BRPI0903009A2 - carotenoid lipid vesicles, composition, process for preparing carotenoid lipid vesicles and use of carotenoid lipid vesicles - Google Patents

Abstract

CAROTENOID LIPID VESICLES, COMPOSITION, PROCESS OF PREPARATION OF CAROTENOID LIPID VESICLES AND USE OF CAROTENOID LIPID VESICLES. The present invention applies in the pharmaceutical, cosmetic, veterinary, biotechnological, dental, medical, oncological, radiopharmaceutical, agronomic, gene therapy, food areas, referring to the constitution of lipid vesicles of carotenoids in which said carotenoids represent the largest proportion molar composition of the vesicular bilayer, as well as a composition containing said carotenoid lipid vesicles, process of preparing carotenoid lipid vesicles and use.

Description

CAROTENOID LIPID VESICULES, COMPOSITION, CAROTENOID LIPID VESICULAR PROCESS, AND USE OF LIPID CAROTENOID VESicles

The present invention is applicable in the pharmaceutical, cosmetic, veterinary, biotechnological, dental, medical, oncological, radiopharmaceutical, agronomic, gene therapy, food fields, referring to the constitution of decarotenoid lipid vesicles in which said carotenoids represent the largest molar proportion of the constitution. of the vesicular bilayer.

TECHNICAL STATE

Liposomes are spherical structures composed of single or multiple concentric bilayers resulting from the self-association of amphiphilic molecules, such as phospholipids, in an aqueous medium. Polar head groups are located on the surface of the membranes in contact with the environment, while fatty acid chains form the hydrophobic center of the membranes, protected from water. These vesicles have part of the aqueous phase inside and therefore can capture and secrete polar molecules; In addition, because of the physicochemical properties of their constituents, they can also dissolve hydrophobic molecules in their bilayers. Liposomes can be prepared in many different sizes, ranging from small unilamellar vesicles (SUVs), whose small diameter can be about 20 nm to giant unilamellar vesicles (GUV) with a diameter of tens of micrometers. Belonging to the first liposome administration are multilamellar vesicles (MLV) with several hundred nanometers in diameter (BANGHAM AD, STANDISHM.M., WATKINS JC. Diffusion of univalent ions across Iamellae of swollen phospholipids.J. Mol. Biol. 13 (1): 238, 1965), and the latest large unilamellar vesicles (LUV-Iarge unilamellar vesicles), characterized by high capture volumes with adjustable diameter (100 or 200 nm) and reduced size by membrane-specific extrusion (SCHUBER). F., KICHLER A., BOECKLER C., FRISCH B. Liposomes: from memory models to gene therapy (Pure & Appl. Chem. 70 (1): 89-96, 1998.).

In essence, liposomes are highly versatile structures whose properties can be modulated by changing parameters such as size, lamellarity, bilayer composition, fillers and surface properties. The chemistry of (phospho) lipids, which are the constituents of liposomes, allows us to design analogues with new properties and derivatives to be attached to the surface of the veins. Liposomes have attracted enormous interest: i) In basic research (chemistry and biology) as membrane models, they also offer attractive possibilities for confining chemical reactions in very small volumes,

(ii) as vehicles for the transport of drugs and recently for the transportation of

iii) In biotechnology, pharmaceutical industry (development of liposome-based anti- tumor formulations and vaccines) and cosmetics (SCHUBER F., KICHLER A., BOECKLER C., FRISCH B. Liposomes: from membrane models to genetherapy. Pure & Appl. Chem. 70 (1): 89-96, 1998).

Liposomes can be rationally designed, resulting in non-reactive (sterically stabilized) liposomes (SLs) 1 as well as polymorphic (fusogenic cationic) liposomes. SLs may be designed to exhibit specific reactivity (targeting), while polymorphic liposomes may exhibit altar reactivity to nucleic acids and cell membranes. Due to their reduced recognition and attack by the immune system, these liposomes may have cancer chemotherapy usefulness. Polymorphic liposomes are promising gene therapy due to the possibility of DNA transfection. In parallel, more efficient release and retention of drugs within liposomes (based on active accumulation through ionic gradients) have contributed to the use of this new generation of liposomes (LASIC D.D., PAPAHADJOPOULOS D. Liposomes revisited. Science. 267: 1275-1276, 1995).

Sterically stabilized liposomes were created when it was known that mechanical or electrostatic stabilization could not form sufficiently stable liposomes in a biological environment such as systemic circulation. Therefore, in SLs, lipid layer contains glycolipids or more recently conjugated cometylene glycol lipids that form a steric barrier external to the membrane. SLs kill blood up to 100 times longer than conventional liposomes and can therefore increase the pharmacological efficacy of encapsulated agents. In addition, SLs allow to target liposomes to specific cells via ligand because they are much less susceptible to nonspecific attack than conventional ones. Antibody-linked or other ligands SLs accumulate much more readily in cells than conventional liposomes (LASIC D.D., PAPAHADJOPOULOS D. Liposomesrevisited. Science. 267: 1275-1276, 1995).

There are techniques that cause profound changes in the pharmacokinetic behavior of SLs (non-reactive but sterically stabilized liposomes). The average life of conventional blood liposomes after intravenous injection, for example, may range from 2 hours to over 40 hours in humans. Long-circulating liposomes have been discovered that are captured more slowly by liver and spleen and passively directed to tumor sites, increasing the "bioavailability" of antitumor drugs. For example, in certain tumors 25-fold higher liposome levels were found even after several days of injection. Another consequence of this phenomenon is the use of SL for imaging (SCHUBERF., KICHLER A., BOECKLER C., FRISCH B. Liposomes: from membrane models to therapy. Pure & Appl. Chem. 70 (1): 89-96, 1998 ).

C26 solid colon carcinoma tumor in mice is practically insensitive to treatments with free doxorubicin or incorporated into conventional liposomes. However, administration of doxorubicin-containing SLs (SL-Dox) resulted in complete remission of tumors in early treatments and significant improvements in late treatments. In a mouse mammary carcinoma tumor model, SL-Dox was much more efficient than conventional liposomes and reduced the incidence of metastases. SL-Dox was able to arrest the growth of human lung cells in severe combined immunodeficiency (SCID) mice, while equivalent doses of free or encapsulated doxorubicin in conventional liposomes were not effective (LASIC DD, PAPAHADJOPOULOSD. Liposomes revisited. Science. 267: 1275- 1276, 1995).

Single bilayer liposomes are usually prepared by one of two sonication methods: a high energy probe is immersed directly in a water dispersion of phospholipids or by dispersing phospholipid in a glass vial suspended in a low energy ultrasonic bath. Both procedures produce vesicles of about 25 nm in diameter, consisting of a single phospholipid bilayer shell. However, the technique has several disadvantages such as high energy sonication causing oxidation and degradation of phospholipid, although it can be minimized by strict control, and can also damage the molecules of the solute to be encapsulated. Another disadvantage is the difficulty in preparing large quantities of liposomes (BATZRI S., KORN E.D. Single bilayer liposomes preparedwithout sonication. Biochim. Biophys. Acta. 298: 1015-1019, 1973).

Because of these limitations on sonication liposome preparation, Batzri eKorn (BATZRI S., KORN ED Single bilayer liposomes prepared without sonication.Biochim. Biophys. Acta. 298: 1015-1019, 1973) developed one of the simplest methods for obtaining liposomes. which is the method called ethanolic injection. This technique allows to obtain simple bilayer liposomes, not distinguishable from those obtained by sonication. The diluted suspension can be easily concentrated by ultrafiltration, not leading to degradation or oxidation of the phospholipid used (BATZRIS., KORN E.D. Single bilayer prepared liposomes. Biochim. Biophys.Acta. 298: 1015-1019, 1973).

As for drug encapsulation, compounds dissolved in the same ethanol as lipid are relatively well encapsulated, while hydrophilic substances have low compartmentalization, as was the case with Pons et al's carboxyfluorescein (PONS M., FORADADA M., STERLRICH J. Liposomes obtained by the ethanol injection method (Int. J. Pharm. 95: 51-56, 1993). However, these same authors reported the advantage of ethanolic injection as the possibility of encapsulating both hydrophilic and lipophilic substances.

The molecular weight and radius of the vesicles produced by the injection method may depend on the injection speed, lipid concentration in the buffer and alcohol, and opH and buffer osmolality. Kremer et al (KREMER JMH, VANDER ESKER MWJ, PATHMAMANOHARAN C., WIERSEM PH PH Vesicles of variable diameter prepared by a modified injection method. Biochemistry. 16: 3032-3935, 1977) showed that injection speed did not influence molecular weight or radius. of lipid particles.

Pons et al (PONS M., FORADADA M., STERLRICH J. Liposomes obtained by the ethanol injection method. Int. J. Pharm. 95: 51-56, 1993) found that lipid concentration is the major factor influencing the size of Hauser (HAUSER H. Methods of preparation vesicles: assessment of their suitability fordrug encapsulation. Trends Pharm. Sci. 3: 274-277, 1982) had established that injection of ethanolic solutions of phospholipids gave rise to SUV-like vesicles (aggregates of SUV), ranging in size from 30 to 60 nm. However, Pons et al (PONS M., FORADADA M., STERLRICH J. Liposomes obtained by the ethanolinjection method. Int. J. Pharm. 95: 51-56, 1993) obtained not only SUVs, but also intermediate and large vesicles, depending of lipid concentration. Ponset al (PONS M., FORADADA M., STERLRICH J. Liposomes obtained by the ethanolinjection method. Int. J. Pharm. 95: 51-56, 1993) concluded that, by varying the concentration of lipid, the method offers the possibility of obtaining Single-layered vesicles of different radii reproducibly and reliably.

Liposomes were proposed and demonstrated by Gregoriadis et al. (GREGORIADIS G. Engineering Iiposomes for drug delivery: progress and problems. Trends in Biotechnology 13: 527-537, 1995) for their ability to carry drugs and used enzymes, anticancer drugs and Many studies have been developed for drug delivery systems due to the considered harmless nature of the liposomal components (which are non-toxic, non-immunogenic, biodegradable lipids), and the versatility of the system structure.

Studies have shown that carotenoids can be carried by traditional liposomal systems to improve the permeability of carrier vesicles (SOCACIU C., LAUSCH C., DIEHL HA. Carotenoids in DPPC vesicles: membrane dynamics. Spectrochimica Acta Part A 55: 2289-2297, 1999) as works demonstrating the use of carotenoids as active ingredients with antioxidant activity (SILVA CR, ANTUNES LMG, BIANCHI MLP Antioxidant action of bixinagainst cisplatin-induced chromosome aberrations and Iipid peroxidation in rats. Pharmacological Research. 43 (6): 561-566, 2001 radiation protective effect against mutagenic elements and induction of cytochrome P450 system (De-Oliveira ACAX, SILVA IB, MORNING-ROCKS DA, PAUMGARTEN FJR Induction of Iivermonooxygenases by annatto and bixin in female rats. Braz J. Med. Biol. Res. 36 (1): 113-118, 2003).

Bixin (cis-bixin, CAS 6983-79-5) is the major oil-soluble pigment of Bixa orellana L. seeds. It is a carotenoid devoid of pro-vitaminA activity with two carboxylic groups, one of which is methylester (Figure 1 THE). Hydrolysis of this methyl ester group gives a corresponding dicarboxylic acid, norbixin (Figure 1B), which is an anate pigment soluble in alkaline aqueous solutions. Deanate extracts, as well as their major carotenoid constituents bixin and norbixin, have been widely used as color additives in food, drugs and cosmetics (De-Oliveira ACAX, SILVA IB, MORNING-ROCKS DA, PAUMGARTEN FJRInduction of Iiver monooxygenases by annatto and bixin in female rats Braz J. Med.Biol Res 36 (1): 113-118, 2003). In northern and northeastern Brazil it is extensively used as a food condiment, known as 'paprika' or 'colorific' (De-Oliveira ACAX, SILVA IB, MORNING-ROCKS DA, PAUMGARTEN FJRInduction of Iiver monooxygenases by annatto and bixin in female rats Braz J. Med.Biol Res 36 (1): 113-118, 2003).

B. orellana seeds have been used in Brazilian folk medicine to prepare aphrodisiac potions as well as medicines to treat fevers, inflammations, parasitic diseases and in Jamaica, folk remedies to treat diabetes mellitus (PAUMGARTEN FJR, DE-CARVALHO RR, ARAÚJO IB, PINTO FM, BORGESO.O., SOUZA CAM, KURIYAMA SN Evaluation of the Developmental Toxicity of Annatto in the Rat. Food and Chemical Toxicology 40: 1595-1601, 2002).

B. orellana leaves are known to exhibit an inhibitory effect against Neisseriagonorrhoea, and leaf extracts a significant antimicrobial activity against standard Gram positive bacterial strains including Bacillus subtilis, Streptococcusfaeealis and Staphylococcus aureus. A recent study of volatile extracts showed several mono and sesquiterpenes (GALINDO-CUSPINERA V., WESTHOFF DC, RANKINS.A. Antimicrobial properties of commercial annatto extracts against selected pathogen, Iactic acid, and spoilage microorganisms. J. Food Protection. 66 (6): 1074-1078, 2003), and other water-soluble compounds (GALINDO-CUSPINERA V., RANKIN SA Bioautography and chemical characterization of antimicrobial compound (s) in commercial water-soluble annatto extracts. J. Agric. Food Chem. 53 (7 ): 2524-2529,2005) with antimicrobial activities. However, no reports of antimicrobial reactivity were found by isolated compounds of B. orellana due to the difficulty in separating the components of the extracts.

The dyes obtained from annatto (Bixa orellana L.) have been used for many years mainly in the cosmetic and food areas. The dyes are found covering the outer surface of annatto seeds and are mostly (about 80%) α-bixin which is a monomethylester of α-norbixin carboxylic acid (FERREIRA VLP, TEIXEIRA NETO RO, MOURA SCSR, SILVA MS). Color degradation kinetics of commercial water-soluble annatto solution, submitted to heat treatments Ciênc. Tecnol. Aliment. 19 (1): 37-42, 1999).

Several countries have banned the use of a number of synthetic food colorings, and most natural colorants, apart from appearance, are important for health improvement, and there is a high demand for food supplements containing an effective dose of these colors (REDDY MK, ALEXANDER-LINDO RL, NAIR MG Relative inhibition of Iipid peroxidation, cyclooxygenaseenzymes.and human tumor cell proliferation by natural food colors (J. Agric. Food Chem. 53: 9268-9273, 2005). Reddy et al (REDDY MK, ALEXANDER-LINDO RL, NAIR MGRelative inhibition of Iipid peroxidation, cyclooxygenase enzymes. And human tumor cell proliferation by natural food colors. J. Agric. Food Chem. 53: 9268-9273, 2005) demonstrated that bixin In combination with ichopene and β-carotene had no expected impact on health improvement, however, inhibition of lipid peroxidation, cyclooxygenase enzymes and tumor cell proliferation was significant.

The present invention aims to elucidate the low cost and ease in the process of preparation of carotenoid lipid vesicles having as main component a low toxicity raw material, even in high dosages, which is quite abundant in Brazil and of low cost. With this, the process becomes an incentive for the national industry to improve the extraction, purification and compound preparation techniques as a nobler input for the pharmaceutical industry, in addition to those already applied to the cosmetic and food industry.

SHORT DESCRIPTION

The present invention is directed to carotenoid lipid vesicles in which ditoscarotenoids represent the largest proportion of the vesicular bilayer constitution. The invention further relates to a composition containing said carotenoid lipid vesicles, process for preparing carotenoid lipid vesicles and use.

DETAILED DESCRIPTION

The present invention relates to carotenoid lipid vesicles in which ditoscarotenoids represent the largest proportion of the composition of the vesicular bilayer, its composition, its process of preparation and use.

According to the invention, the carotenoids used in lipid vesicles may be selected from bixin, zeaxanthin, lutein, cryptoxanthin, alpha carotene, beta carotene, preferably bixin. Optionally, said lipid vesicles may be comprised of single and isolated carotenoids, mixtures of carotenoids, other lipids (carotenoids or non-carotenoids), cholesterol, cholesterol salt, phospholipids, glycolipids, conjugated lipids, other ligands.

Norbixin is a type of carotenoid that can participate in the structural composition of the gallbladder, and also has a pharmacological action alone. Norbixin also forms emulsions due to its surfactant capacity. The advantage is that norbixin is water soluble and can disperse insoluble or poorly water soluble compounds when we add these compounds directly to the norbixin water mixture.

There are no articles or patents presenting vesicles prepared with more than 1% carotenoids compared to the prepared vesicle, as they have been used as adjuvants to improve vesicle stability. According to the present invention, the gallbladder is prepared from carotenoids, optionally it may contain another lipid (carotenoid or non-carotenoid), with adjuvant function or to direct site to some specific organism tissue. The advantage of the gallbladder being prepared from carotenoids is that the cost and adverse effects are very low, and some lipids are very expensive, especially synthetic ones, such as N- (1- (2,3-dioleoyloxy) propyl) -N, N , N-trimethylamoniomethyl sulfate (DOTAP).

Carotenoid lipid vesicles according to the present invention have carotenoids in the concentration of 0.001 to 100 millimolar (mM), preferably between 0.01 to 10 mM, more preferably 0.01 to 1.0mM; and bixinatally also at a concentration of 0.001 to 100 millimolar (mM), preferably between 0.01 to 10 mM, more preferably 0.01 to 1.0mM. These vesicles are characterized by being in the form of liposomes, which may be unilamellar or multilamellar. This same liposome is characterized in that it is preferably in the size between 100 and 200 nm, more preferably 151.9 ± 0.9nm, and may vary from 20 nm to 1500nm, depending on the conditions of preparation with the presence of electrolytes, other non-electrolytic molecular structures, velocity. agitation, solvents used, temperature, lipid concentration, etc.

The molecular structure of the carotenoid bixin (Figure 1 A) has been shown to be very similar to some lipids commonly used for liposome preparation, which may have regions with polar character and long chain support, for example, odioctadecyldimethylammonium (DODA), N- (1- (2 (3-dioleoyloxy) propyl) -N, N, N-trimethylamoniomethyl sulfate (DOTAP). The compound is not water soluble and its solubilization in organic solvents is widely diffused. Its hydrophobic character has advantages to interact with compounds with the same character, being an interesting structure to form vesicles. The use of bixin as the main compound for preparing lipid vesicles has never been demonstrated perhaps because of the difficulty in solubilizing in aqueous solvents.

The carotenoid bixin has been studied under a toxicological aspect and has not shown toxic effects, showing to be very promising as a drug carrier also due to this characteristic. Paumgarten et al (PAUMGARTEN FJR, DE-OAK RR, ARAÚJO IB, PINTO FM, BORGES OO, SOUZA CAM, KURIYAMA S. N. Evaluation of the Developmental Toxicity of Annatto in the Rat. Food and Chemical Toxicology. 40: 1595-1601, 2002 ) did not detect any evidence of toxicity in either rats or offspring when given an amount of 500 mg or more annato (28% bixin) / kg body weight / day orally. This value (> 140 mg bixin / kg weight / day) determined in rats is at least 2153 times the acceptable daily intake recommended by the World Health Organization (0.065 mg debixin / kg weight / day) for humans. Thus it is observed that carotenoid, natural product, competes in efficiency with commercial formulations.

However, another type of innovation found was the interaction of anionic vesicles, because bixin results in negatively charged vesicles and still has the potential to interact with cells with negatively charged membranes. It has been shown that there must be another type of interaction that is stronger than simple electrostatic attraction, so despite classic cases as reported, for example, by Campanhã et al (MTN CAMPAIGN, MAMIZUKA EM, CARMONA-RIBEIRO AM Interactions betweencationic Iiposomes and bacteria J. Lipidres 40: 1495-1500, 1999), where cationic vesicles such as DODAB, which interact with negatively charged bacteria membranes by electrostatic attraction, bixin vesicles have potential negatively charged bacteria that also interact with bacterial membranes by another type of interaction, the so-called hydrophobic one, which has already been demonstrated in other works (KIKUCHI IS, CARMONA-RIBEIRO AM Interactionsbetween DNA and cationic liposomes. J. Phys. Chem. B. 104: 2829 -2835, 2000).

The invention described herein has attributed to the bixin carotenoid the leading decomposing carrier role while at most articles have their role as supporting agents. Hara et al (HARA M., YAMANO Y., SAKAI Y., KODAMA E., HOSHINO T., ITO M., MIYAKE J. Stabilization of Iiposomal membranes by carotenoids: zeaxanthin, zeaxanthinglucoside and thermozeaxanthin. Material Science and Engineering C. 28: 274-279, 2008) demonstrated stabilization of liposomal membranes by other carotenoids, zeaxanthin and its derivatives in a mixture with lipids classically used as dipalmitoyl phosphatidylcholine (DPPC), whereas in the present invention no lipid or other component with supposed membrane stabilizing function.

The application of bixin vesicles or other carotenoids is closely related to the incorporation of components such as essential oils into them, as well as the incorporation of other difficult-to-disperse adjuvants into the formulations, such as amphotericin B and miconazole and others difficult to manipulate. as antineoplastic, radiopharmaceuticals, hormones, vaccines. In all cases, the reduction in dosage applied to the patient is observed beyond cost, since the delivery of the drug to the site of action should be more efficient, reducing also the possible side effects inherent to the drug carried. Carotenoids also lend themselves to the aesthetic effect, giving coloration or scattered particle effect in the formulations, according to size and concentrations to be introduced into the formulations. The major advantage is that elements such as essential oils may remain longer in the vesicles and have a longer lasting effect on the formulation due to this confining "protective" effect, making it difficult to evaporate the active compounds.

Bixin vesicles can also be used in "anti-aging" formulations such as incorporating compounds considered antioxidant, sunscreen, etc., because the bixin molecule may be more avid for free radicals and be "attacked" before the substances incorporated into it are released. It is also achieved because the molecule of the present invention has characteristics that facilitate its oxidation (double bonds) or degradation (by ultraviolet radiation). The fact that hydrophobic interactions are more intense also explains the type of interaction between the amphotericin B drug, cited as preferred when carried, and the debixin molecules, explaining that it is not only positive particles that can interact with negatively charged membranes. Therefore, a disadvantageous feature may be used as an advantage in certain formulations.

In the medical and pharmaceutical fields, bixin and / or norbixin formulations (incorporated drugs) may be mobilized on the surfaces of medical equipment.

Thus, according to the present invention, decarotenoid lipid vesicles have three functions, a carrier function, in which the vesicle acts as a carrier of components within it, such as essential oils, dyes, drugs, nucleic acids, antineoplastics, radiopharmaceuticals, vaccines, antibodies, preferably amphotericin B, producing an activity of potentiating the effect be it chemical, physical, biological, pharmacological effect, etc., the carried component; an active principle function, in which the gallbladder itself has an activity that produces an activity such as antimicrobial activity, preferably gram negative and gram positive overbacteria, antitumor, antibiotic, antilipidemic, antioxidant, antiinflammatory, anti-glycemic agent; and a carrier and principle function concomitantly. The three functions performed by vesicles are very important, since articles cite such activities using solubilized bixin in nonpolar solvents rather than in the form of vesicles.

The present invention further relates to a pharmaceutical composition employing carotenoid lipid particles as an integral part of such composition. Carotenoid lipid veins are made up of a greater proportion of carotenoids. These carotenoids are selected from bixin, zeaxanthin, lutein, cryptoxanthin, alpha carotene, beta carotene, preferably bixin. The composition also employs preservative, fragrance, humectant, emollient, thickener, other actives. Such composition comprises the following pharmaceutical formula and the preferred ranges according to the invention which are described below in Table 1:

<table> table see original document page 11 </column> </row> <table> TABLE 1

The present invention further contemplates a process for preparing carotenoid lipid vesicles wherein said carotenoids represent the largest molar proportion of the composition of the vesicular bilayer. These carotenoids may be chosen from bixin, zeaxanthin, lutein1 cryptoxanthin, alpha carotene, beta carotene, etc., and preferably preferentially bixin. Carotenoid lipid vesicles may present as unilamellar or multilamellar structures.

For the process of preparation of bixin vesicular structures, its solubility in ethanol at room temperature and subsequent addition to water, agout drop, under agitation by swirling or with magnetic bars also at room temperature was explored, allowing the stabilization of the structures by interactions of the molecules. of water with polar portion of bixin molecules. The shaped organization of spherical vesicles is also due to the hydrophobic interactions between apolar long strands of bixin molecules themselves.

The process also employs techniques selected from sonication, organic solvent injection, extrusion, solubilization in evaporative nonpolar solvents, dispersion of the dry layer with other water soluble or aqueous solvents, mobilization of carotenoids on nanoparticle surfaces, preferably organic solvent injection, and other known methods in the field of liposome preparation.

The basic process of preparing lipid vesicles has the following steps:

The. carotenoid solubilization in organic solvent;

B. adding solution obtained in step (a) in water dropwise while stirring;

ç. obtaining the carotenoid lipid vesicle.

According to the present invention, the above mentioned process may further contain intermediate steps according to the solubility of the components to be carried. When the component to be carried is insoluble in water, the lipid vesicle preparation process has the following steps:

The. carotenoid solubilization in organic solvent;

B. organic solvent solubilization of water-insoluble components as carriers;

ç. mixing the solution obtained in step (a) with the solution obtained in step (b);

d. adding the solution obtained in step (c) to water dropwise while stirring;

and. obtaining the carotenoid lipid vesicle. When the component to be carried is soluble in water, the lipid vesicle preparation process has the following steps:

The. carotenoid solubilization in organic solvent;

B. solubilization in water or aqueous solvent of water soluble components to be carried;

ç. mixing the solution obtained in step (a) dropwise with the solution obtained in step (b) under stirring;

d. obtaining the carotenoid lipid vesicle.

The components to be carried by the gallbladder may be essential oils, dyes, drugs, nucleic acids, antibodies, antineoplastics, radiopharmaceuticals, vaccines and adjuvants, preferably amphotericin B drug.

According to the preparation processes, the organic solvent used is selected from chloroform, dimethyl sulfoxide, ether, alcohols such as ethanol and methanol, preferably ethanol, in the preferred concentration of 20%. Ethanol was the solvent used for the process, as well as being a cheap solvent, it has low toxicity, and is easily eliminated from the preparation by dialysis or evaporation process.

The bixin preparation of the present invention was based on the liposome preparation technique by injection into aqueous solutions of previous lipid preparations in organic solvents. All operations mentioned in the process can be performed at room temperature and do not require the carotenoid to reach its melting point, 198 ° C, which would require vortexing preparations with subsequent passage through nanopore filter membranes, which would make the preparation cost more expensive. Possible traces of ethanol used in the first solubilization of bixin can be eliminated by simple dialysis through cellulose membranes using water as the collecting medium.

The processes of carotenoid lipid vesicle preparation and drug incorporation can be performed in an environment with decontamination control. Optionally, the final product may undergo sterilization by means of sterilizing filter membranes according to the size of the selectively formed vesicles.

The present invention also relates to the use of carotenoid lipid vesicles in the pharmaceutical, cosmetic, veterinary, biotechnological, dental, oncological, radiopharmaceutical, agronomic, gene therapy, food, essential oil carrier, dye, drug, nucleic acid, antineoplastic antibody, radiopharmaceutical , vaccines, and other adjuvants and use "per se" as an active ingredient in pharmaceutical, veterinary, cosmetic, biotechnological, dental, medical, oncological, radiopharmaceutical, agronomic compositions for gene therapy.

The following are examples to further illustrate the invention, however, they are not intended to restrict the invention described herein.

EXAMPLES

Bixin dye obtained from annatto (Bixa orellana L.) was used to develop a liposomal preparation and amphotericin B was the model drug for incorporation into this system. In vitro assays were performed to demonstrate the efficacy of this new system from carotene of natural origin, while liposomostraditional ones use lipids (non carotenoids) sometimes synthetic. Results have shown efficacy in inhibiting Candida albicans as well as performance enhancing commercial products such as Ambisome and Amphocil.

1. Materials and methods

Bixin, norbixin and amphotericin B raw materials were obtained from the market and purity and content assays were also performed. Other reagents used were all analytical grade. Ambisome® and Amphocil® were obtained from the market. Candida albicans ATCC 10231 was obtained from the Adolfo Lutz Institute. Culture media Sabouraud Dextrose Agar and medium No. 19 were from Difco. All materials were sterilized by autoclaving, dry heat or sterilizing membrane filtration.

Preparation of bixin liposomes

Sufficient amount of bixin was weighed and solubilized directly in ethanol to prepare 5 mM solution (BX-ETOH). Material was filtered through added filter paper, with stirring and dripping in water at a ratio of 1:10 to obtain dispersion at 0.5 mM.

Preparation of amphotericin B

Amphotericin B stock solution was prepared by direct solubilization in dimethyl sulfoxide (DMSO), ie 10,000 pg active ingredient / mL (ANFO-DMSO solution) and followed by 10-fold dilution in ethanol (ANFO-ETOH solution).

(i) Preparation of dispersion in buffer solution B10 (phosphate)

Dispersions containing 10 pg / ml and 20 pg / ml amphotericin Bem buffer solution # 10 were prepared by diluting the ANFO-ETOH solution directly in buffer. Buffer No. 10 corresponds to 0.2 M potassium phosphate solution, pH 10.5 and constant in The United States Pharmacopeia 32. United States Pharmacopeial Convention. Rockville, Vol. 1, p. 88, 2009.

ii) Preparation of liposomal amphotericin Suitable volumes of the ANFO-ETOH and BX-ETOH solutions were mixed to obtain preparations 10 times more concentrated than desired. From this, 1:10 were added dropwise in water dropwise with stirring. From this latter preparation, serial dilutions can be made in water or other aqueous diluent without loss of the characteristics of the obtained structures.

Commercial Product Preparation

Both Ambisome and Amphocil were reconstituted with water and appropriate dilutions made with the same diluent to obtain final concentrations of 10 and 20 pg / mL of active ingredient.

Candida albicans preparation

About 1 μί of the microorganism was transferred to the agar surface of the Sabouraud Dextrose Agar culture medium and incubated at 30 ° C for 48 hours. On the day of the assay, the microorganism surface was washed with 5 mL 0.9% saline and 1 mL of the suspension was transferred to 99 mL of culture medium No. 19. An 8 mL volume of the inoculated medium was transferred to plates. Petrie's left to solidify. Stainless steel templates were transferred to the surface each of these Petri dishes immediately before applying the samples.

In each hole of the templates, 50 µl of each sample was applied and left for 2 hours at room temperature before incubating at 30 ° C for 24-48 hours and then reading the inhibition halos.

Statistic

For the tests, number of replicates n = 10 and at least 3 independent tests were used. The values presented are mean values and statistical analysis was applied by analysis of variance (ANOVA). Mean values were considered significantly different for P <0.05.

2. Evaluation of bixin and norbixin antimicrobial activity

Preparation of bixin liposomes

Bixin dispersions were prepared as described above at concentrations from 0.1 to 1.0 mM, and more diluted samples were prepared by diluting with water from the 1.0 mM preparation.

Norbixin Solution Preparation

Norbixin solutions were prepared by simple water solubilization at concentrations ranging from 0.1 to 1.0 mM, and more diluted samples were prepared by diluting in water from the 1.0 mM preparation.

Bacterial inoculum preparation

The microorganisms cited in the American Pharmacopoeia (USPXXXII) were challenged: Staphylococcus aureus ATCC 6538, Escheriehia eoli ATCC 8739, Pseudomonasaeruginosa ATCC 9027.

About 1 μ of each microorganism was transferred to the Tryptic Soy Agar (TSA) culture medium agar surface and incubated at 35 ° C for 24 hours. On the day of the assay, the microorganism surface was washed with 5 mL of 0.9% saline and 1 mL of the suspension was transferred to 99 mL of 0.9% saline. Each suspension was counted by serial dilutions in a tube containing 9 mL of saline (dilution to 10-8). Each 1 mL of the last 3 dilutions was plated in duplicate.

Determination of antimicrobial activity

Each preparation of bixin and norbixin in the amount of 9 mL was allowed to interact with 1 mL of each inoculum for one hour at room temperature. After this period, 1 mL of each sample was transferred to Petri dish (in triplicate). A 15 mL volume of Tryptic Soy Agar (TSA) medium was added to each plate and homogenized. After solidification, each sample was incubated at 35 ° C and counted after 48 hours.

2. Results and discussion

The use of carotenoids has been widely explored in the food and cosmetics industries as coloring agents and in some cases as in Farming and Fish Farming as a food supplement. Due to the supportive nature of many carotenoids, scholarly papers have explored their transport by nanoparticles known as nanoparticles, including liposomes and other colloidal systems. No studies were found using bixin and norbixin, the target carcarenoids of this work, with the function of carriers, however, some works cite as active ingredient to be carried by traditional systems such as liposomes (SOCACIU C., LAUSCH C., DIEHL HA Carotenoids in DPPC vesicles: membrane dynamics, Spectroehimica Aeta Part A 55: 2289-2297, 1999).

Bixin was solubilized in ethanol and then added dropwise in water to produce vesicular structures, as can be seen from Figures 2 (A and B) and 3 (A and B). Figure 2 A shows the interior of the microscope where silicon slides were applied to the samples and Figure 2 B shows the vesicles of silicon lamina on the bladder. Figure 3 shows bixin vesicles under scanning electron microscopy, where in A the empty vesicles are observed, and in Bas bixin vesicles with amphotericin B. These are images obtained by scanning electron microscopy. A drop equivalent to a volume of about 100 μl was applied directly to the surface of a silicon slide and taken under a microscope and the samples were observed under vacuum. The particle size of the same drug was measured on the ZetaPIus Zeta_Potential Analyzer (BrookhavenInstruments Co.) apparatus, equipped with a 570 nm laser and 90 ° light angle and with a mean diameter of 151.9 ± 0.9 nm (Figure 4) and Zeta Potential of -14.30 ± 0.95 mV, a negative potential as expected due to the anionic nature of the carotenoid. Figure 4 illustrates the size distribution of bixin vesicles in nanometers.

Amphotericin B was the drug of choice for evaluating the ability to incorporate non-water soluble drugs into bixin vesicles. Amphotericin Ba presents amphiphilic behavior due to the supporting and polar portions of the lactonic ring and amphoteric behavior due to the presence of carboxyl groups and ionizable amines, rendering the compound poorly soluble in aqueous solvents. The negative charge of bixin can interact with the positive amino group of amphotericin B forming a complex and stability being reinforced by hydrophobic interactions between apolar portions of the compounds.

Optical spectrum (Figure 5) was obtained to characterize amphotericin B aggregation in the bixin formulation. Thermo Electron spectrophotometer, model Nicolet Evolution 100, in the region of 300 to 450 nm was used. The spectrum was obtained at room temperature (25 ° C) within up to 1 hour after preparation of the formulations. In Figure 5A, the typical spectrum of amphotericin B solubilized in its best solvent, ie, DMSO and Methanol, while in Figure 5B, a spectrocharacteristic of the drug in lipid vesicles such as those obtained by Fujii et al (FUJII G., CHANG J .AE., COLEY T., STEERE B. The formation of Amphotericin B ionchannels in Iipid bilayers Biochemistry 36: 4959-4968, 1997) using phosphatidylcholine (HSPC), cholesterol (ChoI) and distearoylphosphatidylglycerol (DSPG).

Unlike the article by Silva et al (SILVA CR, ANTUNES LMG, BIANCHIM.LP Antioxidant action of bixin against cisplatin-induced chromosome aberrations and lipid peroxidation in rats. Pharmacological Research. 43 (6): 561-566, 2001), cannot be dissolved bixin in ethanol plus distilled water (1: 9), even immediately before the experiments. Norbixin, another compound of Bixa orellana, has been found to be capable of dissolving in aqueous solvents and the mixture, with different profiles of formulations with bixin.

While most researchers have dispersed bixin in organic solvents such as DMSO, which would limit application in biological systems, the present invention shows the possibility and advantages of aqueous formulations as long as vesicles. Despite the previous use of ethanol for solubilization of bixin and subsequent mixing in water, the amount of this alcohol (up to 20% ethanol) showed no interference with the tested system, ie, did not inhibit Candidaalbicans growth.

Bixin and norbixin contents were determined by spectrophotometry and comparison with absorption coefficient of 2826 in chloroform at 470 nm and 3473 in 0.5% KOH at 453 nm, respectively, according to Tocchini and Mercadante's article (TOCCHINI L., MERCADANTE AZ Extraction and HPLC determination of enorbixin bixin in colorifers (Ciên. Tecnol. Aliment. 21 (3): 310-313, 2001).

Determination of inhibition halo for C. albicans and comparison with commercial formulations

Concentrations of 10 to 20 pg / mL amphotericin B in bixin 0.5 and 1.0 mM were evaluated. Controls for DMSO and ethanol in the proportions used during preparation of the formulations were tested and showed no inhibition halo formation. Controls for carotenoids were also tested without drug addition and showed no inhibition halo formation, showing that bixinasozinha does not interfere with inhibition of inhibition halo. However, according to Figures 6 and 7, bixin potentiates the effect of amphotericin B, since this drug was tested after dilution in ideal solvent, in this case DMSO and methanol, followed by dilution in phosphate buffer solution, as prepared by the American Pharmacopoeia (The United States Pharmacology 32. United States Pharmacopeial Convention, Rockville, vol. 1, p. 88,2009), where Figure 6 illustrates Petri dish containing samples with their inhibiting halos, where A is Anfo 20 pg / mL in B-10, B is Anfo 10 pg / mL in 0.5 mM BX, C is Anfo20 pg / mL in 0.5 mM BX, D is Anfo 10 pg / mL in B-10, E is Ambisome 20 pg / mL and F is Ambisome 10 pg / mL, and Figure 7 illustrates the diameter of the inhibition halos as a function of amphotericin B samples in graph form. According to this figure (Fig. 7) there is a comparison between amphotericin B preparations in phosphate buffer solution (Anfo B10), in liposomal preparations of bixin (BX) and Ambisome (Figure 7 A) and Amphocil (Figure 7 B).

At least 3 independent tests with a number of replicates not less than 5 were performed. Analyzes, Table 2 (Annex), show that the formulation of amphotericin B and bixin is better than amphotericin alone (P <0.05) and better than commercial formulations as Ambisome and Amphocil. Ambisome is the liposomal commercial formulation while Amphocil is a complex with sodium cholesteryl sulfate. These last two showed efficacy in inhibiting growth of C. albicans, but were no better than amphotericin B alone in buffer solution. Table 2 (Annex) shows the comparison between amphotericin preparations and their significance, according to ANOVA statistical analysis. In Figure 6 as in Figure 7, it is observed that amphotericin in the liposomal preparation of bixin has a better inhibition halo than commercial products. and amphotericin B alone. Despite the formation of the halo, the typical coloring of the bixin (Figure 6) did not follow the diameter of the halo.

Thus, it is noticeable that amphotericin B diffused from the vesicles into the agar medium while the vesicle remained at the site of application. Also, the halo corresponding to the template hole size (about 8 mm) is translucent as the default, showing that there was no growth of Candida albicans at the site, whereas if it was positive for growth, there would be turbidity at the site, as the went to the site with application of the bixin formulation alone without the drug (data not shown). Therefore, the bixin vesicles did not diffuse through the agar medium, but the drug could be released, which is expected in a formulation with efficient drug delivery system.

Campanhã et al (MTN CAMPAIGN, MAMIZUKA EM, CARMONA-RIBEIROA.M. Interactions between cationic vesicles and Candida albicans. Journal of Physical Chemistry B. 105: 8230-8236, 2001) and Lincopan et al (LINCOPAN N., MAMIZUKA EM, CARMONA In vivo activity of a novel amphotericin B formulation with synthetic cationic bilayer fragments (J. Antimicrobial Chemotherapy 52: 412-418, 2003) have shown efficacy of amphotericin B formulations in dioctadecyl dimethylammonium bromide (DODAB) synthetic bicamadacatonic fragments and based on these structures, the bixin formulation shows advantages in forming vesicles and being naturally sourced.

Figure 8 illustrates the viable Candida albicans Curve as a function of time of interaction with preparations containing amphotericin B, where CFU / ml means colony forming units per milliliter, (- ■ -) Anfo_gli meaning amphotericin B in 5% glucose solution; (-O-) BX_anfo which means amphotericin B in debixin vesicles; (-A-) Ambisome means aqueous dispersion of the Ambisome commercial product; (-V-) Amphocil which means aqueous dispersion of the commercial Amphocil product. The final concentrations of amphotericin B in the preparations were as follows for the graphs: (a) 5 pg / mL; (b) 2.5 pg / mL; (c) 1 pg / mL. In this figure, it is observed that Amphotericin B when dispersed in 5% glucose solution has the capacity to decrease C. albicans load in 7 logs, as well as the formulation of fungicide incorporated in bixin vesicles and in an interaction time of about 2 hours Commercial formulations such as Ambisome and Amphocil with amphotericin 1, 2.5 and 5.0 pg / mL concentrations failed to completely quench yeast cells, causing an increasing increase in surviving cells 18 hours after the initiation of fungal-fungicide interaction.

Bixin liposomes are expected to make the cheapest formulations marketed today for various drugs, such as amphotericin B, which was the model studied in this study and whose estimated cost per day for a 70 kg patient in the United States was $ 790 to $ 1,316 with Ambisome (TOWER JJ, SWORD R., BALLESTEROS MP, TOWER-SANTIGAGO S.Amphotericin B formulations and drug targeting. Journal of Pharmaceutical Sciences.97 (7): 2405-2425, 2008).

Antimicrobial activity of each of the compounds alone was evaluated: norbixin (Figure 9 A) and bixin (Figure 9 B), where each symbol represents the microorganisms tested as follows: (■) S. aureus, (·) E. coli and (A) P.aeruginosa. As many articles cite, norbixin, which is the water-soluble compound, showed antimicrobial activity when interacting with S. aureus, a Gram positive bacterium, while the results obtained with bixin vesicles were the most surprising. It has been shown that this compound in the form of water-dispersed galls has antimicrobial activity on Gram negatives, as well as E. coli and P. aeruginosa, as well as on S. aureus (Gram positive) at concentrations tested between 1 and 0.5 mM. even better, leaving no viable cells, while norbixin left viable cells residual, which leaves the possibility of proliferation after a longer time with depletion of the principles.

Norbixin preparations in water fail to reduce the burden of Gram negative bacteria, as they still contribute to fungal growth, demonstrating innocuity on eukaryotic cells due to the presence of cytochrome P450 system enzymes that allow biotransformation in innocuous metabolites. It acts by another mechanism which may be by interaction of the debixin vesicles on the bacterial cell membrane, as suggested by Campanhãet al. (CAMPANHÃ MTN, MAMIZUKA EM, CARMONA-RIBEIRO AM Interactionsbetween cationic Iiposomes and bacteria: the physical chemistry of the bactericidalaction. J. Lipid Res. 40: 1495-1500, 1999), may be by occlusion of purines or action on the proteins of membrane transport, not allowing exchanges with the extracellular medium for metabolite clearance and / or nutrient entry, but there is formation of a layer of vesicles on the surface of bacteria.

3. ConclusionIn the present invention, bixin has been shown to be capable of forming vesicles when using the ethanolic injection technique in water, making it possible to incorporate drugs, including support as amphotericin B. Inhibition halide, however, in combination with amphotericin B, allowed its release and interaction with yeast, promoting greater inhibition halo (P <0.05) than commercial products such as Ambisome and Amphocil, when they were tested at the same final concentrations of the active principle.

Bixin vesicles were anionic in character, but can be electrostatically linked to cationic regions of other compounds. Because they have a long supporting chain as the lipids used for liposome preparation, they allow hydrophobic interactions with nonpolar regions of other compounds, with active supporting principles.

Therefore, what can be expected as an alternative to preparing debixin vesicles or other similar carotenoids such as zeaxanthin, lutein, cryptoxanthin and others with close structures is the use of an appropriate organic solvent (preferably non-toxic or which can be eliminated later by simple techniques such as dialysis, evaporation or pumping of inert gas such as nitrogen), and then be transferred dropwise to aqueous solutions. <table> table see original document page 22 </column> </row> <table>

Claims (38)

1. Carotenoid lipid vesicles, characterized in that they comprise carotenoids in which said carotenoids represent the largest molar proportion of the vesicular dabicamate constitution. Carotenoid lipid vesicles according to claim 1, characterized in that the carotenoid pellets represent above 10% in molar proportion to the constitution of the vesicular bilayer. Carotenoid lipid vesicles according to claim 1, characterized in that the carotenoid pellets represent above 50% in molar proportion to the constitution of the vesicular bilayer. Carotenoid lipid vesicles according to claim 1, characterized in that the carotenoid pellets are selected from bixin, zeaxanthin, lutein, cryptoxanthin, alpha carotene, beta carotene. Carotenoid lipid vesicles according to claim 4, preferably comprising bixin. Carotenoid lipid vesicles according to claim 1, characterized in that they are in liposome form. Carotenoid lipid vesicles according to claim 6, characterized in that the liposome phage is unilamellar or multilamellar. Carotenoid lipid vesicles according to claim 6 or 7, characterized in that the liposome is in the size of 20 nm to 1500 nm, preferably between 100 and 200 nm, more preferably 151.9 ± 0.9 nm. Carotenoid lipid vesicles according to claim 1, characterized in that they present the carotenoid at a concentration of from 0.001 to 100 millimolar (mM), preferably from 0.01 to 10 mM, more preferably from 0.01 to 1.0 mMem relative to the liposomal formulation. total. Carotenoid lipid vesicles according to claim 5, characterized in that they exhibit bixin at a concentration of from 0.001 to 100 millimolar (mM), preferably from 0.01 to 10 mM, more preferably from 0.01 to 1.0 mMem relative to the liposomal formulation. total. Carotenoid lipid vesicles according to claim 1, characterized in that they have a carrier function within them. Carotenoid lipid vesicles according to claim 1, characterized in that they have an active principle function. Carotenoid lipid vesicles according to claim 1, characterized in that they have concomitant carrier and active principle function. Carotenoid lipid vesicles according to claim 11, characterized in that they comprise components within them such as essential oils, dyes, drugs, nucleic acids, antineoplastics, radiopharmaceuticals, vaccines, antibodies, adjuvants, among others. Carotenoid lipid vesicles according to claim 14, characterized in that they preferably comprise antimicrobial drugs such as amphotericin B. 16. Carotenoid lipid vesicles, according to claim 11, characterized by the fact that it has activity potentiating the biological, pharmacological, chemical, physical effect of the carreate component. Carotenoid lipid vesicles, according to claim 12, characterized by the fact that they have activity such as antimicrobial, antitumor, antibiotic, antilipidemic, antioxidant, antiinflammatory, anti-glycemic activity, among others. Carotenoid lipid vesicles according to claim 17, characterized in that they preferentially exhibit antimicrobial activity, more preferably on Gram negative and Gram positive bacteria. Carotenoid lipid vesicles, according to claim 13, characterized by the fact that it has activity enhancing the biological, pharmacological, chemical, physical effect of the carried component and antimicrobial, antitumor, antibiotic, antilipidemic, antioxidant, antiinflammatory, anti-glycemic activity, among others. Carotenoid lipid vesicles according to Claims 1 to 19, characterized in that they optionally comprise isolated and single carotenoids, mixtures of carotenoids, other lipids (carotenoids or non-carotenoids), cholesterol, cholesterol salt, phospholipids, glycolipids, conjugated lipids, other binders. Composition characterized in that it comprises carotenoid lipid vesicles according to claims 1 to 20. Composition according to Claim 21, characterized in that the carotenoid lipid vesicles are composed of carotenoids in which ditoscarotenoids represent the highest molar ratio in relation to the vesicular bilayer constitution. Composition according to Claim 22, characterized in that the carotenoids are selected from bixin, zeaxanthin, lutein, cryptoxanthin, alpha carotene, beta carotene. Composition according to Claim 23, characterized in that the carotenoid is preferably bixin. Composition according to Claim 22, characterized in that it comprises aindaglycerine, aqueous plant extracts, olive oil, glyceryl stearate, carboxymethylcellulose, preservative and fragrance. Process for the preparation of carotenoid lipid vesicles, according to claims 1 to 20, characterized by employing carotenoids in which ditoscarotenoids represent the highest molar ratio in relation to the vesicular bilayer constitution. 27. Carotenoid lipid vesicle preparation process according to claim 26, characterized in that the carotenoids are selected from bixin, zeaxanthin, lutein, cryptoxanthin, alpha carotene, beta carotene, preferably bixin. 28. Carotenoid lipid vesicle preparation process according to claim 26, characterized by employing techniques chosen from sonication, organic solvent injection, extrusion, solubilization in non-evaporating polar solvents, dispersion of the dry layer with other water-soluble or aqueous solvents, mobilization of liposoluble pigments in nanoparticle surfaces, as well as other methods. 29. Process for the preparation of carotenoid lipid vesicles, according to claim 28, characterized in that it preferably employs the organic solvent injection technique. Process for the preparation of carotenoid lipid vesicles, according to claim 26, characterized in that it comprises the following steps: a. solubilization of carotenoid in organic solvent b. adding solution obtained in step (a) in water, dropwise, under agitation c. obtaining the carotenoid lipid vesicle. Process for the preparation of carotenoid lipid vesicles according to claim 30, characterized in that it comprises the following steps when the component to be carried is insoluble in water: a. solubilization of carotenoid in organic solvent b. solubilization of water-insoluble components to be carried in organic solvent c. mixing the solution obtained in step (a) with the solution obtained in step (b); adding the solution obtained in step (c) in water, dropwise, under agitation; obtaining the carotenoid lipid vesicle. 32. Process for the preparation of carotenoid lipid vesicles according to claim 30, characterized in that it comprises the following steps when the component to be carried is water soluble: a. solubilization of carotenoid in organic solvent b. solubilization of water soluble components to be carried in water or aqueous solvent c. mixing the solution obtained in step (a), dropwise with the solution obtained in step (b), under stirring; obtaining the carotenoid lipid vesicle. Carotenoid lipid vesicle preparation according to claim 31 or 32, comprising components to be carried as essential oils, dyes, drugs, nucleic acids, antibodies, antineoplastics, radiopharmaceuticals, vaccines, and adjuvants, preferably the drug amphotericin B .. Process for the preparation of carotenoid lipid vesicles according to claims 30 to 32, characterized in that the organic solvent is chloroform, dimethyl sulfoxide, ether, alcohols such as ethanol and methanol, preferably ethanol. 35. Process for the preparation of carotenoid lipid vesicles, according to claim 34, characterized in that ethanol is preferably employed and the concentration of up to 20% of the final preparation is advisable to eliminate it by dialysis or evaporation of these organic solvents. 36. Use of carotenoid lipid vesicles according to claims 1 to 35 in the pharmaceutical, food, veterinary, cosmetic, basic research, dental, oncological, radiopharmaceutical, agronomic, and gene therapy areas. Use of carotenoid lipid vesicles according to claims 1 to 35 as a carrier of essential oils, dyes, drugs, nucleic acids, antibodies, antineoplastics, radiopharmaceuticals, vaccines and adjuvants. Use of carotenoid lipid vesicles according to claims 1 to 35 as an active ingredient in pharmaceutical, veterinary, cosmetic, biotechnological, dental, oncological, radiopharmaceutical, agronomic, gene therapy formulations.