that can be hydrolysed very easily and therefore poorly stable such as esters, carbonates, silyl ethers, etc ... a barrier against coalescence and sintering of the nanoconstructs, increasing their stability to aggregation. The lack of crosslinking (linear polymer adsorbed on the SLN) causes an increase in the polydispersity of the nanoconstructs over time which is an index of disintegration.

In the production of solid lipid nanoconstructs (SLNs) to be inserted into nanoconstructs, the inventors of the present invention, by analogy with what is reported by Sanna and colleagues [V. Sanna, A.

M. Roggio, S. Siliani, M. Piccinini, S. Marceddu, A. Mariani, and M. Sechi Int J Nanomedicine.

2012; 7: 5501–5516], had considered applicable the method of nano precipitation described by Fessi in US 7,348,031. Consequently, nanoconstructs - in the specific case described by Fessi in US 7,348,031 of lipid (solid) - containing resveratrol and covered with a layer of cross-linked chitosan would have been obtained. It was experimentally observed that the presence of at least one lipophilic component necessary for the creation of the outer shell was not compatible with the presence of solid lipids and therefore ultimately with the obtaining of SLN.

However, the inventors have surprisingly discovered that by operating the mixing at a temperature higher than the melting temperature of the lipid used for the production of SLN and lower than the boiling temperature of the solvent used, it was possible to overcome the described problem. Compared to US 7,348,031, the present invention also differs in that it is also possible to use polymers of biological origin, biocompatible, biodegradable and coming from renewable sources in the composition of the shell.

Furthermore, compared to the method used by Garcia-Fuentes [M. Garcia-Fuentes, C. Prego, D. Torres, M.J. Alonso, Eur J Pharmaceutical Sci, Volume 25, Issue 1, May 2005, Pages 133-143], the present invention does not require the use of extremely lipophilic and toxic solvents such as dichloromethane, nor the use of sophisticated equipment such as ultrasound, making the process more easily applicable by industry.

The presence of a cross-linked polymer shell in the nanoconstructs according to the present invention, instead of a linear polymer shell, entails greater stability of the nanoconstructs with respect to disintegration and re-aggregation phenomena. These phenomena occur spontaneously during the conservation of the nanoconstructs, both at room temperature and in the case of refrigeration, in particular in suspension, and contribute to the deterioration of the nano structure and therefore of its content.

The production of the nanoconstructs object of the invention involves the preparation of an organic solution and an aqueous solution and their mixing. The organic solution contains one or more organic solvents miscible with water, one or more lipids and one or more active ingredients (pharmacologically active substances and, or nutrients), one or more surfactants, one or more crosslinkers, monomers, oligomers or polymers (component lipophilic content of the shell) and may contain one or more preservatives and adjuvants. The aqueous solution contains one or more surfactants and one or more water-soluble crosslinkers, monomers, oligomers or polymers (hydrophilic component of the shell). In an alternative solution of the invention, such one or more water-soluble crosslinkers, monomers, oligomers or polymers (hydrophilic component of the shell) are added after mixing the organic and aqueous solutions. Both after mixing the organic and aqueous solution, and after the separate addition of the hydrophilic component of the shell in the case of the alternative solution, the mixing is maintained for 1 -5 hours. These crosslinkers are water-soluble compounds capable of having crosslinking reactions (for example through polycondensation) with the crosslinking agents of the lipophilic solution. The ratio between the volumes of the organic solution and the aqueous solution can vary between 2: 1 and 1:10.

The production procedure involves mixing the organic solution with the aqueous solution described above at a temperature higher than the melting temperature of the lipids contained in the organic solution and below the boiling temperature of the solvent used in order to obtain a nanometric emulsion of the type oil in water. As said, an alternative solution to the problem of the present invention is to add such one or more water-soluble crosslinkers after mixing the organic and aqueous solutions.

As organic solvents miscible with water, acetone, acetonitrile, acetic acid, formic acid, pyruvic acid, bis- (2-hydroxypropyl) -ether, 1,4-butanediol, 2,3-butanediol, butanedione, 2- ( 2-butoxyethoxy) -ethanol, butyrolactone, N, N-diethylformamide, N, N-dimethylacetamide, 2- (dimethylamino) ethanol, N, N-dimethylformamide, dimethyl sulfoxide, 1,4-dioxane, 1,3-dioxolane, 1, 2-ethanediol, ethanol, 2-ethoxyethanol, 2- (2-ethoxyethoxy) ethanol, 2- (2-ethoxyethoxy) ethyl acetate, ethyl lactate, N-ethylmorpholine, hexamethylphosphoramide, 2,5-hexanedione, glycerol, hydroxyacetone, 3- hydroxy-2-butanone, isopropanol, methanol, N-methylformamide, 2-methyl-2-propanol, N-methylpyrrolidine, N-methyl-2-pyrrolidinone, 3-methoxy-1-butanol, 2-methoxyethanol, 2- (2 -methoxyethoxy) ethanol, 2-methoxyethyl acetate, 1,3-propanediol, tetraethylene glycol, tetrahydrofuran, tetrahydropyran, 1,1,3,3-tetramethyl-urea, triethylene glycol, tripropylene glycol methyl ether. The boiling temperature of these solvents is between 55 and 290 ° C.

The solvents foreseen in the production can be partially or completely eliminated by dialysis, evaporation, lyophilization or centrifugation of the nanoconstructs. The choice of suitable solvents is preferably oriented towards those with low toxicity and a high vapor pressure. The preferred solvents are acetic acid, acetone, dimethyl sulfoxide, ethanol and tetrahydrofuran. Particularly preferred is acetone.

After the shell formation at the emulsion interface and then after the complete formation of the nanoconstruct, the temperature is brought back to room temperature.

The organic solution comprises one or more solid lipids at room temperature and immiscible with water which have a melting temperature lower than the boiling point of the organic solvent miscible with water used. By way of example, mono-di- and tri-glycerides can be mentioned; fatty acids; long-chain alcohols, aldehydes or ketones, fatty acid esters, waxes, sterols, and the like. Examples of said lipid compounds are: monocaprine, monocaprilin, monolaurin, monomyristin, monopalmitin, monostearin, monoelaidine, monoolein, monoerucine, glycerol behenate, dilaurin, dimyristine, dipalmitin, stearopalmitin, stearidomyristin, oleicoleomyristin, oleicoleomyristin, behenopalmitina, arachidobehenina, dicapropalmitina, dicaprostearina, dilaurocaprina, dilauromiristina, dilauropalmitina, dilaurostearina, sorbodimiristina, dimiristocaprina, laurodimiristin, dimiristopalmitina, dimiristostearina, dipalmitocaproina, dipalmitocaprina, dipalmitolaurina, dipalmitostearina, dipalmitoelaidina, dipalmitobehenina, distearocaproina, distearocaprina, distearolaurina, distearomiristina, distearooleina, distearopalmitina, distearoelaidin, dielaidopalmitin, dielaidostearin, palmitomyristolaurin, trilaurine, trimyristin, tripalmitin, tristearin, trimargarin, trielaidin, tribrassidine, trinonadecanoin, triparinarin, triarachidine, tribehenin, tripentadecanoin, tripetroselaidine, trilignocerin, tritridecanoin, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, methylpalmitic acid, margaric acid, stearic acid, isostearic acid, anteisononadenachecanoic acid, beecanoic acid , lignoceric acid, cerotic acid, heptacosanoic acid, montanic acid, nonacosanoic acid, melissic acid, parinaric acid, petroselaidic acid, nervonic acid, cyclohexanundecanoic acid, angelic acid, tiglic acid, vaccenic acid, calendic acid, jacaric acid, cibaric acid, acid lumequic acid, margarolic acid, punicic acid, traumatin, ximenic acid, cerebronic acid, hydroxypalmitic acid, arachidyl alcohol, melissil alcohol, montanil alcohol, sapiol, stenol, behenil alcohol, eicosanol, heptadecanol, ceryl alcohol, lignoceryl alcohol, heptacosanediol, 15, lacceril -entriacontanol, heptacosandione, tritriacontyl octacosanoate, octac osyl triacontanoate, tetratriacontyl octacosanoate, ethyl lignocerate, methyl stearate, methyl behenate, methyl lignocerate, lauryl stearate, cetyl palmitate, dotriacontyl hydroxycinamate, methyl hydroxymyristate, eugenyl palmitate, cetyl myristoleate, 3-glyceride-glycerides, sitosterol 3-sterol glycerides stigmasterol, ergosterol, stigmastanol. Particularly preferred are: trilaurine, trimyristin.

The organic solution comprises one or more active ingredients such as a drug, a prodrug, a parapharmaceutical or a mixture thereof, a nutrient or a mixture of nutrients, a plant extract, all cumulatively indicated as pharmacologically active substances and / or nutrients (PA) , dissolved in an organic solvent as indicated above. The pharmacologically active and / or nutritive substance selected must be soluble in the selected organic solvent (or mixture) and scarcely soluble in water. The structuring of the encapsulated solid lipid nanoconstructs makes it possible to use easily hydrolyzable and therefore highly bioreversible derivatives. The PA does not have to react with the shell precursors. In the case of highly hydrophilic and / or reactive PAs, it is desirable to use bioreversibly protected derivatives, in this case: alcohols, phenols and carboxylic acids in the form of esters; amines in the form of amides; alcohols, phenols and amines in the form of carbamates and all those derivatives known to those with proven technical experience in the pharmaceutical / nutraceutical sector. The encapsulation of plant extracts can be done easily when the extraction from the plant is carried out in a water-miscible solvent, as this extract can be used directly as a base for the organic solution. In the present description, applications are presented by way of example in which the PAs are resveratrol and / or its derivatives, among which the trisethoxymethoxy derivative, triacetate and tristearate are preferred.

The organic solution comprises one or more surfactants soluble in the selected organic solvent. Surfactants can be anionic, cationic, zwitterionic or non-ionic, for example: sodium / potassium / magnesium / calcium / aluminum caprate, sodium / potassium / magnesium / calcium / aluminum laurate, sodium / potassium / magnesium / calcium stearate, sodium oleate , sodium stearoyl 2-lactylate, lauroyl arginate, betaines, phosphatidylcholine and lecithins, sorbitan trioleate, sorbitan tristearate, polyethyleneoxide sorbitan stearate, polypropylene glycol stearate, sorbitan monooleate, polypropylene glycol monoxide, polypropylene glycol monolithene propane, polypropylene glycol monolithene propane, polypropylene glycol monolithene propane, polypropylene glycol monolithene propanate, polypropylene glycol monolithene propan . Particularly preferred is phosphatidylcholine.

The organic solution comprises one or more crosslinkers, monomers, oligomers or polymers (lipophilic component of the shell) characterized by two or more functional groups per molecule; chosen so as not to react with the organic solvent, with the active principle and with the lipid (or mixtures of them) used and with each other. The functional groups of the lipophilic component of the shell can be identical or different and must be selected to react selectively and under mild conditions with the crosslinkers, monomers, oligomers or polymers of the aqueous solution (hydrophilic component of the shell). The lipophilic component of the shell is composed of one molecule or more molecules carrying two or more electrophilic functional groups such as isocyanate, isothiocyanate, acyl halide, activated ester, aldehyde or acetal, alkyl halide, epoxide, anhydride, carbonate groups. The lipophilic components of the preferred group are: 1,1,3,3-tetraethoxypropane, isophthalic aldehyde, adipic aldehyde, muconic aldehyde, genipin, isophorone diisocyanate, lysine disocyanate, poly (propylene glycol) terminated with toluene 2,4-diisocyanate (CAS # 9057-91-4), poly (ethylene adipate) terminated with toluene 2,4-diisocyanate (CAS # 9019-92-5), bis (4-isothiocyanatebutyl) disulfide, tetraglycidyl 4,4'diamino diphenylmethane, resorcinol-diglycidyl- ether, bisepossimentene, limonene dioxide. Particularly preferred crosslinkers for the lipophilic component of the shell are poly [(phenyl isocyanate) -co-formaldehyde], poly (ethylene adipate) terminated with toluene 2,4-diisocyanate, bromoacetylbromide, tetraglycidyl 4,4'diamino diphenylmethane, resorcinol-diglycidyl- ether. The hydrophilic component of the shell, capable of reacting with the compounds of the lipophilic component of the shell just reported, is composed of one molecule or more molecules that carry two or more nucleophilic functional groups such as amino, hydrazide, alcohol and thiol groups. The hydrophilic components of the preferred group are: diamines, oligoamines, peptides, proteins and oligomeric or polymeric amino sugars. Particularly preferred are tetraethylenepentaamine, cystamine sulfate hydrate, spermine, lysine, arginine, polylysine, polyarginine, albumin, bovine serum albumin, casein, gelatin, lactalbumin and chitosan.

Alternatively, the lipophilic component of the shell can be composed of a molecule or molecules carrying two or more nucleophilic functional groups such as the amino or hydrazide group. Preferred lipophilic components of the shell are 1,6-diamino-hexane, adipic dihydrazide, zein. Particularly preferred is adipic dihydrazide. Said lipophilic component is able to react with a hydrophilic component composed of one molecule or more molecules carrying two or more electrophilic functional groups such as aldehyde, activated ester, epoxy groups. Preferred hydrophilic components of the shell are: periodate oxidized polysaccharides, epichlorohydrin modified polysaccharides, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide activated pectin. Particularly preferred is pectin activated with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide.

The aqueous solution must contain one or more hydrophilic surfactants commonly known as for example: poloxamers (PEO-PPO-PEO copolymers), polyethylene oxide monostearate, polyethylene oxide lauryl ether, polyethylene oxide monooleate, polyvinyl alcohol (PVA), polyvinyl dextridone, modified starch and starch , alginate, cellulose, methylcellulose, carboxymethylcellulose, furcellerano, guar gum, karaya gum, albumin, gelatin, casein. Preferred surfactants are Pluronix and polyvinylpyrrolidone.

Examples

Example 1: Preparation of nanoconstructs comprising encapsulated solid lipid nanoparticles

The aqueous phase is prepared by dissolving 12 mg of Pluronic <®> F127 in 8 mL of demineralized water and heating to a temperature of 53 ° C. The organic solution is prepared by dissolving 5 mg of poly [(phenyl isocyanate) -co-formaldehyde] (Mn≈ 340), 22 mg of poly (ethylene adipate) terminated with toluene 2,4-diisocyanate (Mn≈ 2700), 10 mg of L-α-phosphatidylcholine (from egg yolk) and 50 mg of trilaurine in 4 mL of acetone and heating the whole to 53 ° C. The two solutions are mixed together and immediately afterwards 200 microliters of tetraethylenepentaamine (25% in water) are added while stirring at 53 ° C for at least 3 hours. The Dinamic Light Scattering measurements of an encapsulated SLN dilute dispersion indicate an average diameter of 217 nm with a polydispersity index (PDI) of 0.128 as can be seen from the graph in figure 1.

Example 2: Preparation of nanoconstructs comprising solid lipid nanoparticles encapsulated with a cross-linked chitosan shell

The aqueous phase is the same as in example 1. The organic solution is prepared by dissolving 140 mg of bromoacetylbromide, 10.2 mg of L-α-phosphatidylcholine (from egg yolk) and 43 mg of trilaurine, in 4 mL of acetone and heating all at 53 ° C. After mixing the two solutions, 4 mL of 0.5% chitosan in 1.0% acetic acid in water are added and everything is kept under stirring at 53 ° C for at least 3 hours. Encapsulated SLNs have an average diameter of 176 nm with a polydispersity index (PDI) of 0.100.

After ten months of storage in aqueous suspension at room temperature and in the absence of additives, the nanocapsules demonstrate a high structural integrity, their average diameter is 189 nm with a polydispersity index (PDI) of 0.071.

Example 3: Preparation of nanoconstructs comprising solid lipid nanoparticles encapsulated with a cross-linked serum albumin shell

The aqueous phase is the same as in example 1. The organic solution is prepared by dissolving 137 mg of bromoacetylbromide, 10 mg of L-α -phosphatidylcholine (from egg yolk) and 43 mg of trilaurine, in 4 mL of acetone and heating all at 53 ° C. After mixing the two solutions, 4 mL of 1.3% bovine serum albumin in water are added and everything is kept under stirring at 53 ° C for at least 3 hours. Encapsulated SLNs have an average diameter of 145 nm with a polydispersity index (PDI) of 0.094.

Example 4: Preparation of nanoconstructs comprising solid lipid nanoparticles encapsulated with a cross-linked pectin shell

The aqueous phase is prepared by dissolving 12 mg of Pluronic <®> F127, 21 mg of pectin (from citrus peel) and 40 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide in 8 mL of demineralized water and heating the temperature of 53 ° C. The organic solution is prepared by dissolving 15.5 mg of adipic dihydrazide, 13 mg of L-α-phosphatidylcholine (from egg yolk) and 46 mg of trilaurine, in 4 mL of acetone and heating everything to 53 ° C. The two solutions are mixed together and the mixture is kept under stirring at 53 ° C for at least 3 hours.

Encapsulated SLNs have an average diameter of 238 nm with a polydispersity index (PDI) of 0.226.

Example 5: Preparation of nanoconstructs comprising encapsulated solid lipid nanoparticles containing a resveratrol derivative

The aqueous phase is the same as in example 1. The organic solution is prepared by dissolving 25 mg of poly [(phenyl isocyanate) -co-formaldehyde] (Mn≈ 340), 11 mg of L-α-phosphatidylcholine (from yolk of egg), 42 mg of trilaurine and 21 mg of resveratrol tristearate in 4 mL of acetone and heating the whole to 53 ° C. The two solutions are mixed together and immediately afterwards 400 microliters of tetraethylenepentaamine (25% in water) are added. It is mixed at 53 ° C for at least 3 hours. The encapsulated SLNs have an average diameter of 301 nm with a polydispersity index (PDI) of 0.150. The encapsulation efficiency, measured by UV spectrometry, is 85%.

Example 6 Preparation of nanoconstructs comprising encapsulated solid lipid nanoparticles containing beta-sitosterol and a resveratrol derivative

The aqueous phase is the same as in example 1.

The organic solution is the same as in example 5 except for the use of 30 mg of resveratrol tristearate and the addition of 23 mg of beta-sitosterol. The two solutions are mixed together and immediately afterwards 400 microliters of tetraethylenepentaamine (25% in water) are added. It is mixed at 53 ° C for at least 3 hours.

Encapsulated SLNs have an average diameter of 284 nm with a polydispersity index (PDI) of 0.229. The encapsulation efficiency, measured by UV spectrometry, is 96%.

Example 7: Preparation of nanoconstructs comprising solid lipid nanoparticles encapsulated with a cross-linked chitosan shell containing a resveratrol derivative - I

The aqueous phase is the same as in example 1.

The organic solution is prepared by dissolving 8,5 mg of 1,1,3,3-tetraethoxypropane, 15 mg of L-α-phosphatidylcholine (from egg yolk), 25,3 mg of resveratrol triacetate and 35 mg of trilaurine, in 4 mL of acetone and heating everything to 53 ° C. After mixing the two solutions, 4 mL of 0.5% chitosan in 1.0% acetic acid in water are added and everything is kept under stirring at 53 ° C for at least 3 hours.

Encapsulated SLNs have an average diameter of 228 nm with a polydispersity index (PDI) of 0.257. The encapsulation efficiency, measured by HPLC, is 90%.

Example 8: Preparation of nanoconstructs comprising solid lipid nanoparticles encapsulated with a cross-linked chitosan shell containing a derivative of resveratrol - II

The aqueous phase is the same as in example 1.

The organic solution is prepared by dissolving 26.1 mg of resveratrol triacetate, 3.1 mg of isophthalic aldehyde, 10 mg of picoline-borane complex, 10 mg of L-α-phosphatidylcholine (from egg yolk) and 43 mg of trilaurine , in 4 mL of ethanol and heating the whole to 53 ° C. After mixing the two solutions, 4 mL of 0.5% chitosan in 1.0% acetic acid in water are added and all kept under stirring at 53 ° C for at least 3 hours. Encapsulated SLNs have an average diameter of 220 nm with a polydispersity index (PDI) of 0.148. The encapsulation efficiency, measured by HPLC, is 97%.

Example 9: Pharmacokinetic study

The nanocapsules of example 7 and example 8 were administered intragastrically to male Wistar rats in such quantities as to provide 0.088 mmol / kg. Blood samples were taken at set times, treated and analyzed by HPLC to quantify the presence of resveratrol and resveratrol sulfate, the main metabolite. The data obtained are shown in table I (example 7) and in table II (example 8).

TABLE I

time (hours) 0 0.17 0.5 1 2 4 8 24

resveratrol 0 0.343 1.34 1.77 1.21 1.08 0.115 0 sulphate

(microM)

TABLE II

time 0 0.17 0.5 1 2 4 8 10 14 17 24

(hours)

resveratrol 0 0.124 0.286 1.03 1.24 0.468 0.223 0.162 0.185 0.155 0.0943 sulphate

(microM)

resveratrol 0 0.0203 0.0383 0.0680 0.0744 0.029 0 0 0 0 0

(microM)

The results of Table I and II are compared with the plasma concentrations of resveratrol e

resveratrol sulfate (metabolite of resveratrol which exhibits significant biological activity

albeit lower than that of resveratrol itself) obtained by administering an equivalent amount

of resveratrol in the graphs of figure 2 and figure 3. Summing the concentrations of resveratrol e

resveratrol sulfate an area under the curve is obtained for the formulations of resveratrol triacetate

encapsulated of Examples 7 and 8 equal to 8.17 µM • h and 7.44 µM • h, both considerably higher

to that for the simple administration of resveratrol in solution, which is equal to 3.55 µM • h.

Furthermore, a shift of the plasma peak at plus times is evident in both examples

long.

The administration through the nanocapsules object of the invention therefore allows respect

upon administration in solution, both the absorption of higher doses of the active ingredient and a

slowing of relative pharmacokinetics.

Example 10: Preparation of nanoconstructs comprising solid lipid nanoparticles

encapsulated with a cross-linked chitosan shell containing a resveratrol derivative

- III

The aqueous phase is the same as in example 1.

The organic solution is prepared by dissolving 27 mg of trisethoxymethoxy-resveratrol, 10 mg of

dodecandioyl-dichloride, 13 mg of L-α-phosphatidylcholine (from egg yolk) and 41 mg of trilaurine, in 4 mL of acetone and heating everything to 53 ° C. After mixing the two solutions, 4 mL of 0.6% chitosan in 1.0% acetic acid in water are added and all kept under stirring at 53 ° C for at least 3 hours.

Encapsulated SLNs have an average diameter of 654 nm with a polydispersity index (PDI) of 0.590. The encapsulation efficiency, measured by HPLC, is 76%.

Example 11: Preparation of nanoconstructs comprising solid lipid nanoparticles encapsulated with a cross-linked chitosan shell containing quercetin

The aqueous phase is the same as in example 1.

The organic solution is prepared by dissolving 18 mg of tetraethoxypropane, 9 mg of L-αphosphatidylcholine (from egg yolk), 17 mg of quercetin and 44 mg of trilaurin, in 4 mL of acetone and heating everything to 53 ° C. After mixing the two solutions, 4 mL of 0.5% chitosan in 1.0% acetic acid in water are added and everything is kept under stirring at 53 ° C for at least 3 hours. Encapsulated SLNs have an average diameter of 309 nm with a polydispersity index (PDI) of 0.294.

Example 12: Preparation of nanoconstructs comprising solid lipid nanoparticles encapsulated with a cross-linked serum albumin shell II

The aqueous phase is the same as in example 1.

The organic solution is prepared by dissolving 100 microliters of tetraglycidylmethylenedianiline solution at 16% in tetrahydrofuran, 14 mg of L-α-phosphatidylcholine (from egg yolk) and 54 mg of trilaurine, in 3.9 mL of acetone and heating everything at 53 ° C. After mixing the two solutions, 4 mL of 1.3% bovine serum albumin in water is added and everything is kept under stirring at 53 ° C for 20 hours.

Encapsulated SLNs have an average diameter of 178 nm with a polydispersity index (PDI) of 0.161.

Example 13: Preparation of nanoconstructs comprising solid lipid nanoparticles encapsulated with a cross-linked chitosan II shell

The aqueous phase is the same as in example 1 and the organic solution is the same as in example 12.

After mixing the two solutions, 4 mL of 0.6% chitosan in 1.0% acetic acid in water are added and the whole is kept under stirring at 53 ° C for 20 hours.

Encapsulated SLNs have an average diameter of 254 nm with a polydispersity index (PDI) of 0.255.

Example 14: Comparison between the behavior of solid lipid nanoparticles encapsulated with a cross-linked chitosan shell and solid lipid nanoparticle with chitosan adsorbed on the surface

Two different nanoconstructs were prepared.

Lot A: the same as in example 13.

Lot B: the same as in example 13 omitting the crosslinker tetraglycidylmethylenedianiline.

After measuring the polydispersity index (PDI) of the two constructs, the two batches were stored in aqueous suspension away from light and at a temperature of 4 ° C for 44 days. After this time the PDI measurement was repeated. In the case of lot A no variations were observed, while in the case of lot B the PDI underwent an increase of 39.5%, which may be associated with phenomena of disintegration and re-aggregation of the particles with non-cross-linked shells.

Example 15: Preparation of nanoconstructs comprising solid lipid nanoparticles encapsulated with a cross-linked chitosan III shell

The aqueous phase is prepared by dissolving 3 milligrams of Pluronic F127 in 2 milliliters of water. The organic solution is prepared by dissolving 2.1 milligrams of L-α-phosphatidylcholine (from egg yolk), 9.6 milligrams of trilaurine, 1.6 milligrams of resorcinol-diglycidyl-ether in 1 milliliter of acetone. The two solutions are heated to 56 ° C and mixed together.

After mixing the two solutions, 1 mL of 0.5% chitosan in 1.0% acetic acid in water is added, also at 56 ° C. Encapsulated SLNs have an average diameter of 187 nm with a polydispersity index (PDI) of 0.139.

Example 16: Preparation of nanoconstructs comprising solid lipid nanoparticles encapsulated with a cross-linked chitosan shell IV

The aqueous phase is prepared by dissolving 10 milligrams of polyvinylpyrrolidone (average molecular weight of 58 kDa) in 2 milliliters of water. The organic solution is the same as in example 15. The two solutions are heated to 56 ° C and mixed together. After mixing the two solutions, 1 mL of 0.5% chitosan in 1.0% acetic acid in water is added, also at 56 ° C. Encapsulated SLNs have an average diameter of 194 nm with a polydispersity index (PDI) of 0.162.

Example 17: Preparation of nanoconstructs comprising solid lipid nanoparticles encapsulated with a cross-linked chitosan shell V

The aqueous phase is the same as in example 15. The organic solution is prepared by dissolving 2.1 milligrams of L-α-phosphatidylcholine (from egg yolk), 9.6 milligrams of trilaurine, 1.9 milligrams of resorcinol-diglycidyl - ether in 1 ml of tetrahydrofuran. The two solutions are heated to 56 ° C and mixed together. After mixing the two solutions, 1 mL of 0.5% chitosan in 1.0% acetic acid in water is added, also at 56 ° C. Encapsulated SLNs have an average diameter of 334 nm with a polydispersity index (PDI) of 0.162.

Example 18: Preparation of nanoconstructs comprising solid lipid nanoparticles encapsulated with a cross-linked chitosan and cystamine shell

The aqueous phase is the same as in example 16. The organic solution is prepared by dissolving 3.2 milligrams of L-α-phosphatidylcholine (from egg yolk), 4.7 milligrams of trilaurine, 4.5 milligrams of trimyristin, 5 , 4 milligrams of resorcinol-diglycidyl-ether in 1 milliliter of tetrahydrofuran mixture: dimethyl sulfoxide 50:50 v / v. The two solutions are heated to 65 ° C and mixed together. After mixing the two solutions, 1 mL of solution containing 5 mg of chitosan, 10 mg of acetic acid, 18 mg of cystamine sulfate hydrate (CAS # 16214-16-7) in water is added, also at 56 ° C . Encapsulated SLNs have an average diameter of 430 nm with a polydispersity index (PDI) of 0.237.

Claims (1)

CLAIMS 1. Nanoconstructs formed by a cross-linked polymer shell enclosing at least one solid lipid nanoparticle containing at least one pharmacologically active substance and / or at least one nutrient obtainable by the process comprising the following operations: to. preparation of an organic solution comprising at least one pharmacologically active substance and / or at least one nutrient substance, at least one organic solvent miscible with water, at least one solid lipid at room temperature, at least one fat-soluble compound with at least two functional groups capable of having reactions of crosslinking, at least one lipophilic surfactant; b. preparation of an aqueous solution comprising at least one hydrophilic surfactant; c. mixing said solutions at a temperature higher than the melting temperature of said at least one lipid, and lower than the boiling temperature of said at least one water-miscible organic solvent; d. addition of at least one water-soluble compound with at least two functional groups capable of having cross-linking reactions with at least two functional groups of said at least one fat-soluble compound And. mixing and crosslinking reaction, f. cooling to room temperature 2. Nanoconstructs formed by a cross-linked polymer shell enclosing at least one solid lipid nanoparticle containing at least one pharmacologically active substance and / or at least one nutrient obtainable by the process comprising the following operations: to. preparation of an organic solution comprising at least one pharmacologically active substance and / or at least one nutrient substance, at least one organic solvent miscible with water, at least one solid lipid at room temperature, at least one fat-soluble compound with at least two functional groups capable of having reactions of crosslinking, at least one lipophilic surfactant; b. preparation of an aqueous solution comprising at least one hydrophilic surfactant and at least one water-soluble compound with at least two functional groups capable of having cross-linking reactions with said at least two functional groups of said at least one fat-soluble compound; c. mixing of said solutions at a temperature higher than the melting temperature of said at least one lipid and lower than the boiling temperature of said at least one water-miscible organic solvent, d. and crosslinking reaction And. cooling to room temperature 3. Nanoconstruct according to claim 1 or 2, wherein the ratio between the volumes of said organic solution and said aqueous solution is comprised between 2: 1 and 1:10. 4. Nanoconstruct according to at least one of the preceding claims, wherein said at least one solid lipid at room temperature is selected from the class consisting of triglycerides, diglycerides, monoglycerides, fatty acids, long-chain alcohols, waxes, sterols, stanols. 5. Nanoconstruct according to at least one of the preceding claims, wherein said at least one fat-soluble compound capable of undergoing cross-linking reactions is selected from the class formed by compounds containing two or more isocyanate, isothiocyanate, acyl halide, activated ester, aldehyde or acetal groups, alkyl halide, epoxide, anhydride, carbonate and said at least one water-soluble compound capable of having cross-linking reactions with said at least one fat-soluble compound is selected from the class of compounds containing two or more amino, hydrazide, alcohol and thiol groups. 6. Nanoconstruct according to claim 5, wherein said at least one fat-soluble compound capable of undergoing cross-linking reactions is selected from the class formed by compounds containing an amino or hydrazide group: 1,6-diamino-hexane, adipic dihydrazide, zein and said at least one water-soluble compound capable of having cross-linking reactions with said at least one fat-soluble compound is selected from the class: polysaccharides oxidized with periodate, polysaccharides modified with epichlorohydrin, pectin activated with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide. 7. Nanoconstruct according to claim 5, wherein said at least one fat-soluble compound capable of undergoing cross-linking reactions is selected from the class formed by 1,1,3,3-tetraethoxypropane, isophthalic aldehyde, adipic aldehyde, muconic aldehyde, genipin, isophorone diisocyanate, lysine disocyanate, toluene 2,4-diisocyanate terminated poly (propylene glycol) (CAS # 9057-91-4), toluene 2,4-diisocyanate terminated poly (ethylene adipate) (CAS # 9019-92-5), bis (4-isothiocyanatebutyl) disulfide, tetraglycidyl 4,4'diamino diphenylmethane, resorcinol-diglycidyl-ether, bisepoximentene, limonene dioxide, poly [(phenyl isocyanate) -co-formaldehyde], poly (ethylene adipate) terminated with toluene 2,4 -diisocyanate, bromoacetylbromide, tetraglycidyl 4,4'diamino diphenylmethane, resorcinolodiglycidyl-ether and said at least one water-soluble compound capable of having cross-linking reactions with said at least one fat-soluble compound is selected from the class formed by diamines, oligoamines, peptides, proteins and oligomeric or polymeric amino sugars, tetraethylenepentaamine, cystamine sulfate hydrate, spermine, lysine, arginine, polylysine, polyarginine, albumin, bovine serum albumin casein, gelatin, lactalbumin and chitosan. 8. Nanoconstruct according to at least one of the preceding claims in which said pharmacologically active substance and / or said nutrient substance is selected from the class formed by resveratrol and its derivatives.