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    • 专利摘要:
    The present invention relates to a purified pollen particle that retains its intine and exine layer, the compositions that include it, and its uses. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2613586A1
    申请号:ES201730152
    申请日:2017-02-09
    公开日:2017-05-24
    发明作者:Noemi Csaba；Diego PAN DELGADO；Sonia REIMONDEZ TROITIÑO；Marcos Garcia Fuentes
    申请人:Universidade de Santiago de Compostela；
    IPC主号:
    专利说明:

    Field of the Invention
    The present disclosure is directed to purified pollen particles, their manufacturing methods and their uses in the administration of encapsulated active ingredients. State of the Art
    Transmucosal administration of active ingredients, for example drugs, is one of the most accepted forms of administration by patients. It includes, for example, nasal, oral (eg, absorption through the small or large intestine), or ocular administration.
    The transmucosal route for drug administration has certain limitations inherent in its physicochemical characteristics. In this sense, the most used route of administration is parenteral, which however offers disadvantages. For example, the preparation of injectables requires sterile production conditions, their management usually involves medical personnel, is more uncomfortable for the patient, and implies greater risks of infection caused by the improper use of the needles. For all these reasons, transmucosal administration is in most cases an attractive alternative.
    One of the most frequent pharmaceutical routes of administration is oral administration. This also occurs in the field of food, where ways of administration of different additives and nutrients that improve existing technology are constantly being sought. The epithelium of the human intestine is highly absorbent, and is composed of a large number of microvilli with a total absorption area of up to 350 m2, ideal for efficient absorption.
    This route is not without problems, however. Many macromolecular components may exhibit low permeability and poor stability due to the aggressive environment of the digestive tract. In addition to being stable in this environment, they must overcome the mucosal barrier to reach their destination and, before disposal, be absorbed.
    The intestinal mucosa is a complex hydrogel that comprises proteins, carbohydrates, lipids, salts, and other components. It is secreted and continuously renewed to prevent the entry of pathogens, lubricate and protect the digestive tract, but at the same time allows the passage of nutrients. Due to the presence of this intestinal mucosa barrier and its dynamic nature, the particles administered orally may not have an adequate residence time for absorption, and be eliminated without fulfilling their objective. Mucoadhesive polymers like some


    Cellulose derivatives, polyacrylate, starch or chitosan, have been used to improve the residence time of the particles in the intestinal tract. However, so far most nanosystems developed based on mucoadhesive polymers are prone to remain anchored in the less adherent mucus layer without penetrating the intestinal mucosa barrier, thus being exposed to rapid removal.
    When nanoparticles are used, oral administration can be considered a complex stepwise process. The nanoparticles must first reach the intestinal mucosa without degrading, and without releasing the active substance prematurely. Next, it must adhere to the outermost mucosa. This adhesion must be strong enough to prevent its rapid removal, but at the same time allow penetration into the deeper layers of the mucosa. Once the deeper layers of the mucosa are reached, closer to the epithelium, the nanoparticles must yield the active substance with the required release profile.
    To achieve adequate administration, the administration of therapeutic drugs through the digestive tract has been evaluated using platforms that also use physical methods (Traverso G, Schoellhammer CM, Schroeder A, Maa R, Lauwers GY, Polat BE, Anderson DG, Blankschtein D , Langer RJ Pharm Sci 2015, 104, 362–367). For example, the robotic pill is one of the most recent platforms for oral delivery of large molecules. Although this strategy is promising, it does not cease to involve perforation and alteration of the mucosa, with the risks that this entails, especially in the case of chronic diseases, where repeated administration is necessary, sometimes for years.
    On the other hand, several efforts have been made in the use of spores or pollen as vehicles for transporting molecules. The spores are produced by lower plants or cryptogams, also known as sporaphytes. Pollen is produced by plants with seeds (spermatophytes), and contains a microgametophyte (male gametophyte). Like their biology, the structure and composition of spores and pollen vary. A pollen particle or a spore is essentially composed of genetic material contained in a cytoplasm, which is covered by a first inner layer called intin in the case of pollen, and endospores in the case of spores. These in turn are coated with a second layer called exina and exospora, respectively. The composition of the cytoplasm, intin and endospora vary.
    In the literature you can find numerous works based on the use of spores as vehicles for the administration of molecules.
    Several documents of the UNIVERSITY OF HULL describe the use of Lycopodium clavatum spores (pteridophytes) for the encapsulation of low molecular weight oils and drugs. For example,


    WO2005000280 describes hollow spores, that is, emptied of its cytoplasm and its intin, and free of the lipid layer, prepared from Lycopodium clavatum, which are loaded with dietary or pharmaceutical ingredients and have a protein content of less than 0 ,5%. These hollow spores are prepared by a complex process of aggressive washing with acidic, basic and organic media. The possibility of using hollow spores as a vehicle for nanosystems is not indicated in this document. WO2006064227 describes the use of this same technology based on the spores of Lycopodium clavatum for the creation of magnetic formulations. Other applications in the name of the same authors describe hollow spore compositions of Lycopodium clavatum following the same principles: WO2007012856 (antioxidant activity), WO2007012857 (topical compositions), WO2009077749 (compositions comprising protective additives), and WO2010004334 (improved whiteness) . In none of the cases is the possibility of incorporating nanosystems to the hollow spores mentioned, and it is always a hollow exine of Lycopodium clavatum spores, free of the outer lipid layer, and prepared from a complex sequence of acidic washes and Basic, among others. These hollow spores have a limited ability to control the administration profile, showing very variable release times. In addition, Lorch, M., et al. Chem. Comm., 2009, 6442-6444 have described the behavior of these spores in blood plasma, and in said biological environment it is observed that they are not stable and collapse.
    Following essentially the same procedures, other research groups have developed technologies around the hollow spores of Lycopodium clavatum, for example, as an antigen vehicle for oral vaccines (Shashwti U. Atwe, Yunzhe Ma, Harvinder Singh Gill, Journal of Controlled Release, 2014, 194, 45-52).
    In Shwan A. Hamad, Amro F. K. Dyab, Simeon D. Stoyanovb, Vesselin N. Paunov J. Mater. Chem., 2011,21, 18018-18023 describes a procedure for encapsulation of a combination of cells and magnetic nanoparticles within hollow spores of Lycopodium clavatum. Said method comprises fracturing the spores by compression and subsequent incubation in the presence of a mixture of magnetic cells and nanoparticles. Despite considering the possibility of incorporating inorganic nanoparticles, the procedure described in this document obliges to fracture the spore, compromising its physicochemical properties and therefore its usefulness in biological systems as a vehicle of administration.
    The exine of the pollen particles has on its surface an additional lipid layer complex mixture of proteins, lipids and other molecules (known as "pollenkitt"). Intin is generally formed by cellulose, while exine is composed of a protein material called sporopolenin, and whose exact composition is unknown. Exina is a layer


    extremely resistant, stable to acidic and basic conditions, and has a high porosity. Given these properties, various technologies have been tested to isolate the exine, that is, empty the inside of the exina from its intin and genetic material, as well as clean the outer surface of the lipid or pollenkitt layer. Once isolated, its use is tested as a vehicle for administration of molecules of interest.
    Raghavendra C. Mundargi, Michael G. Potroz, Soohyun Park, Hitomi Shirahama, Jae Ho Lee, Jeongeun Seo, Nam-Joon Cho, small 2016, 12, No. 9, 1167–1173 describe the use of pollen particles Helianthus annuus (sunflower) previously washed with ethyl ether to encapsulate BSA, and the possibility of using different pollen spores for the encapsulation of small molecules, proteins, peptides, growth factors or biosimilars is suggested. The BSA load comprises incubation or incubation under vacuum. The preparation process described in this document does not include washing with aqueous solutions.
    A unique example can be found in W. Brandon Goodwin, Ismael J. Gomez, Yunnan Fang, J. Carson Meredith, Kenneth H. Sandhage, Chem. Mater. 2013, 25, 4529-4536, where Helianthus annuus (sunflower) pollen particles are washed with a mixture of chloroform and methanol for later use as templates in the preparation of iron oxide-based replicas. The resulting product is subjected to pyrolysis, leaving only the "shell" of iron oxide in the form of the original pollen particle.
    Studies that focus on the study of the composition of pollen particles can also be found in the literature, although they do not describe possible uses of them. Some of these studies can be found in, for example, Doughty, J .; Hedderson, F .; McCubbin, A .; Dickinson, H. Proc. Natl Acad. Sci., 1993, 90, 467-471, where Brassica oleracea spores are washed with cyclohexane to study the coating proteins thus extracted. In Mohamed Elfatih H. Bashir, Jason M. Ward, Matthew Cummings, Eltayeb E. Karrar, Michael Root, Abu Bekr A. Mohamed, Robert M. Naclerio, Daphne Preuss, PLoS ONE 8 (1): e53337 the proteins of the pollen bark of the herbs of the Poaceae family, including, Cynodon dactylon (Bermuda), Phleum pratense (Timothy), Poa pratensis, or Dactylis glomerata. In this document the pollen is washed with cyclohexane to extract the lipophilic proteins from its surface. It is reported that, as a result of the treatment, the exina's microchannels collapse, thus losing the pollen particle its structure.
    The use of pollen is not without problems. Although pollen can have similar sizes, its morphology and its biocompatibility once treated is not guaranteed, especially in view of the aggressiveness of some of the treatments to which it is subjected. Also consider whether this


    Treatment will keep your morphology intact, and will be able to incorporate and then protect, and release the products that can be incorporated, for example, nanosystems.
    Nasal and ocular administration face similar problems to those described above for oral administration, and it is still a challenge to administer active ingredients by these routes so that they have adequate stability and residence time.
    Thus, it would be desirable to find alternative methods for the administration of nanosystems that can overcome the limitations indicated above, and can be obtained by simple methods. Brief Description of the Invention
    The authors of the present disclosure now provide an administration platform that solves the problems posed in the prior art. Said platform is based on purified pollen particles. They are obtained by a pollen purification method that improves its biocompatibility and allows the incorporation of nanosystems in a reproducible way.
    Thus, a first aspect of the invention is a process for preparing purified pollen particles comprising the steps of
    (to)  washing a pollen particle in a buffered aqueous medium at a pH between 4.5 and 9; Y
    (b) wash with an organic solvent;
    to obtain the purified pollen particle that preserves the intin layer and the exine layer, but whose lipid layer (pollenkitt) has been totally or partially removed.
    This simple process avoids putting the pollen particles in contact with basic means or strong acids, contrary to the processes disclosed in the state of the art, and comprising multiple aggressive washes with different acids and bases (see for example, WO2005000280). With the method of the invention, the intin is partially or totally conserved, and the resulting purified pollen particle is more stable in biological fluids (see examples below) than the hollow spores that only retain the exine layer.
    A further aspect of the invention is therefore the purified pollen particles obtainable by this process, which, as evidenced by the examples, show differentiated properties of biological fluid stability and transportability to, and within, the mucous membranes
    Although in other studies hollow spores have been used as containers of molecules, the possibility of conserving exina and intin together has not been reported, nor its possible use


    as an administration platform for nanosystems, and in particular for nanosystems through mucous channels. In addition, researchers have proven, both in ex vivo experiments and in vivo, that the simple purification method used results in purified pollen particles with surprising adhesion capacity and, which is fundamental, penetration into the intestinal mucosa , even after the incorporation of nanosystems into its surface and / or its absorption, which significantly improves current knowledge.
    The purified pollen particles of the invention can be incorporated into different compositions, which constitutes a further aspect of the invention. An additional aspect is also the possibility of its use as a medicine. This opens the possibility of a new platform for use in the manufacture of medicines for the treatment of diseases or medical conditions such as, for example, metabolic, immune, gastrointestinal, cardiovascular, joint, rare, tropical, oncological diseases, among others, and also to prevent infectious diseases, such as vaccines. Preferably, in the case of chronic and / or metabolic diseases that require repeated administrations, in particular mucosal routes. Additional aspects are also its use in the manufacture of a medicament for the controlled release of a pharmaceutically acceptable active ingredient. Given the excellent adhesion to the intestinal mucosa and its stability in vivo, the capsules of the invention also have excellent use as a dietary supplement in the administration of a dietary acceptable principle, and for use in the manufacture of a medically administered oral administration, nasal or ocular Even the possibility of designing treatments in these ways that were not possible before.
    The purified pollen particles obtained by this washing procedure can incorporate nanosystems by a simple incubation process. Thus, a further aspect of the invention is a purified pollen particle, preferably equine, comprising an intin layer and an exine layer, but whose lipid layer (pollenkitt) has been removed. A further aspect of the invention is a composition comprising a nanosystem and a purified pollen particle, preferably equine, comprising an intin layer and an exine layer, but whose lipid layer (pollenkitt) has been removed.
    The purified pollen particles described herein provide a new strategy for the administration of nanosystems, especially for their transmucosal administration. They allow a high number of nanosystems to be administered simultaneously, and improve their chances of adhesion and penetration into the mucosa.
    Brief Description of the Figures


    Figures 1A, 1B, 1C and 1D: Photographs showing the size and shape of the purified pollen of the invention in different simulated biological media: A) before incubation; B) after incubation at 37 ° C in simulated intestinal fluid; C) after incubation at 37 ° C in simulated gastric fluid; and D) after incubation at 37 ° C in blood.
    5 Figure 2: Measurement of autofluorescence in pollen particles washed with water (a), and in capsules that have undergone additional washing with chloroform / methanol (b) or with cyclohexane (c). See example 1. The axis of the abscissa shows the wavelength in nm, the axis of ordinate the absorption units.
    Figure 3: SEM photographs of the pollen particles that associate A) chitosan nanoparticles; B) 10 protamine nanocapsules and C) protamine nanocapsules after lyophilization.
    Figure 4: Photographs of confocal ex vivo at 120 minutes. In the red channel (Figure 4A) the nanosystems are seen and in the green channel (Figure 4B) the pollen particles. It is appreciated how the nanosystems have been deposited on the purified pollen particles of the invention.
    Figure 5: Microscopy photograph showing that pollen grains keep their integrity and morphology in vivo. Detailed description of the invention
    Purified Pollen Particles of the Invention
    In the present disclosure, "purified pollen particle" means those particles of
    20 pollen that have been treated to remove the lipid layer that covers its exterior ("pollenkitt"). Preferably, the treatment also totally or partially empties the cytoplasm inside the particle. Thus, the treatment preferably leaves the intin and exine substantially free of the cytoplasm and the lipid layer. Such purification may not be total, and still partially retain the lipid layer.
    It is understood in the present invention that purified pollen particles do not retain more than 50%, preferably not more than 40%, for example, not more than 20%, not more than 10%, more preferably not more than 5% in weight of the lipid layer, with respect to the original weight of the lipid layer. Preferably, at least 10%, for example at least 20%, for example at least 30%, for example at least 40%, has also been removed from the purified pollen particle by
    For example at least 50%, for example at least 60%, for example at least 70%, for example at


    less than 80%, for example at least 90% by weight of the cytoplasm, with respect to the original weight of the cytoplasm.
    These are purified pollen particles that comprise the intin layer and the exine layer, but do not comprise its lipid layer (pollenkitt), and preferably neither the cytoplasm. The purified pollen particles are obtained through the simple pollen purification method described above and comprising, first washing the pollen particles with aqueous media, and then with an organic solvent, preferably, without at any time the Pollen particles come into contact with basic or acidic media (that is, without pH less than 4.5 or greater than 9).
    Therefore, in the process of purifying the pollen particles the water is at an essentially neutral pH, for example, at a pH between 5 and 9, preferably between 6 and 8, more preferably between 6.5 and 7, 5. Nor is it necessary that the purification process involves washing at high temperatures. The average expert can adjust the conditions, and it is preferred that the water in the first stage is at a temperature between 15 ° C and 60 ° C, preferably between 20 ° C and 45 ° C. Depending on the type of pollen, it is also possible to perform a second wash with water.
    Various organic solvents are available to the person skilled in the art to carry out the second wash. They can be cyclic or linear hydrocarbons, for example, of the formula CnH2n + 2 (non-cyclic, linear or branched) or CnH2n (cyclic), of 5 to 20 carbon atoms (n = 5-20). Examples that can be used in the present invention are pentane, hexane, heptane, octane, nonane, decane, cyclopentane, or cyclohexane, or mixtures thereof, for example, cyclohexane. The solvent used in the second stage may also be an alcohol, for example, from 1 to 12 carbon atoms, for example, from 1 to 4 carbon atoms. Examples that the average person skilled in the art can use in the present invention are methanol, ethanol, propanol, tert-butanol, or mixtures thereof. Non-limiting examples are also halogenated hydrocarbons, that is, hydrocarbons as defined at the beginning of this paragraph, but in which at least one of the hydrogen atoms has been replaced by a halogen (fluorine, chlorine, bromine or iodine, preferably chlorine). The halogenated hydrocarbon used in the present invention may have the formula CnH (2n + 2-z) Xz (non-cyclic, linear or branched) or CnH2n-zXz (cyclic) where z is an integer (equal to or less than 2n +2 or 2n, as the case may be) and X is fluorine, chlorine, bromine or iodine, preferably chlorine. Examples of this type of solvents are dichloromethane and chloroform. It is also possible to wash using mixtures of organic solvents in different proportions. For example, using a mixture of halogenated hydrocarbon: alcohol in


    ratios between 1:20 and 20: 1. An example may be the mixture of chloroform and methanol.
    Other solvents that can be used in the present invention are those that include a carbonyl or ester group, and that have a low molecular weight, for example, below 250 Da. Extended examples of these solvents are acetone or ethyl acetate.
    As in the case of water washing, more than one washing with organic solvents can be performed. It is also preferred that the organic solvent wash be carried out at low or moderate temperatures, for example, at a temperature between 15 ° C and 60 ° C, preferably between 20 ° C and 45 ° C.
    The method of the invention also thus includes the possibility of repeating one or more times the aqueous wash of step (a) and / or the organic solvent wash of step (b). For example, it may be convenient to perform more than one aqueous wash to facilitate the removal of the cytoplasm. The method of the invention optionally includes one or more enzyme treatments (eg, cellulases and / or amylases). These methods manage to pierce the intin and thus improve its porosity.
    Therefore, the conditions under which it is possible to prepare the purified pollen particles of the invention are not very aggressive and are easy to implement at the industrial level. They do not preferably comprise high pressures so that the process does not include any stage in which the particles are subjected to pressures greater than 10 bar (106 Pa). For example, all stages of the process are performed at pressures below 5 bar (0.5 x 106 Pa), preferably at pressures below 2 bar (0.2 x 106 Pa), usually at atmospheric pressure (substantially 1 bar (105 Pa)). This makes the process more attractive at the industrial level and prevents fractures in pollen particles. It is also preferred that the method of production does not involve any washing in aqueous medium whose pH is less than 4.5, preferably not less than 5, or greater than 9, preferably greater than 8.
    Since the purified pollen particles of the invention may have applications in the pharmaceutical and food industry, it is preferred that the solvents be pharmaceutically acceptable. This is not critical to obtain the desired properties, and it must also be taken into account that these purified pollen particles are going to undergo subsequent incubation and / or purification steps before being administered.
    Each of the washes is performed according to the usual techniques in this field and comprises the formation of a suspension of the pollen grains in water or in organic solvent, usually followed by stirring. The suspension is usually incubated and centrifuged to then remove the supernatant. The resulting particles are usually dried, usually in


    moderate temperature conditions, for example, between 4 ° C and 60 ° C, for example, between 20 ° C and 40 ° C.
    The purification method of the invention a priori admits the incorporation into the purified pollen particle of any nanosystem. Such incorporation can occur as a result of adsorption of the nanosystem on the surface of the purified pollen particle, or as a consequence of the absorption of the nanosystem inside the purified pollen particle. In addition, either as a component of the nanosystem itself, or as an additional component, the purified pollen particle can also incorporate a pharmaceutically acceptable active ingredient, a dietary acceptable principle or mixtures of both. The purified particles of the invention can thus incorporate the nanosystems, which in turn can comprise a pharmaceutically acceptable active ingredient, a dietary acceptable principle or mixtures of both.
    The incorporation of the nanosystem simply requires putting it in contact with the purified pollen particle obtained after step (b). The method of incorporating nanosystems can therefore comprise incubating the nanosystem in the presence of the purified pollen particle. Said incubation can be done dry or in the presence of a liquid.
    Therefore, the preparation procedure may include the steps of
    (to)  washing a pollen particle in a buffered aqueous medium at a pH between 4.5 and 9;
    (b) wash with an organic solvent to obtain the purified pollen particle that preserves the intin layer and the exine layer, but whose lipid layer (pollenkitt) has been totally or partially removed, preferably also including the total or partial removal of the cytoplasm; Y
    (C) incubate the purified pollen particle in the presence of a nanosystem, for example, in the presence of a solvent.
    Alternatively, the preparation procedure may include the steps of
    (to)  washing a pollen particle in a buffered aqueous medium at a pH between 4.5 and 9;
    (b) wash with an organic solvent to obtain the purified pollen particle that preserves the intin layer and the exine layer, but whose lipid layer (pollenkitt) has been totally or partially removed;
    (C)  drying the purified pollen particle from step (b); Y


    (d) impregnate the purified pollen particle with the nanosystems, optionally, by applying a vacuum.
    Additionally, the purified pollen particles thus obtained can be lyophilized. If necessary, a cryoprotectant, for example glucose or trehalose, can be added before proceeding with such lyophilization.
    This method of purification thus allows the purified pollen particles to subsequently incorporate organic nanosystems, and be able to transport them to, and through, the mucous membranes, for example, in the intestine, the buccal, nasal or ocular mucosa.
    10 Pollen particles that are used as raw material to be purified can come from different species. Although the pollen particles have different sizes and morphologies, depending on the original species, the purification method of the present invention can be applied to anyone. Preferably, the pollen particles are equine, that is, they comprise needles with a length of at least one micron, for example, between 1 micron and 10 microns, or between 1
    15 microns and 7 microns, usually between 1 micron and 5 microns or between 1.5 microns and 2.5 microns, or between 1.5 microns and 2 microns. For example, they can be particles of the Helianthus family, for example, Helianthus annuus (Sunflower).
    Due to its size, availability and morphology, the pollen particle may come from, for example, the
    20 Angiosperms or Magnoliophytas, that is, flowering plants. Within the Angiosperms or Magnoliophytas the pollen particles may belong to the Monocotiledóneas, Chloranthaceae, Ceratophyllaceae, Magnoliidae or Eudicotiledóne clades, especially Eudicotiledoneas, equine species being preferred. The families of Eudicotiledoneas that are most appropriate are Annonaceae, Malvaceae, Meliaceae, Tamaricaceae, Asteraceae, Oleaceae or
    25 Caprifoliaceae.
    For example, equine species especially useful for the purposes of the invention are one or more selected from the group consisting of
     Magnoliophyta Eudicotiledóneas Asterales Asteraceae Ambrosia sp.


     Magnoliophyta Eudicotiledóneas Asterales Asteraceae Helianthus sp.  Magnoliophyta Eudicotiledóneas Asterales Asteraceae Pectis sp.  Magnoliophyta Eudicotiledóneas Asterales Asteraceae Tagetes sp.  Magnoliophyta Eudicotiledóneas Asterales Asteraceae Stevia sp.
    5  Magnoliophyta Eudicotiledóneas Asterales Asteraceae Calendula sp. Magnoliophyta Eudicotiledóneas Asterales Asteraceae Parthenium sp. Magnoliophyta Eudicotiledóneas Asterales Asteraceae Balsamorhiza sp. Magnoliophyta Eudicotiledóneas Asterales Asteraceae Cirsium sp. Magnoliophyta Eudicotiledóneas Asterales Asteraceae Balsamorhiza sp.
    10  Magnoliophyta Eudicotiledóneas Asterales Asteraceae Arnica sp.  Asteral Eudicotyledonous Magnoliophyta Asteraceae Ambrosia sp  Asteral Asteral Magnoliophyta Asteraceae Asterceae Aster sp.  Magnoliophyta Eudicotiledóneas Asterales Asteraceae Bidens sp.  Magnoliophyta Eudicotiledóneas Asterales Asteraceae Clibadium sp.
    15  Magnoliophyta Eudicotiledóneas Asterales Asteraceae Cosmos sp.  Magnoliophyta Eudicotiledóneas Asterales Asteraceae Doronicum sp.  Magnoliophyta Eudicotiledóneas Asterales Asteraceae Echinacea sp.  Magnoliophyta Eudicotiledóneas Asterales Asteraceae Erechtites sp.  Magnoliophyta Eudicotiledóneas Asterales Asteraceae Erigeron sp.
    20  Magnoliophyta Eudicotiledóneas Asterales Asteraceae Gaillardia sp.  Magnoliophyta Eudicotiledóneas Asterales Asteraceae Inula sp.  Magnoliophyta Eudicotiledóneas Asterales Asteraceae Leucanthemopsis sp.  Magnoliophyta Eudicotiledóneas Asterales Asteraceae Liatris sp.  Magnoliophyta Eudicotiledóneas Asterales Asteraceae Pulicaria sp.
    25  Magnoliophyta Eudicotiledóneas Asterales Asteraceae Scorzonera sp.  Magnoliophyta Eudicotiledóneas Asterales Asteraceae Tetragonotheca sp.  Magnoliophyta Eudicotiledóneas Alismatales Araceae Ulearum sp.  Alismatal Eudicotiledóne Magnoliophyta Araceae Zomicarpa  Eisicotyledonous Magnoliophyta Alismatales Araceae Pinellia sp.
    30  Magnoliophyta Eudicotiledóneas Magnoliales Annonaceae Annonaceae sp.


     Magnoliophyta Eudicotiledóneas Sapindales Meliaceae Melia sp.
     Magnoliophyta Eudicotiledóneas Caryophyllales Tamaricaceae Tamarix sp.
     Magnoliophyta Eudicotiledóneas Lamiales Oleaceae Olea sp.
     Magnoliophyta Eudicotiledóneas Dipsacales Caprifoliaceae Lonicera sp.
    5  Magnoliophyta Eudicotiledóneas Malvales Malvaceae Malope sp.
     Magnoliophyta Eudicotiledóneas Malvales Malvaceae Abutilon sp.
     Magnoliophyta Eudicotiledóneas Malvales Malvaceae Hibiscus sp.
     Magnoliophyta Eudicotiledóneas Malvales Malvaceae Lavatera sp.
     Magnoliophyta Eudicotiledóneas Malvales Malvaceae Sphaeralcea sp. 10  Magnoliophyta Eudicotiledóneas Malvales Malvaceae Malva sp.
     and mixtures thereof.
    The pollen particles used as raw material in the invention can have different sizes, for example, it can have an average diameter between 1 micron and 400 microns, 1 microns and 300 microns, or between 1 microns and 200 microns, or between 1 microns and 100 microns, for example, between 10
    15 microns and 50 microns, for example, between 15 microns and 40 microns, or between 20 microns and 30 microns, or between 25 microns and 30 microns.
    Both purified pollen particles, such as nanosystems, the pharmaceutically acceptable active ingredient, or the dietary acceptable principle, may be associated with a label, for example, a fluorescent label. Non-limiting examples of this type of
    20 fluorescent markers are amines reactive dyes (for example, Alexa Fluor®), TAMRA or Cy Dyes (Cy-dyes). This allows you to track each of them together or separately.
    Nanosystems
    The purified pollen particles of the invention have the ability to incorporate nanosystems, preferably organic nanosystems. In the present disclosure the term "nanosystem" in the


    term that we use here is a colloid, that is to say a particle in which at least one of its dimensions is in the range between 1 and 1000 nm, preferably, it has an average diameter between 1 nm and 500 nm, more preferably, between 40 nm and 400 nm, measured by photonic correlation spectroscopy in a Malvern Instruments Nanosizer, and which has a lyophobic character, that is, it is not dissolved in its external phase (Paul Hiemenz, Raj Rajagopalan, Principles of colloid and Surface chemistry, 3rd Ed., Marcel Dekker, Inc., New York, 1997). In the context of this invention the colloidal system is intended to have in its composition at least one fluorescence marker or a pharmacologically active molecule. Said term is considered in the present invention to include, for example, "nanocapsule", "nanoparticle", "vesicle", "micelle", "nanoemulsion", "liposome" or "utra-fine particle". The average expert in the field also understands that organic nanosystems are those nanosystems whose components are at least partly organic, that is, based on molecules comprising carbon and hydrogen, for example, proteins, carbohydrates or lipids. See, for example, Kumar R, Lal S J Nanomater Mol Nanotechnol 2014, 3, 4. The nanosystems of the invention can be for example polymeric nanosystems. Polymeric nanosystems are widely described in the literature and are known to those skilled in the art, as described for example by Pinto Reis et al., In Nanomedicine: Nanotechnology, Biology, and Medicine 2 (2006) 8-21.Pinto Reis, et to the. 2006
    The average diameter polydispersity index of the polymeric nanosystems that can be used in the present invention is between 0.1 and 0.5, where the polydispersity index is measured by the photonic correlation spectroscopy technique measured in a Nanosizer from Malvern Instruments. For the same reasons, it is preferable that the nanosystem be substantially biodegradable and of low or no toxicity. Thus, the nanosystem is considered to be biodegradable when at least one of its components complies with commonly accepted standards, for example, the biodegradability standards written by different standards bodies (ISO, CEN, ASTM, DIN, etc.), for example, it is biodegradable in 90% after 6 months according to the UNE-EN-ISO 14852: 2005 standard (determination of the final aerobic biodegradability of plastic materials in aqueous medium), that is to say 90% of the carbon atoms ( C) present in the component were converted to carbon dioxide after six months under the conditions defined in the standard.
    The person skilled in the art can recognize that there are various kinds of nanosystems that can be used in the present invention, many of them commercially available, or that can be prepared by methods described in the state of the art. They can be of the matrix type, that is, polymeric networks containing ionic crosslinks, for example, which contain water soluble polymers. Non-limiting examples of this type of nanosystems are those based on


    polysaccharides, for example, chitosan (chitosan nanoparticles) or in polyamino acids, for example, protamine (protamine nanocapsules). See for example the method described in Calvo P, Remuñán-López C, Vila-Jato JL, lonso MJ J Appl Polym Sci 1997, 63, 125-132 or in Thwala L (2016) Protamine nanocapsules as carriers for oral peptide delivery.
    Nanosystems comprising a liquid core surrounded by a coating layer can also be used in the present invention. The core may incorporate different oils, lipid material (for example, fatty acids or phospholipids or mono-, di- or tri-glycerides), in combination with non-ionic surfactants. The coating layer may be a polymer, for example, protamine. See for example the methods described in patent application PCT / ES2013 / 070885.
    Other nanosystems suitable for use in the present invention are solid nanoparticles, such as chitosan nanoparticles, in particular those described in ES2481940B1, ES2093562, Csaba et al, Journal of Controlled Release, 2017, 245, 62-69; Marcos García-Fuentes, M.J.A, Journal of Controlled Release, 2012, 161 (2), 496–504. A person skilled in the art can know how to prepare these and other polymeric nanosystems according to Nanomedicine: Nanotechnology, Biology, and Medicine 2 (2006) 8-21.Pinto Reis, et al. 2006
    It is preferred that the nanosystems be pharmaceutically acceptable for the applications proposed in the present invention.
    Applications
    Researchers have proven that the purified pollen particles of the invention are stable in vivo, and have a surprising ability to penetrate the intestines, making them an excellent platform for transmucosal administration of nanosystems, especially for oral administration. . Thus, the present invention includes the use of purified pollen particles of the invention, especially those incorporating nanosystems, for the manufacture of a medicament. That is, a purified pollen particle of the invention, especially those incorporating nanosystems, for use as a medicament. The purified pollen particles of the invention are therefore especially suitable for use in the manufacture of a transmucosal administration medicament, such as oral administration, ocular administration or nasal administration.


    For example, purified pollen particles of the invention can be used for ocular administration, including their use for the manufacture of a medicament for the treatment of ocular diseases, for example, for the cure of ocular wounds or macular disease.
    The purified pollen particles of the present invention can also be used for the
    5 administration of nasal antigens, especially interesting for the administration of vaccines, which would alleviate the burden that parenteral administration poses to health services, especially in developing countries.
    Given its good absorption and penetration into the intestinal mucosa, a particularly suitable use for purified pollen particles of the invention is the manufacture of a medicament for oral administration and / or for the manufacture of a medicament for the controlled release of a principle. active, preferably of release in the digestive tract. The term "digestive tract" means in the present invention the system through which any active ingredient administered orally from its intake to excretion passes. It therefore includes the mouth, throat, esophagus, stomach, small intestine and large intestine, among others. For example, the particles
    Purified of the invention may be useful for the administration of molecules for which only parenteral administration means are known, for example for the administration of antidiabetic peptides. The incorporation of these active substances in nanosystems, and that of these in turn in purified pollen particles, allows transport to the epithelium and its controlled release.
    A situation in which transmucosal administration can offer unique advantages is the
    20 administration of antigens. Mucosal surfaces such as the respiratory or gastrointestinal tract represent the main route of entry of many pathogens and play an integral role in the development of effective defense mechanisms against them. In fact, a combined immune response that involves systemic and mucosal immunity is best achieved by transmucosal administration of antigens.
    25 The term "active substance" refers to a substance used to treat, cure or prevent a medical condition or disease. It also encompasses in the context of the present invention those substances that are used in diagnostic tests. Said active ingredient is part of the nanosystem, and can be, for example, a contrast agent or a vaccine. In an embodiment of


    The invention, the active ingredient is a high molecular weight compound, for example, a protein, a peptide, a lipid, an antibody or a nucleic acid.
    Therefore, the present invention also relates to a composition comprising the purified pollen particle of the invention and a pharmaceutically acceptable excipient. In another aspect, the invention relates to a composition comprising the purified pollen particle of the invention and a dietary acceptable excipient.
    The term "excipient" refers to a diluent or adjuvant with which the active substance is administered. Such pharmaceutical excipients may be sterile liquids, such as water or oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical excipients are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such excipients can also be considered as the auxiliary substances necessary to manufacture the desired pharmaceutical form. Its nature and quantities depend, among other factors, on the pharmaceutical form of administration chosen. Said pharmaceutical forms of administration of the composition
    Pharmaceuticals will be manufactured according to conventional methods known to those skilled in the art. A review of different methods of administration of active ingredients, excipients to be used and procedures to produce them can be found in “Treaty of Farmacia Galenica”, C. Faulí i Trillo, Luzán 5, S.A. of Editions, 1993.
    The term "pharmaceutically acceptable" refers to molecular entities and compositions that
    20 are physiologically tolerable and do not normally produce an allergic or similar unwanted reaction, such as gastric discomfort, dizziness and the like, when administered to a human being. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a federal government or state regulatory agency or listed in the US Pharmacopoeia. or other pharmacopoeia generally recognized for use
    25 in animals and more particularly in humans.
    Similarly, the term "dietary acceptable" refers to molecular entities and compositions that are physiologically tolerable, preferably approved for human consumption by a regulatory agency for use with food purposes in animals and more particularly in humans. That is, the use of purified pollen particles from the


    The present invention for the administration of a dietary acceptable principle excludes therapeutic uses, and may serve, for example, for the administration of a taste masker.
    The present disclosure thus includes methods for the treatment of a subject in need of treatment by administering a therapeutically effective amount of the purified pollen particles of the invention. The term "treatment" or "treat" refers in the context of the present invention to the administration of the pharmaceutical compositions of the invention to prevent, reduce or eliminate one or more symptoms associated with a medical condition or disease. The term "treat" also covers the elimination, reduction or prevention of the sequelae of said disease or medical condition. The term "reduce" is understood in the context
    10 of the present invention as the improvement in the patient's situation, either evaluated by subjective means (perception of the patient as to some particular aspect or in terms of its general condition) or by objective means, for example, biological parameters, for example , levels of an analyte in certain fluids.
    The term "therapeutically effective amount" refers to the amount of active ingredient calculated.
    15 to produce the desired effect and will generally be determined, among other reasons, by the characteristics of the active ingredient used and the therapeutic effect to be obtained. In a particular embodiment, the dose of active ingredient administered to a subject in need of treatment for the treatment is in the range of 10-10 to 1010 mg / kg of body weight, usually between 10-3 and 103 mg / kg of body weight. .
    The medicament comprising the purified pollen particles of the invention can be found in any form suitable for administration to humans and / or animals, preferably humans, including infants, children and adults and can be prepared by standard procedures known to those skilled in the art. subject, for example, “Pharmaceutics: The Science of Dosage Forms, second edition, Aulton, ME (ed.) Churchill Livingstone, Edinburgh (2002); “Encyclopedia of
    25 Pharmaceutical Technology ”, second edition, Swarbrick, J. and Boylan J.C. (eds.), Marcel Dekker, Inc. New York (2002); "Modern Pharmaceutics", fourth edition, Banker G.S. and Rhodes C.T. (eds.) Marcel Dekker, Inc. New York 2002 and "The Theory and Practice of Industrial Pharmacy", Lachman L., Lieberman H. and Kanig J. (eds.), Lea & Febiger, Philadelphia (1986). The composition of the medication may vary depending on the route of administration.


    The pharmaceutical composition of the invention can be administered in a plurality of pharmaceutical forms of administration, for example, solid, liquid, etc. Illustrative, non-limiting examples of said pharmaceutical forms of administration of the pharmaceutical composition of the invention include oral drops (suspension, emulsion, etc.); oral formulations (liquids, suspension, emulsion, gel, paste, powder, etc.); oral lyophilisate; oral gum; powder for oral suspension; granules; gastro-resistant granules; prolonged release granules; modified release granules; granules for oral suspension; powder and solvent for solution or oral suspension; syrup; syrup powder; syrup granules; tablets (for example, soluble tablet, dispersible tablet, coated tablet, film-coated tablet, effervescent tablet, orodispersible tablet, gastro-resistant tablet, prolonged-release tablet, modified-release tablet, oral tablet, chewable tablet, etc.); effervescent powder or granules; envelope, capsule; pills; intra-continuous continuous release device; pulsatile release intrarruminal device; block to suck; premix for medicated feeding staff; dragees; suspension, drops, spray, gel, paste, capsule or bucomucosos etc .; spray, sublingual tablet, etc .; mouthwash; gel, gingival paste, etc .; tablet to suck; tablet; gel, stick, insert, powder, suspension, dental emulsion, etc .; toothpaste; cream; gel; ointment; eye cream; eye gel; eye ointment; eye drops (powder and solvent for suspension, lotion, solvent to reconstitute a lotion, etc.); ophthalmic insert; ear cream; ear gel; ear ointment; ear drops (suspension, emulsion, powder, etc.); ear spray (suspension, etc.); irrigation for the ears (emulsion, etc.); ear buffer; ear stick; nasal cream; nasal gel; nasal ointment; nasal drops (suspension, emulsion, etc.); nasal powder; nasal spray (suspension, emulsion, etc.); nasal irrigation, nasal stick; vaginal cream; vaginal gel; vaginal ointment; vaginal foam; vaginal suspension; vaginal emulsion; hard or soft vaginal capsule; vaginal tablet; 25 vaginal effervescent tablet; vaginal administration system; rectal cream; rectal gel; rectal ointment; rectal foam; rectal suspension; rectal emulsion; powder for rectal dissolution; powder for rectal suspension; tablet for rectal suspension; suppository; rectal capsule; nebulizer suspension; powder for nebulizer suspension; nebulizer emulsion; pressurized inhalation (suspension, emulsion, etc.); inhalation powder; inhalation powder (hard capsule); powder for inhalation, predispensed; injection gel; suspension for injection; emulsion for injection; powder for suspension for injection; powder and solvent for suspension for injection; infusion solution; infusion emulsion; implantation tablet; bladder irrigation; powder for


    bladder irrigation; urethral gel; urethral stick; endotracheopulmonary instillation (dissolution); endotracheopulmonary instillation; endotracheopulmonary instillation (suspension); endocervical gel; powder and solvent for endocervical gel; intramarine suspension; intramarine emulsion; intramammary ointment; nipple stick; intrauterine administration system; etc. 5 Examples
    Specific examples of pollen purification (example 1), of preparation of suitable nanosystems for the present invention (example 2) and their incorporation into purified pollen particles to form delivery vehicles (example 3) are described below. The results obtained in an ex vivo example (example 4) designed to evaluate the results are also shown.
    10 purified pollen particles of the invention, as well as in vitro and in vivo experiments showing the stability of the purified pollen particles of the invention (examples 5 and 6, respectively). These examples serve to illustrate embodiments of the invention, but which in no case should be considered limiting.
    Example 1: Purification of purified pollen particles
    15 Purification
    For the purification of the pollen particles, sunflower pollen particles, Helianthus annuus, were started.
    Stage 1
    10 mg of pollen particles were suspended in 20 ml of miliQ water previously heated to 37 ° C
    20 for 30 seconds with stirring. The suspension was kept under horizontal stirring overnight at room temperature, and centrifuged the next day at 2500 rpm at 15 ° C for 10 minutes (Hettich Zentrifugen with 1689 rotor). The supernatant was discarded and the process was repeated, washing with 10 ml of miliQ water at 37 ° C. The suspension was centrifuged again at 2500 rpm, at 15 ° C for 5 minutes. The supernatant was discarded and the resulting particles dried at 37 ° C a
    25 night
    Stage 2
    The material from Step 1 was then re-suspended in miliQ water to prevent the formation of aggregates, and the organic solvent was washed. Here are two examples, the first using cyclohexane, and the second a mixture of chloroform / methanol.


    In the first case, 800 microliters of cyclohexane were added to 75 mg of the material from Step 1. The resulting suspension was stirred 30 seconds, centrifuged at 14000 rpm at 15 ° C for 1 minute (Hettich Zentrifugen with rotor 1689), and then discard the supernatant. The resulting particles were dried at 37 ° C overnight.
    In the second case on 50 mg of the material of Step 1, 10 ml of a chloroform / methanol mixture (3: 1; v / v) was added. The suspension was stirred 30 seconds and filtered on a 0.22 micron pore size membrane, which was washed with 5 ml of the chloroform / methanol mixture. After 2 hours the filtered material was recovered from the filter.
    Characterization
    The stability of the material thus obtained was evaluated against in vivo conditions. Specifically, it was incubated for 2 hours at 37 ° C in the following media: simulated intestinal fluid at pH 6.8, simulated gastric fluid at pH 1.2, and blood and post-pandial conditions in the upper small intestine, that is, simulation of feeding situation in the intestinal fluid (pH 5.8). After the various tests, the material was evaluated by SEM, without observing significant variations, thus confirming that the purified pollen particles prepared by the simple purification method of the invention are stable in in vivo conditions, in such aggressive media such as gastric and intestinal. Only in the case of gastric fluid incubation was the formation of salt materials and other products observed on the surface of purified pollen particles. Photos of the particles obtained after incubation in simulated intestinal fluid at pH 6.8, simulated gastric fluid at pH 1.2, and blood, are shown in Figures 1B, 1C and 1D, respectively, and can be compared with the photo obtained before incubation (Figure 1A).
    To evaluate the results of this procedure, the morphology of the material obtained from Stage 1, washed only with water, was compared with the material resulting from Stage 2, according to the invention, washed with water and organic solvent. The SEM photographs showed poorly defined pores in the first case, showing that the layer covering the exine is not completely removed only with water, and it is necessary to perform an additional wash with an organic solvent.
    For this comparison, the autofluorescence of the material obtained from Stage 1, washed only with water, and that of the material resulting from Stage 2, according to the invention, washed in aqueous medium at approximately neutral pH and organic solvent was also measured. In all cases the emission occurs around 500 nm, but in the case of the material prepared by the process of the invention, the level of autofluorescence was significantly lower, approximately 8000 units in the case of further washing with the chloroform / mixture. methanol, and of


    approximately 4000 units in the case of additional washing with cyclohexane. See Figure 2. This shows the different modification made to the system.
    Example 2: Some examples of organic nanosystems that can be encapsulated
    Two examples of non-limiting preparation of organic nanosystems that can be encapsulated are given below.
    Example 2.1: formulation of chitosan nanoparticles / sodium triphosphate (CS / TPP)
    Chitosan / sodium triphosphate nanoparticles were obtained using a CS / TPP ratio
    (5: 1) (w / w), by the ionic gelation technique, according to patent application WO9804244). A solution of 2 mg / ml of CS and 1.2 mg / ml of TPP in milliQ water was prepared. To obtain the nanoparticles 0.5 ml of TPP were poured onto 1.5 ml of CS under magnetic stirring and left under stirring for 10 min at 25 ° C. The nanoparticles form spontaneously.
    Example 2.2: protamine nanocapsules
    Protamine nanocapsules were prepared by the solvent diffusion technique, according to the ES2481940 patent, according to the following steps:
    one. An aqueous phase (10 ml) of protamine was prepared with a final concentration of 1.5 mg / ml. This solution was kept under magnetic stirring at room temperature.
    2.  An organic phase of 3 ml of PEG-stearate 40 (5,333 mg / ml), 20 uL sodium glycocholate (250 mg / ml) was prepared, mixed with vortex (5 seconds) and then 62.5 µL of Miglyol® oil was added and the The mixture was stirred again by vortex and finally 1.98 ml of acetone are added. Optionally, for the preparation of fluorescent nanocapsules, at this stage 20 microliters of the 1,1'-Dioctadecyl-3,3,3`, 3`- marker are added. Tetramethylindodicarbocianine perchlorate (DiD; 2.5 mg / ml) to the organic phase.
    3. The organic phase was quickly transferred to the previously prepared aqueous phase. The nanocapsules form spontaneously.
    Four. After holding the suspension for 10 minutes, the organic solvents were removed by rotary evaporation. After this step, they can optionally be isolated (e.g. by centrifugation: 30,000 rpm, 1h, 15 ° C)
    Characterization
    The properties of nanoparticles and nanocapsules are summarized in Table 1 below.


    Example Size (nm)Polydispersion Index -PdIPotential Z (mV)DiD fluorescent agent (%)
    Example 2.1 1870.30-
    Example 2.2 1910.1+3-
    285 0.2+396
    Table 1
    Example 3: Association between purified pollen particles and organic nanosystems
    About 5 mg of the purified pollen particles of example 1 (0.1) of the CS / TPP nanoparticles (0.4 mg / ml) were added; or (ii) 0.1 ml of protamine nanocapsules (10 mg / ml). The 5 suspensions in each case were kept under horizontal stirring for 30 minutes at 37 ° C in order to obtain administration vehicles according to the invention.
    As an optional additional step, lyophilization of the capsules was evaluated for 50 hours at -40 ° C (Labconco Corp.), which were then re-dispersed in a medium of interest, for example, water or buffered water.
    10 Both in the case of using lyophilization, and in the case of applying only one incubation, an effective association between the organic nanosystems of example 2 and the purified sporopolenin capsule could be verified (SEM photo and SP5 Leica AOBS-SP5 confocal microscope) obtained in example 1. All this without the need to apply aggressive media such as acidic or basic media, to the need to apply high pressures that could compromise integrity and
    15 capsule properties. Figure 3 shows the SEM photographs of the pollen particles that associate A) chitosan nanoparticles; B) protamine nanocapsules and C) protamine nanocapsules after lyophilization.
    Example 4: Ex vivo evaluation of purified pollen particles of the invention
    20 Model Preparation
    Male Sprague-Dawley rats weighing approximately 250 g were used. They fasted 18 hours before the experiment, leaving them only with access to water. Rats were sacrificed using CO2 using the appropriate conditions in the hypoxia chamber. The intestines were removed immediately through an abdominal incision and washed with Krebs solution (pH 6.5) 25 before the preparation of the sacks. This step is done for the disposal of food waste


    that may remain in the intestine. The intestine was divided into different parts. For administration, the samples were suspended in 1 ml of Krebs solution and introduced into the bags with the help of a syringe. One sample was used to evaluate the anatomy and physiology of the intestine after treatment with hematoxylin-eosin and the others were used for the analysis of the mucointeraction capacity of the nanosystem complex and the biomaterial with the intestine by confocal microscopy.
    Results
    It highlights the fact that the integrity of the intestine was maintained during the tests performed, indicating that the vehicles of administration of the invention are safe and do not cause side effects on tissue morphology.
    After incubation periods of the purified pollen particles associated with organic nanosystems, the intestinal tissue samples were observed by confocal microscopy. In all cases it has been found that incubations of 30 or 120 minutes lead to an improvement in the interaction and retention of nanosystems on the intestinal mucosa. In Figures 4A, 4B and 4C it can be seen in the confocal photographs at 120 minutes how the nanoparticles are maintained on the purified pollen particles of the invention. It is seen how the red channel (Figure 4A), which highlights the nanoparticles, coincides with the green channel (Figure 4B), which highlights pollen particles. It is thus appreciated how the nanosystems are kept deposited on the purified pollen particles of the invention.
    In contrast, the administration of free nanosystems did not lead to the retention of appreciable amounts on the intestinal mucosa, and no significant fluorescence was observed by the nanoparticles in the tissue (as can be seen in Figure 4 in the photographs). which refer to high concentration NCs and low concentration NCs), the adhesion promotion effect that is obtained when the purified pollen particles are combined with the nanosystems.
    Example 5: In vivo evaluation of purified pollen particles of the invention
    The experiment was performed with Sprague Dawley rats, which were fasted 12 hours before carrying out the experiment.
    The samples used were 75 milligrams of pollen per 1 milliliter of water, were administered by oral gavage to each of the study rats.
    The observation was carried out after periods of 1 hour and 3 hours respectively.


    For tissue extraction, the rat was sacrificed by using a CO2 chamber. Subsequently, an incision was made in the abdomen of the animal through which different parts of the digestive tract were removed. Specifically in these studies we focus on different parts of the small intestine (duodenum and jejunum).
    5 After tissue removal and without any treatment, direct observation was performed by an optical microscope at different magnifications.
    The conclusions drawn from these experiments were that pollen grains are stable in in vivo conditions since we can observe their characteristic morphology by microscopy and on the other hand we see that oral administration of pollen in vivo can be effective since grains
    10 pollen reach different regions of the small intestine without difficulty. See Figure 5.

    权利要求:
    Claims (32)
    [1]
    1. Procedure for the preparation of a purified pollen particle, comprising the steps of
    (to)  washing a pollen particle in a buffered aqueous medium at a pH between 4.5 and 9; Y
    (b)  wash with an organic solvent
    to obtain the purified pollen particle that preserves the intin layer and the exine layer, but whose lipid layer (pollenkitt) has been totally or partially removed.
    [2]
    2. The method according to claim 1, wherein the particle is not contacted with a medium of pH less than 4.5 or pH greater than 9.
    [3]
    3. The method according to any one of claims 1 or 2, wherein the exine of the purified pollen particle is not fractured.
    [4]
    Four. The process according to any one of claims 1 to 3, wherein the purified pollen particle is not subjected to pressures greater than 5 atmospheres.
    [5]
    5. The method according to any one of claims 1 to 4, comprising contacting a nanosystem with the purified pollen particle obtained after step (b).
    [6]
    6. The method according to claim 5, comprising the steps of
    (to) washing a pollen particle in a buffered aqueous medium at a pH between 4.5 and 9y;
    (b) wash with an organic solvent to obtain the purified pollen particle that preserves the intin layer and the exine layer, but whose lipid layer (pollenkitt) has been totally or partially removed; Y
    (C)  incubate the purified pollen particle in the presence of a nanosystem.
    [7]
    7. The process according to claim 6, wherein the incubation is carried out in a solvent.
    [8]
    8. The method according to claim 5, comprising the steps of
    (a) washing a pollen particle in a buffered aqueous medium at a pH between 4.5 and 9;

    (b) wash with an organic solvent to obtain the purified pollen particle that preserves the intin layer and the exine layer, but whose lipid layer (pollenkitt) has been totally or partially removed;
    (C)  drying the purified pollen particle from step (b); Y
    (d) impregnate the purified pollen particle with the nanosystems, optionally, applying a vacuum.
    [9]
    9. The method according to any of claims 1 to 8, comprising an additional lyophilization step.
    [10]
    10. Purified pollen particle obtainable by the method defined in any of the preceding claims.
    [11]
    eleven. The purified pollen particle according to claim 10, wherein said pollen is equine.
    [12]
    12. The purified pollen particle according to any of claims 10 or 11, wherein said pollen is an Eudicotyledonous Magnoliophyta.
    [13]
    13. The purified pollen particle according to any of claims 10 to 12, wherein said pollen is from the Helianthus family.
    [14]
    14. The purified pollen particle according to any of claims 10 to 13, having an average diameter between 1 and 400 microns.
    [15]
    fifteen. The purified pollen particle according to any one of claims 10 to 15, which comprises a nanosystem.
    [16]
    16. The purified pollen particle according to claim 15, wherein said nanosystem is an organic nanosystem.
    [17]
    17. The purified pollen particle according to any of claims 15 or 16, wherein the nanosystem has an average particle diameter between 1 nm and 500 nm, wherein the average diameter is measured by photonic correlation spectroscopy.
    [18]
    18. The purified pollen particle according to any of claims 15 to 17, wherein the polydispersity index of the average diameter of the organic nanosystem is between 0.1 and 0.5, wherein the polydispersity by photonic correlation spectroscopy.
    [19]
    19. The purified pollen particle according to any of claims 10 to 18, wherein the nanosystem is biodegradable.

    [20]
    twenty. The purified pollen particle according to any of claims 10 to 20, wherein the nanosystem comprises a pharmaceutically acceptable active ingredient, a dietary acceptable principle or mixtures of both.
    [21]
    twenty-one. Composition comprising the purified pollen particle defined in any one of claims 10 to 20.
    [22]
    22 The composition according to claim 21, comprising a nanosystem, wherein said nanosystem comprises a pharmaceutically acceptable active ingredient.
    [23]
    2. 3. The composition according to claim 22, which comprises a pharmaceutically acceptable excipient.
    [24]
    24. The composition according to claim 21, comprising a nanosystem, wherein said nanosystem comprises a dietary acceptable principle.
    [25]
    25. The composition according to claim 24, which comprises a dietary acceptable excipient.
    [26]
    26. The purified pollen particle defined in any of claims 10 to 20 for use as a medicament.
    [27]
    27. Use of the purified pollen particle defined in any of claims 10 to 20 for the manufacture of a transmucosal administration medicament.
    [28]
    28. The use according to claim 27 for the manufacture of a medicament for oral administration, ocular administration or nasal administration.
    [29]
    29. The use according to any of claims 27 or 28 for the manufacture of a medicament for controlled release a pharmaceutically acceptable active ingredient.
    [30]
    30 The use according to claim 29 wherein said active ingredient is a protein, a peptide, a lipid, an antibody, or a nucleic acid.
    [31]
    31. Use of the purified pollen particle defined in any one of claims 10 to 20 to administer a dietary acceptable principle.
    [32]
    32 Use according to claim 31, as a taste masker.

    Figure 1A
    Figure 1B

    Figure 1C
    Figure 1D

    Figure 2
    Figure 3

    Ex live 120 minutes
    Red GreenTransmitted
    High concentration NCs (900µL)
    NCs low concentration
    NCs + Pollen 4A4B4C
    Figure 4

    Figure 5
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2009077749A1|2007-12-18|2009-06-25|University Of Hull|Formulations comprising exine shells|
    WO2017010945A1|2015-07-16|2017-01-19|Nanyang Technological University|Microencapsulation of compounds into natural spores and pollen grains|
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