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    • 专利摘要:
    The present invention relates to biocide zinc oxide microparticles, preparation method and use thereof. More particularly, the present invention relates to zinc oxide microparticles comprising platelets, wherein the platelets are prisms with hexagonal type base, have an edge length less between 30 nm and 200 nm, are stacked in direct contact with each other along the stacking c-axis by means of their [0001] or [0001] crystalline planes, and are rotated by a non-zero angle with respect to their adjacent platelets along said stacking c-axis, wherein said zinc oxide microparticles are characterised by a specific surface area which is less than or equal to 4 m2/g. The invention also relates to a method for preparing the zinc oxide microparticles of the invention. The invention further relates to a cosmetic product, paint, ink, paper, cardboard, textile, food, agricultural, home care, air conditioning, animal care, personal and work hygiene, contact lens, chromatography material, medical equipment, dermatological, lacquer, coating and/or plastic product containing the zinc oxide microparticles of the invention and to a biocide composition comprising the zinc oxide microparticles as defined above. Finally, the invention further relates to the use of the biocide composition of the invention for the elimination, inhibition of the growth, or inhibition of progeny of microorganisms and to the zinc oxide microparticles of the invention or pharmaceutical composition comprising them for use in the treatment or prevention of an infection.
    公开号:ES2724825A1
    申请号:ES201990046
    申请日:2017-11-29
    公开日:2019-09-16
    发明作者:Lozano José Francisco Fernandez；Lucas Gil Eva De；Marcos Fernando Rubio
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；
    IPC主号:
    专利说明:

    [0001]
    [0002] ZINC OXIDE MICROPARTICLES, METHOD OF PREPARATION AND USE OF THE
    [0003]
    [0004] FIELD OF THE INVENTION
    [0005]
    [0006] The present invention relates to the field of zinc oxide particles and the method of preparation. More particularly, the present invention relates to the field of biocidal zinc oxide microparticles.
    [0007]
    [0008] BACKGROUND
    [0009]
    [0010] Zinc oxide (ZnO) materials find industrial applications in many technological fields. For example, ZnO materials show excellent optoelectronic properties (M. Bitenc et al., Cryst. Growth Des. 2010, 10, 830-837, Z. Hou et al., Nanoscale Res. Lett., 2012, 7, 507 -513) due to its large prohibited broadband energy of 3.37 eV and large exciton binding energy (K. Foe et al., Thin Solid Films, 2013, 534, 76-82, K. He et al. , Cryst Eng Comm, 2014, 16, 3853-3856). Not only have they been widely studied as catalyst supports, such as in the synthesis of methanol or in the decomposition of industrial and experimental processes (Phuruangrat, A. et al., Journal of Nanomaterials, 2014 (2014), but they have also been shown to excel in other applications such as semiconductors in solar cells (P. Li et al., Mater. Chem. Phys.
    [0011] 2007, 106, 63-69; M. Klaumünzer et al., Cryst Eng Comm, 2014, 16, 1502-1513), or for antimicrobial applications, of which antifungal applications have been reported (Patra P. et al., Langmuir, 2012, 28, 16966-16978 ; L. He et al., Microbiol. Res. 2011, 166, 207-215) and antibacterials (Talebian N. et al., J. Photochem. Photobiol. B: Biology, 2013, 120, 66-73; N. Padmavathy and R. Vijayaraghavan, Sci. Tech. Adv. Mater. 2008, 9)
    [0012]
    [0013] In addition, ZnO is a particularly interesting oxide in materials engineering because its self-assembly can be manipulated, allowing the formation of meso-, micros- and nanostructures with precise control at the molecular level that in turn governs its structure, properties and function. When acting on the growth mechanisms of ZnO materials, different structures, shapes and morphologies have been described more or less complex. Examples of ZnO particles in 1D configuration are nanovarillas (Schlur L. et al., Chemical Communications, 2015, 51, 3367-3370), nanoagujas (Park WI et al., Adv Mater, 2002, 14, 1841-1843), nanowires (Yang P. et al., Advanced Functional Materials, 2002, 12, 323-331) and nanocints (Pan ZW et al., Science, 2001, 291, 1947 1949). The most common 2D structures are nano sheets (Pan A. et al., J. Cryst. Growth, 2005, 282, 165-172) and nanogranules (Chiu WS et al., Chem. Eng. J., 2010, 158, 345 - 352). As for 3D structures, these include cauliflower (Lin L. et al., RSC Advances, 2015, 5, 25215-25221), snowflakes (Li C. et al., Nanoscale, 2010, 2, 2557- 2560) and nanoboxes (Gao PX and Wang ZL, JACS., 2003, 125, 11299-11305). The self-assembly of inorganic basic nanocomponents in ordered one-dimensional, two-dimensional and three-dimensional nanostructures is attractive because the tuning of the way in which the basic components are organized provides a method for adjusting the final properties of the resulting material.
    [0014]
    [0015] K. Shingange et al., Materials Research Bulletin, 2016, vol. 85, 52-63, discloses ZnO nanostructures that consist of flower-like structures of a group of nanovarillas composed of small particles that are nucleated from a center.
    [0016]
    [0017] In WO 2010/018075 A1, biocidal ZnO nanoparticles are disclosed. Other documents disclose nanometric ZnO particles such as Liu et al., Journal of Materials Processing Technology, 2007, vol. 189 (1-3), 379-383; Srinkanth CK et al., Journal of Alloys and Compounds, 2009, vol. 486 (1-2), 677-684 or CN 1192991 A. CN 102 079 540 A discloses 3D porous particles of ZnO composed of aggregated nanoparticles. None of these documents refers to the ZnO microparticles of reduced toxicity.
    [0018]
    [0019] When it comes to the antimicrobial applications ZnO materials, most of the prior art relates only to its antibacterial applications (Li, M. et al., Environ. Sci. Technol., 2011, 45, 1977- 1983, Jones, N. et al., FEMS Microbiol. Lett., 2008, 279, 71-76) with little mention of their antifungal properties (Sharma, D., Thin Solid Films, 2010, 519, 1224-1229). In general, researchers look for factors such as particle concentration, size and specific surface area as decisive factors in the antimicrobial activity of ZnO. A clear common trend in the state of the art is that the smaller nanometric ZnO particles show increased antibacterial efficacy. Although little is known about the antifungal activity of ZnO materials, published research reveals a similar trend for ZnO antifungal activity: nanometric ZnO materials possess an increased antifungal activity with respect to micro- or mesoparticles. This effect is usually associated with an increase in the specific surface area of smaller materials compared to bulkier ones. In summary, when it comes to the antimicrobial activity of ZnO, the consensus so far published is that the smaller the better. However, a disadvantage of nanometric ZnO materials is their toxicity: not only are nanometric ZnO particles readily available to penetrate membrane cells or generate reactive oxygen species (ROS), but it is also known that ZnO materials undergo leaching of toxic Zn2 + ions into the environment.
    [0020]
    [0021] Therefore, there is a need for ZnO materials that show improved antimicrobial activity (both antibacterial and antifungal) without the inconvenience of the aforementioned toxicity phenomena.
    [0022]
    [0023] SUMMARY OF THE INVENTION
    [0024]
    [0025] The authors of the present invention have cultivated ZnO microstructures with low specific surface area and surprisingly increased antimicrobial activity with respect to nanometric commercial ZnO particles. The low specific surface area and the micrometric size allow the particles of the invention to show very little toxicity associated with reduced levels of leaching of Zn2 + and limited ROS production and show high antimicrobial activity. In a broad sense, the particles of the invention comprise conical shaped structures joined by their base, which in turn consist of ZnO platelets. This configuration provides to the particles of the invention the advantageous properties of ZnO particles of less toxic micrometer size and of increased antimicrobial activity, typical of ZnO particles of nanometric size.
    [0026]
    [0027] Accordingly, in a first aspect, the present invention relates to zinc oxide microparticles comprising platelets, wherein platelets,
    [0028] - they are prisms with hexagonal base,
    [0029] - have an edge length between 30 and 200 nm, and
    [0030] - they are stacked in direct contact with each other along the stacking axis c, by means of their crystalline planes [000T] or [0001], and
    [0031] - they are rotated by a non-zero angle with respect to their adjacent platelets along said stacking axis c,
    [0032]
    [0033] wherein said zinc oxide microparticles are characterized by a specific surface area that is less than or equal to 4 m2 / g.
    [0034] A second aspect of the present invention relates to a method for preparing the zinc oxide microparticles as defined above, which comprises the steps of: i) adding urea to an aqueous solution of zinc salt; ii) heating the solution resulting from step (i); iii) isolate the product resulting from step (ii); and iv) subject the product isolated from step (iii) to an annealing treatment.
    [0035]
    [0036] The inventors have surprisingly discovered that the microparticles of the present invention show intense antimicrobial activity and at the same time are less toxic than commercial compositions of zinc oxides of nano- and micrometric size.
    [0037]
    [0038] Therefore, a third aspect of the present invention relates to a cosmetic product, paint, ink, paper, cardboard, textiles, food, agriculture, home care, air conditioning, animal care, personal and work hygiene, contact lenses, chromatography material, medical equipment, dermatological product, lacquer, coating and / or plastic product containing the zinc oxide microparticles as defined above.
    [0039]
    [0040] In yet another aspect, the present invention is directed to a biocidal composition comprising the zinc oxide microparticles as defined above and optionally a vehicle.
    [0041]
    [0042] Finally, another aspect of the present invention is directed to the use of the biocidal composition as defined above for the elimination, growth inhibition or inhibition of the progeny of microorganisms.
    [0043]
    [0044] DESCRIPTION OF THE FIGURES
    [0045]
    [0046] These and other features and advantages of the invention will be clearly understood in view of the detailed description of the invention that is apparent from preferred embodiments, given only as an example and not limited thereto, with reference to the drawings.
    [0047]
    [0048] Figure 1: Representative growth mechanism of the microparticles of the present invention. Panel a shows a single plate of ZnO (1) and panel b shows the microstructure of the microparticles of the invention comprising at least three platelets of ZnO (3) stacked along the axis c and has a conical shaped morphology. He Thickness of the short dimension of a single ZnO plate is characterized by its edge length (2). Panel c shows a representation of a flower-like microparticle (4) comprising groups of conical shaped platelets (3) joined by its base.
    [0049]
    [0050] Figure 2: Micrographs by scanning electron microscopy of low resolution (af panels) and high resolution (gl panels) for the morphological characterization of the microparticles of examples 1 and 2 obtained before and after the thermal annealing treatment for samples: A ( no T, panels a, gym); B (350 ° C, panels b, h and n); C (400 ° C, panels c, I and o); D (500 ° C, panels d, j and p); E (600 ° C, panels e, k and q) and shows F (700 ° C, panels f, l and r). Panels m to r show the groups of platelets stacked conically.
    [0051]
    [0052] Figure 3: Crystallite size (panel a) and statistical study of the microstructural parameters of the microparticles of Figure 2: platelet diameter (panel b), platelet thickness defined as edge length (panel c) and rotation angle between platelets separated by a domain wall (panel d).
    [0053]
    [0054] Figure 4: Scanning electron microscopy micrograph of a flower-like microparticle of the invention (panel a) heat treated at 500 ° C (sample D) in which different groups of conical platelets are signaled (5) (6) (7) and (8) with respect to the x and y axes of reference. Panel b shows the Raman spectra of each conical platelet group that reveals the orientation of the crystal related to the intensity of the ZnO E2high Raman mode. The 532 nm laser used in the Raman experiment was a polarized laser along the x-axis. Panel c shows a transmission electron micrograph of a thermally treated microparticle at 500 ° C (sample D) showing the crystallographic direction parallel to the side of the platelet edge length and therefore along the stacking direction of the platelets of ZnO.
    [0055]
    [0056] Figure 5: Specific surface values (SSA) of two commercial samples, nanoZnO (square without filling) and microZnO (triangle without filling), as well as the particles of the invention (filled circles) as a function of annealing temperature.
    [0057]
    [0058] Figure 6: Zn2 + leaching test in peptone water of example 5 (panel a). The data were obtained by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) that produced the concentration of Zn2 + in peptone water for each of the samples tested: commercial microZnO and nanoZnO samples and samples from A to F (Examples 1 and 2). Panel b shows a graphic representation in which the ordinate axis (corresponding to the concentration of Zn2 +) is limited to a value of 1 mg / l.
    [0059]
    [0060] Figure 7: Photocatalytic degradation of methyl orange (MO) exposed to UV light irradiation in the presence of commercial samples microZnO (triangle without filling) and nanoZnO (square without filling) as well as exemplary particles of the present invention (sample D, circle filling). The graphic area is divided into two regions according to the photocatalytic degradation capacity. The gray zone (9) corresponds to a region in which the ZnO particles produce a high photocatalytic degradation and the white zone (10) indicates that the ZnO particles produce a low photocatalytic degradation.
    [0061]
    [0062] Figure 8: Antimicrobial activity of the particles of the present invention represented by samples A to F, compared to commercial references (microZnO and nanoZnO). Panels a and b represent the antibacterial activity (R) against E. coli and S. aureus. Panel c shows the antifungal activity of said samples against A. niger.
    [0063]
    [0064] Figure 9: Panel a shows a unit of three platelets (3) of the microparticles of the present invention in which each platelet is stacked along the direction [0001]. Adjacent platelets form a domain wall interface (11) in which adjacent platelets are rotated along the direction [0001]. The accumulation of charge generated on the domain walls forms a potential barrier called the Schottky barrier (12). Panel b shows the zeta potential and conductivity values of the ZnO microparticles of the present invention (sample D) measured in an aqueous suspension at different pH values.
    [0065]
    [0066] Figure 10: Micrographs by scanning electron microscopy of ZnO microparticles of the present invention (sample D) attacking bacteria E. coli (a), S. aureus (b) and fungus A. niger (c) by electrostatic action. Panel d shows the cathode luminescence of sample D against A. niger. The dotted line in Figure 10 (a) delimits the E. coli bacteria and in Figure 10 (b) delineates the S. aureus bacteria.
    [0067]
    [0068] Figure 11: Survival of HeLa cells with 0.1 mg / ml, 0.2 mg / ml and 0.5 mg / ml of nanoZnO and microparticles of the present invention heat treated at 500 ° C (marked 500 ° C in the x axis of the figure). Cell survival was assessed by the MTT assay after 24 h of contact with the particles.
    [0069] DETAILED DESCRIPTION OF THE INVENTION
    [0070]
    [0071] Unless otherwise defined, all technical and scientific terms and expressions used herein have the same meaning commonly understood by an expert in the field to which this description belongs.
    [0072]
    [0073] A first aspect of the invention is directed to zinc oxide microparticles comprising platelets, wherein platelets,
    [0074] - they are prisms with hexagonal base,
    [0075] - have an edge length between 30 and 200 nm, and
    [0076] - they are stacked in direct contact with each other along the stacking axis c, by means of their crystalline planes [000T] or [0001], and
    [0077] - they are rotated by a non-zero angle with respect to their adjacent platelets along said stacking axis c;
    [0078]
    [0079] wherein said zinc oxide microparticles are characterized by a specific surface area that is less than or equal to 4 m2 / g.
    [0080]
    [0081] The specific surface area (SSA) of a solid should be understood as the total surface area of said solid per unit mass, and is expressed in m2 / g. Under this definition, it is clear to the expert that a flat surface has a lower SSA value than an accordion type surface. In this regard, SSA values for solids can be easily measured by Brunauer-Emmett-Teller (BET) isothermal adsorption curves.
    [0082]
    [0083] It is considered a nanomaterial when 50% or more of the particles in their numerical size distribution have one or more external dimensions in the size range between 1 nm and 100 nm. The minimum value of the specific surface area (SSA) for different particle morphologies (sphere, fiber and plate) having a dimension of 100 nm for reference purposes was calculated. To calculate the SSA values it is necessary to determine the particle volume and area for each morphology, as well as the number of particles per gram. To express the data as a mass function, instead of using the number of particles, the density value of 5.61 g / cm3 of ZnO material was used. The results obtained for the sphere, fiber and plate morphologies were 10.7 m2 / g, 7.5 m2 / g and 4.3 m2 / g, respectively. The minimum SSA calculations allow the particles to be categorized into two groups: nanoscale group (SSA more than 10.7 m2 / g) and microscale group (SSA less than 4.3 m2 / g). Potential ranges of particle toxicity are defined according with the minimum SSA as unsafe (more than 10.7 m2 / g), safety limit (between 10.7 and 4.3 m2 / g) and safe (less than 4.3 m2 / g).
    [0084]
    [0085] The widely used term "microparticle" should be interpreted in the context of the present invention as any particle having at least one micrometric dimension, that is, less than 100 ^ m but greater than 100 nm.
    [0086]
    [0087] The term "stack" should be interpreted as an ordered set or stack of, in the present context, platelets.
    [0088]
    [0089] In the context of the present invention, the term "platelet" should be associated with any ZnO particle characterized by being plate-shaped (1 in Figure 1) or a hexagonal-based prism.
    [0090]
    [0091] The terms "edge length" (2 in Figure 1) when used to refer to platelet thickness refers to the short edge between the two closest vertices of each polygonal base of the crystalline particle. The terms "lateral length" refer to the large edge of the two closest vertices of the base of the polygon. The terms "diameter" or "equivalent diameter" of the platelet refer to the distance between the two vertices furthest from the polygonal base. To exemplify the latter, if the polygon in question is a hexagonal prism, then the edge length would be the height of that prism, that is, the length of one of the six edges formed between two closest vertices of each hexagonal base that form the hexagonal prism. The term "vertex" refers to the union of two adjacent edges. The term rotation angle between two adjacent platelets refers to the angle formed by the line that goes from the center of a first platelet to a vertex of the first platelet and the line that goes from the center of a second platelet (adjacent to the first platelet) to a vertex of the second platelet (and equivalent to the vertex considered for the first platelet). The angle of rotation occurs along the c axis of the ZnO platelets.
    [0092]
    [0093] In a particular embodiment of the invention, the platelets are monocrystals. In another particular embodiment of the invention, each platelet creates a single domain.
    [0094]
    [0095] In the context of the present invention, the term "domain" refers to a region within a ZnO crystal in which the crystal is homogeneous in a crystallographic direction.
    [0096] In addition, zinc oxide microparticles, as defined above, comprise platelets having an edge length between 30 and 200 nm.
    [0097]
    [0098] In a particular embodiment of the invention, platelets are characterized by an edge length between 40 and 180 nm.
    [0099]
    [0100] In another particular embodiment of the invention, platelets are characterized by a diameter between 100 and 500 nm.
    [0101]
    [0102] The terms "crystallite size" should be interpreted as the length of coherence of the crystalline structure and is determined by X-ray diffraction.
    [0103]
    [0104] In a particular embodiment of the invention, the ZnO microparticles are characterized by a crystallite size between 10 nm and 100 nm, preferably between 40 and 70 nm.
    [0105]
    [0106] The zinc oxide microparticles of the invention comprise platelets stacked along the hexagonal c axis.
    [0107]
    [0108] In a particular embodiment of the invention, stacked platelets form domain walls or Schottky barriers.
    [0109]
    [0110] The inventors have surprisingly discovered that the microparticles of the invention are formed by platelets that are stacked in direct contact with each other along the stacking axis c by means of their crystalline planes [0001] or [0001], that is, their faces polar, and are rotated by a non-zero angle with respect to their adjacent platelets along said stacking axis c. The angle of rotation formed between two vertices of adjacent platelets joined by domain walls is the result of the conical stacked structure in which the adjacent platelets have different sizes and therefore the six vertices are not aligned as the diameter of platelets decreases along the growth axis, the c axis of the crystalline structure of ZnO.
    [0111]
    [0112] In a particular embodiment of the invention, the non-zero angle is an angle between 1 ° and 40 °.
    [0113]
    [0114] Without pretending to be limited by any particular theory, it is believed to be direct contact between adjacent platelets at an angle other than 0 ° along the c axis of stacking which results in the generation of domain walls in each interplate layer, accumulating electrical charge and generating an electrical potential distributed throughout the structure. A probable explanation follows of the mounting mechanism and the electrical phenomena behind the particles of the invention.
    [0115]
    [0116] Under ambient conditions, zinc oxide (ZnO) crystallizes mainly in the most stable form of hexagonal wurtzite , a polar crystal that is characterized by a positively charged surface of Zn-polar ([0001] crystalline plane) and an O-polar surface negatively charged ([0001] crystalline plane) in addition to the six non-polar faces. The crystalline plane [0001] or [0001] is the crystallographic direction of the c axis of the ZnO structure. In terms of the crystalline molecular structure of ZnO, polar surfaces are associated with the atoms Zn and O placed in terminal positions. Schematically, it is believed that these positively and negatively charged surfaces play an important role in the mechanism behind the formation of stacked platelets along the c axis: it is the electrostatic attraction between said polar surfaces that brings them together and allows stacking of ZnO nanoplates (Wang S. and Xu A., CrystEngComm, 2013, 15, 376).
    [0117]
    [0118] Once the ZnO microparticles comprising stacked platelets are formed, a heat treatment or thermal annealing compresses said platelets against their neighboring adjacent platelets resulting in platelets that are in direct contact with the adjacent platelets. As a consequence of heat treatment, adjacent platelets form a domain wall at the interface. In addition, the heat treatment improves the crystallinity of the structure that results in a crystallite size between 10 nm and 100 nm, preferably between 40 and 70 nm. Large crystallite sizes of stacked platelets result in greater immobilization of zinc cations, that is, heat treatment reduces the release of Zn2 + as demonstrated in leaching experiments.
    [0119]
    [0120] In addition, the stacking of ZnO platelets along the c axis occurs with a non-zero rotation angle, that is, a rotation angle that is> 0 °, relative to its adjacent platelets, which means that each Platelet has a degree of rotation along the c axis with respect to its two adjacent platelets. It is believed that, under these conditions, each platelet is a unique domain, and it is the boundaries formed by each platelet that gives rise to finite interfacial phenomena and the appearance of walls of domain (11) (Figure 9). As can be seen in Figure 9 (a), the domain walls lead to a generation of Schottky type barriers, where a negative charge to the depletion of the ZnO conduction band accumulates. Therefore, these Schottky barriers involve the generation of an electrical potential. Schottky barriers can be asymmetric due to the relative rotation, size, among others, of each platelet. In addition, the presence of multiple stacked platelets results in the appearance of multidomains that could be considered as a particle or structure comprising more than, or at least, two domains.
    [0121]
    [0122] In the context of the present invention, the terms "domain wall" refer to the crystallographic domains that separate the interfaces, where, as defined above, each domain is a region within a ZnO crystal in which the crystal It is homogeneous in a crystallographic direction. With respect to the axis of preferential growth, the platelets are oriented along the axis c giving rise to a structure of platelets stacked conically. The domain wall is produced in the plane perpendicular to the main axis of the ZnO crystal system, the c axis and the symmetry of the crystal is broken by the rotation of adjacent platelets along the c axis.
    [0123]
    [0124] The zinc oxide microparticles of the present invention are characterized by a specific surface that is less than or equal to 4 m2 / g.
    [0125]
    [0126] In a preferred embodiment of the invention, the zinc oxide microparticles defined above are characterized by a specific surface area that is less than or equal to 2 m2 / g, more preferably less than or equal to 1.5 m2 / g.
    [0127]
    [0128] It is believed in the context of the present invention that one of the reasons behind the toxicity of ZnO nanoparticles refers to the fact that the particle size is of nanometric dimensions. Similarly, it is also believed in the context of the present invention that the toxicity of ZnO nanoparticles is also related to their specific surface area (SSA). Taking SSA as a criterion for assessing the toxicity of ZnO, it was determined that the particles of the invention are much safer than commercial samples of nanoparticles of spherical ZnO (nanoZnO, Evonik) and micrometric plates (microZnO, Asturiana Zinc).
    [0129]
    [0130] The zinc oxide microparticles of the invention can be microparticles where the stacked platelets form a conical structure and in which at least two of said conical structures are joined by the base of each cone. This structure can be schematically represented by panel c of Figure 1.
    [0131]
    [0132] In a particular embodiment, each conical structure of the microparticles of the invention is formed by at least three platelets. For example, a conical structure having a length of 500 nm could be formed by about 10 stacked platelets having an edge length of about 50 nm.
    [0133]
    [0134] In a particular embodiment, the conical structures of the invention are characterized by a diameter between 0.1 and 0.5 ^ m, more preferably between 0.1 and 0.3 ^ m.
    [0135]
    [0136] In a preferred embodiment of the invention, the zinc oxide microparticles of the invention are characterized as flower-shaped microparticle structures. The SEM images of Figure 2 show non-limiting representatives of these flower-like microparticles of the invention. In this way, the terms "flower type" refer to a shape that resembles a flower, or similar figures such as a bouquet, a tree, a star, a cactus, among others.
    [0137]
    [0138] In a particular embodiment, the flower-like microparticles of the invention are characterized by a diameter between 0.1 and 20 m, more preferably between 1 and 10 m.
    [0139]
    [0140] Another aspect of the invention relates to a method for preparing the zinc oxide microparticles as defined above, which comprises the steps of: i) adding urea to an aqueous solution of zinc salt, ii) heating the resulting solution of step (i), iii) isolate the product resulting from step (ii), and iv) subject the product isolated from step (iii) to an annealing treatment.
    [0141]
    [0142] In another preferred embodiment, the method for preparing the zinc oxide microparticles as defined above is a method in which step (i) is at a concentration between 1 and 10 M, more preferably between 4 and 6 M, even more preferably 5 M. In yet another preferred embodiment, the zinc salt solution of step (i) is at a concentration between 3 and 10 M, more preferably between 4 and 10 M, even more preferably between 3 and 7 M. In yet another preferred embodiment, the method for preparing the zinc oxide microparticles as defined above is a method in which the solution in step (ii) is heated to a temperature between 80 ° C and 140 ° C for a period of time. period of time between 1 and 3 hours.
    [0143] In a more preferred embodiment of the invention, the solution in step (ii) is heated to a temperature between 100 ° C and 120 ° C.
    [0144]
    [0145] In yet another particular embodiment of the invention, the solution in step (ii) is heated for 2 hours.
    [0146]
    [0147] The term "isolation" refers to any process capable of producing the main reaction product of step (ii) deprived of most of the liquid material. For example, product isolation may refer to the process of extraction, centrifugation, filtration, evaporation, crystallization and other processes known in the art.
    [0148]
    [0149] In a preferred embodiment of the invention, the method for preparing the zinc oxide microparticles as defined above is a method in which step (iii) comprises cooling the solution resulting from step (ii) to room temperature and adding a hot aqueous solvent to obtain a precipitated product; and isolate the precipitated product. Once the precipitated product is isolated, it can be optionally washed with a solvent selected from polar or non-polar solvents such as the group of water, ethanol, methanol, isopropanol, acetone, methyl acetate, ethyl acetate, dimethylformamide, acetonitrile, pentane, hexane, toluene, turpentine, tectraclorethylene.
    [0150]
    [0151] The terms "hot aqueous solvent" in the context of the present invention should be quickly identified by the expert as an aqueous solvent that is not at or below room temperature (20-25 ° C). It is an aqueous solvent that is subjected to heat but is still in the liquid phase and therefore is an aqueous solvent at a temperature higher than room temperature but below its boiling point.
    [0152]
    [0153] In a preferred embodiment, the hot aqueous solvent is at a temperature from 30 to 120 ° C, from 30 to 100 ° C, from 40 to 100 ° C, from 50 to 100 ° C, from 60 to 100 ° C and from 70 up to 100 ° C. Preferably, the hot aqueous solvent is at a temperature from 30 to 80 ° C.
    [0154]
    [0155] In a particular embodiment, the hot aqueous solvent of step (iii) is water.
    [0156] In another preferred embodiment, the method for preparing the zinc oxide microparticles of the invention is a method in which the annealing treatment (step iv) is performed at a temperature between 350 ° C and 700 ° C.
    [0157] In yet another preferred embodiment, said annealing treatment of step (iv) is performed for a period of time between 1 and 24 hours.
    [0158]
    [0159] The term "annealing" is widely used in metallurgy and materials science, and refers to a heat treatment that alters the physical and sometimes chemical properties of a material to increase its ductility and reduce its hardness, making it more feasible. It involves heating a material above its recrystallization temperature, maintaining a suitable temperature and then cooling.
    [0160]
    [0161] It has been found that the particles of the invention exert a biocidal effect when in contact with microorganisms. Therefore, another aspect of the invention relates to a biocidal composition comprising the zinc oxide microparticles as defined above and optionally a vehicle.
    [0162]
    [0163] Although any suitable vehicle known to those skilled in the art can be employed in the biocidal compositions of this invention, the type of vehicle will vary depending on the type of application. The biocidal compositions of the present invention may be formulated comprising a liquid carrier in which examples of liquid carriers include, but are not limited to, a liquid, such as water, saline, alcohol, a fat, a wax or a buffer, or A solid carrier Examples of solid carriers include, but are not limited to, a solid such as a polymer, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate which can both Liquid vehicles such as solids, carrying a dispersion, suspension or solution. Other suitable vehicles include emulsions, pastes, ointments, gels, creams, lotions, powders, oils, pencils, deodorant-creams, gels, lotions, emulsions, deodorant sticks, roll-ons, sprayers, pump sprayers or lacquers.
    [0164]
    [0165] The inventors have discovered that the microparticles of the invention have excellent antimicrobial properties, being suitable as biocidal agents for any type of microscopic unicellular or multicellular living organisms such as bacteria, archaea, protozoa, fungi and algae. In addition, the microparticles of the invention act on living microorganisms by a mechanism that, surprisingly, is not based on the leaching of Zn2 + or the generation of reactive oxygen species (ROS). This is evident in Figures 6 and 7, as well as in Examples 5 and 6, and is a paradigm shift finding that goes against the teachings of the prior art.
    [0166] The term "biocide", used in the current context when referring to the microparticles of the invention or a composition and / or material comprising said microparticles, should be interpreted as the quality inherent to the microparticles of the invention to destroy, deter, harmless, inhibit growth, exert a control effect or induce apoptosis in any microorganism. In the context of the present invention, the term "dose" refers to the required concentration of ZnO microparticles of the present invention to produce said biocidal effect. The term "biocide" should also be interpreted as having a similar meaning as a disinfectant, pesticide or preservative. In accordance with the present description, it is clear to the expert that the biocidal ZnO microparticles as defined above can be incorporated into any suitable material to be brought into close contact with any surface that needs to be disinfected.
    [0167]
    [0168] Therefore, in another aspect of the invention, the invention is directed to a cosmetic product, paint, ink, paper, cardboard, textiles, food, agricultural product, home care, air conditioning, animal care, personal and work hygiene , contact lenses, chromatography material, medical equipment, dermatological product, lacquer, coating and / or plastic product containing the zinc oxide microparticles of the invention.
    [0169]
    [0170] In a particular embodiment, the invention is directed to a product selected from paints, inks, agricultural product, home care products, air conditioning, animal care, products for occupational hygiene, chromatography materials, medical equipment, lacquers, coatings and / or plastics containing the zinc oxide microparticles of the invention.
    [0171]
    [0172] In another particular embodiment, the invention is directed to a cosmetic product, food, agricultural product, animal care, personal hygiene products, contact lenses, medical equipment and / or dermatological product containing the zinc oxide microparticles of the invention.
    [0173]
    [0174] In yet another particular embodiment of the present invention, the microparticles as defined above can be used in the manufacture of materials that are in close contact with human, animal or plant organisms. This application is possible due to the low dose of particles required by the present invention in addition to the advantageous properties of the particles of the invention: low species generation Oxygen reagents (ROS) and low release of Zn2 + cations. Therefore, the present invention also relates to compositions comprising the zinc oxide microparticles of the invention.
    [0175]
    [0176] In another aspect, the invention is directed to a biocidal composition comprising the zinc oxide microparticles of the invention and optionally a vehicle.
    [0177]
    [0178] In still another aspect, the invention is directed to the use of a biocidal composition comprising the zinc oxide microparticles of the invention and optionally a vehicle, for the elimination, growth inhibition or inhibition of the progeny of microorganisms. In a particular embodiment, said use excludes any diagnostic treatment or methods in the human or animal body.
    [0179]
    [0180] In a preferred embodiment of the invention, the invention is directed to the use of the biocidal composition of the invention in which microorganisms are selected from bacteria and fungi.
    [0181]
    [0182] In a particular embodiment, the biocidal composition of the invention is:
    [0183] - a pharmaceutical composition, or
    [0184] - a personal care composition, or
    [0185] - a home care composition, or
    [0186] - a material protection composition, or
    [0187] - a composition used for industrial disinfection, or of institutions or hospitals, or
    [0188] - a plant protection composition, preferably a fungicidal composition.
    [0189]
    [0190] In one embodiment, the invention is directed to a pharmaceutical composition comprising zinc oxide microparticles of the invention and optionally a pharmaceutically acceptable carrier.
    [0191]
    [0192] In a particular embodiment, the invention is directed to the zinc oxide microparticles of the invention or the pharmaceutical composition of the invention for use in the treatment or prevention of an infection. Preferably, for use in the treatment or prevention of an infection in an animal, more preferably a human being.
    [0193]
    [0194] In another particular embodiment of the invention, the infection to be treated or prevented is selected from infections of the skin, mucosa or mucous membranes, surfaces. Dental, nails, hair or lesions on the skin and / or mucous membranes.
    [0195]
    [0196] In a preferred embodiment, the invention is directed to the zinc oxide microparticles of the invention or the pharmaceutical composition of the invention for use in the treatment or prevention of a skin infection, mucous membranes, nails, skin lesions. and / or mucous membranes.
    [0197]
    [0198] In a particular embodiment, the invention is directed to the use of the zinc oxide microparticles of the invention or the pharmaceutical composition of the invention, in the treatment or prevention of an infection. Preferably, in the treatment or prevention of an infection in an animal, more preferably a human being.
    [0199]
    [0200] In a particular embodiment, the invention is directed to the zinc oxide microparticles of the invention or the pharmaceutical composition of the invention, in the manufacture of a medicament for the treatment or prevention of an infection. Preferably, in the treatment of an infection in an animal, more preferably a human being.
    [0201]
    [0202] In another embodiment, the invention is directed to a method for treating or preventing an infection, the method comprising administering the zinc oxide microparticles of the invention or the pharmaceutical composition of the invention. Preferably, the infection is in an animal, more preferably in a human being.
    [0203]
    [0204] The terms "pharmaceutically acceptable carrier" as used in the present invention include any solvent, dispersion medium, coating, isotonic agents and absorption retardants and the like that are compatible with the activity of the microparticles and are physiologically acceptable to the subject.
    [0205]
    [0206] In the context of the present invention, the term "bacteria" (plural "bacteria") refers to both gram-negative and gram-positive prokaryotic microorganisms, typically a few micrometers in length and having a large number of shapes, which They range from spheres to rods and spirals. Non-limiting examples of bacteria suitable for the use of the biocidal composition of the present invention are Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Aspergillus niger (A. niger).
    [0207]
    [0208] In the context of the present invention, the term "fungus" (plural "fungi") refers to eukaryotic microorganisms that include unicellular microorganisms such as yeasts and molds, as well as multicellular fungi that produce familiar fruiting forms known as fungi.
    [0209]
    [0210] Non-limiting examples of fungi suitable for the use of the biocidal composition of the present invention are brown rot (Poria Sp.), White rot (Polyporous Sp.), Wood decay fungus (Poria placenta) and mold.
    [0211]
    [0212] EXAMPLES
    [0213]
    [0214] The present invention will now be described by way of examples that serve to illustrate the construction and testing of illustrative embodiments. However, it is understood that the present invention is not limited in any way to the following examples.
    [0215]
    [0216] Example 1 Synthesis of an exemplary microparticle of the invention
    [0217]
    [0218] All chemicals were used directly without further purification. A solution of zinc nitrate hexahydrate (Zn (NO3) 2-6H2O) 5M was prepared by diluting 297.2 g Zn (NO3) 2-6H2O (0.888 mol) in 166 ml of water. To begin the procedure, 218 g of urea (3.6 mol, CO (NH2) 2) was added under stirring at room temperature to the zinc nitrate solution hexahydrate (Zn (NO3) 2-6H2O) 5M.
    [0219]
    [0220] The reaction was heated in an oil bath at 100-120 ° C and stirred at 300 rpm for 2 h. After cooling naturally to room temperature, hot water was added to obtain a white suspension. Subsequently, the precipitate was isolated by filtration and washed with water and ethanol to remove impurities. The white powder product was dried at 80 ° C for 24 h to provide an exemplary sample A (Figure 2, panels a, g and m).
    [0221]
    [0222] Example 2 Heat annealing treatment of an exemplary microparticle of the invention.
    [0223]
    [0224] The dry product obtained in example 1 (sample A) was further subjected to an annealing treatment at temperatures between 350 ° C and 700 ° C, giving rise to samples B (350 ° C), C (400 ° C), D (500 ° C), E (600 ° C) and F (700 ° C). The annealing treatment consisted of placing a sample of ZnO obtained from a method such as that of example 1, in an electric oven (Nabertherm) for 1 to 24 hours under an air atmosphere. Figure 2 shows SEM images of the exemplary microparticles of samples B to F.
    [0225] Regarding the characterization of the present invention, a statistical study of the main morphological parameters has been carried out (Figure 3). Figure 3 (a) shows that heat treatment plays an important role in the crystallite size of the present invention. As can be seen in Figure 3 (b), the platelet diameter is between 100 and 500 nm, where the average value is 231 nm. Figure 3 (c) shows the histogram of edge length between 30 and 180 nm and whose average is 76 nm. The characteristic non-zero angle histogram of this invention is depicted in Figure 3 (d). The average angle is 13 °, but it can vary between> 0 ° and 35 °.
    [0226]
    [0227] Example 3. Raman characterization of the microparticles of the invention
    [0228]
    [0229] Panel a of Figure 4 shows the scanning electron micrograph of a single exemplary microparticle of the invention (sample D), comprising groups of different conical shape arranged in different spatial directions. For simplicity, an x-axis of reference was drawn on the micrograph. Each conical structure comprised a collector of stacked ZnO platelets. This exemplary microparticle is characterized by a flower-like shape. The same microparticle was observed under the optical microscope of the Raman Confocal microscope and the x-axis reference was aligned accordingly. A polarized laser at 534 nm was used along the x-axis for Raman spectroscopy. As can be seen in panel b (Figure 4), the intensity of the E2high signal varies with the orientation of each stacked conical shaped group that forms the flower-shaped microparticle. In the case of ZnO, the E2high Raman mode has a higher Raman intensity when the polarized light is aligned perpendicular to the crystallographic plane [0001]. The Raman spectra represented in panel b of Figure 4 are identified with the corresponding conical platelet groups shown in panel a of Figure 4. The most intense Raman peak (5) corresponds to the stacking of vertically oriented platelets. However, the conical shaped groups oriented in the xy plane (6, 7, 8) show less intense E2high Raman peaks according to the projection on the axis and which is also perpendicular to the direction of the polarized light. The previous results support that platelets are stacked along the crystallographic direction [0001]. Transmission electron microscopy (TEM) confirms this result (panel c of Figure 4). The TEM micrograph shows the alignment of the crystalline structure [0001] parallel to the edge length of a ZnO platelet. In this case, the crystalline plane observed in the TEM micrograph corresponds to the surface [1010], and the direction of preferential growth of the group of stacked platelets is [0001], that is, the c axis.
    [0230] Example 4. Determination of the specific surface for the particles of the invention.
    [0231]
    [0232] The SSA of the exemplary ZnO flower-shaped microparticles is determined by the Brunauer-Emmett-Teller (BET) method. For comparison purposes, two commercial products of different sizes are used in this study: microZnO (Zinc Asturiana) and nanoZnO (Evonik). The data is represented in Figure 5. The SSA value corresponding to the nanoZnO reference is in the unsafe range (greater than 10.7 m2 / g), corresponding to the nanoscale behavior. The SSA value for the microZnO commercial reference is within the safety limit (between 10.7 and 4.3 m2 / g), close to the microscale behavior. With respect to flower-like ZnO structures such as samples from A to F (see Examples 1 and 2), the experimentally determined SSA values are lower than commercial microZnOs. These samples are within the microscale behavior (less than 4.3 m2 / g), and therefore their potential toxicity is established in the safe range. In addition, the evolution of SSA values with annealing treatment (arrow in Figure 5) reveals that for higher temperature treatments, the specific surface area decreases (see the following table). Therefore, it is suggested that by increasing the annealing temperature, the stacked platelets of the particle of the invention can be increasingly compacted together, blocking some pores that are possibly created during synthesis. Therefore, the annealing temperature plays an important role in the process of compacting stacked platelets, which allows adjusting the SSA values in the particles of the invention, reaching values below 1 m2 / g (sample F).
    [0233]
    [0234]
    [0235]
    [0236]
    [0237] Example 5. Leaching of cationic zinc for ZnO samples in peptone water.
    [0238]
    [0239] The inventors have surprisingly discovered that the particles of the invention do not leach cationic zinc in the environment when compared to commercial samples of nanoZnO and that the amount of Zn2 + ions leached by the particles of the invention falls well below the values of commercial microZnO samples. In this regard, a Zn2 + leaching test was performed for samples A to F, as well as for comparative commercial samples microZnO and nanoZnO in peptone water. The test consisted of preparing a suspension of 10 g / L of ZnO (commercial samples and samples of the invention) in 25 mL of peptone water (PW) for 24 hours at 37 ° C and collecting the colorless solution obtained. Figure 6 shows the results of the leaching test representing the concentration of Zn2 + measured by ICP-AES in peptone water for each of the samples. The Zn2 + release concentration for the flower-like ZnO particles of the invention is compared with commercial references (microZnO and nanoZnO). Figure 5a shows that the thermally untreated particle (sample A) and the nanoZnO sample are responsible for a greater leaching of Zn2 + in solution, while the commercial microZnO sample and the flower-like ZnO samples from B to F they are well below 1 mg / l. In addition, Figure 6b shows in detail the relationship between the amount of Zn2 + leachate and annealing treatments. The leached Zn2 + decreases with increasing temperature, possibly due to the stabilization and compaction of the stacked platelet structure of ZnO. At 700 ° C (sample F) the measured released Zn2 + is equal to values as low as 0.02 mg / l. Therefore, the use of heat treatment allows to control the release of Zn2 +.
    [0240]
    [0241] Example 6 Production of reactive oxygen species.
    [0242]
    [0243] The inventors have surprisingly discovered that the microparticles of the invention are not involved in a significant production of reactive oxygen species (ROS) when subjected to light, contrary to commercial samples of nanoZnO and microZnO. In this regard, the photocatalytic performance of the particles of the invention (sample D in example 2) as well as comparative commercial samples were studied by studying the photodegradation of the organic methyl orange (MO) dye under UV light. The results of the photodegradation of MO in the presence of an exemplary microparticle of the invention, commercial samples of nanoZnO and commercial samples of microZnO as a function of time are shown graphically in Figure 7. As can be seen, the commercial sample microZnO (triangle without filling ) causes the complete degradation of MO at 2 h and the commercial sample of nanoZnO (square without filling) photodegrates the MO in 3h. Surprisingly, the flower-like ZnO microparticles (sample D, filled circle) are only capable of inducing the photodegradation of 30% of the MO dye, and this after 5 h of radiation. This result clearly indicates two zones in Figure 7, one where The generation of Reactive Oxygen Species predominates, ROS (9) (Figure 7) and another where ROS (10) are not produced (Figure 7). Therefore, the generation of ROS in commercial microZnO and nanoZnO, found in the gray region (9) (Figure 7), is much larger than the flower-like ZnO microparticles (sample D). In addition, this behavior of lower ROS production extends to other samples such as B or F as can be seen in the following table.
    [0244]
    [0245]
    [0246]
    [0247]
    [0248] Example 7. Biocidal Activity
    [0249]
    [0250] The extensive biocidal activity of the ZnO microparticles of the invention, exemplified by samples from A to F (examples 1 and 2) against three microorganisms, two bacteria, Escherichia coli (CECT 516) and Staphylococcus aureus (CECT 240) was studied ) and a fungus, A. niger (CECT 2807). With respect to the two bacteria, E. coli is a rod-shaped Gram-negative bacterium, while S. aureus is a round-shaped Gram-positive bacterium. The study of antibacterial activity was carried out using the macrodilution method according to the National Committee for Clinical Laboratory Standards (NCCLS) with slight modifications. In Figure 8 (panels a and b) show the values of antibacterial activity (designated as R) calculated from colony forming units (CFU) of bacteria after adding 3 ppm of the ZnO sample under study (commercial samples and AF samples ) and subsequent incubation for 24 h at 37 ° C. As can be seen from panels a and b of Figure 8, commercial microZnO and nanoZnO induce a decrease in the bacterial population, reaching R values of approximately 2 for E. coli and approximately 1.5-2.3 for S. aureus On the contrary, the presence of the exemplary particles of the AF samples shows a dramatic decrease in the bacterial population that reaches R values of approximately 3-3.7 for E. coli and approximately 3-3.3 for S. aureus. These values mean that the particles of the present invention are capable of enhancing the antibacterial activity of traditional ZnO by approximately 70% for Gram-negative bacteria and 50% for Gram-positive bacteria. Returning to fungal growth, the study was conducted using the Kirby-Bauer method. The A. niger fungus (CECT 2807) is grown in petri dishes containing the particles of the present invention, exemplified by samples from A to F, heat treated and incubated at 37 ° C for 3 days. For comparison purposes, commercial references of different sizes were used as before. Figure 7 (panel c) shows the reduction of fungal growth in the presence of samples A to F, as well as microZnO and nanoZnO references. The results showed that the particles of the present invention increase the antifungal activity by approximately 42% with respect to microZnO and 26% with respect to nanoZnO. These results showed again that the particles of the present invention significantly inhibit the growth of A. niger and were more effective than commercial ZnO products. In all cases, the tests were carried out in the dark, that is, without the presence of UV light, which means that no photoexcited phenomena occur and, therefore, the biocidal activity does not depend on the formation of ROS.
    [0251]
    [0252] An advantage of the microparticles of the present invention is that the antibacterial and antifungal activity is achieved at a very low dose, only 3 ppm or 3 mg / l, without ROS generation and low Zn2 + release that is highly relevant for practical applications. The dose of ZnO used in the prior art is in the range of 50-500 mg / l (A. Sirelkhatim et al. Review on Zinc Oxide Nanoparticles: Antibacterial Activity and Toxicity Mechanism. Nano-Micro Lett. (2015) 7 : 219), and as a consequence 10 mg / l of ZnO is considered as a low dose and preferably 3 ppm is also considered a low dose.
    [0253]
    [0254] Example 8. Accumulation of charge in the microparticles of the present invention and their effect on microorganisms.
    [0255]
    [0256] Figure 9a shows a schematic representation of the microstructure of the present invention. Platelets (1) are organized in conically stacked groups (3) joined by their base along the c axis, as mentioned in Figure 1. Each platelet (1) attached to adjacent platelets generates a domain and each border of two platelets a domain wall (11). These domain walls create possible energy barriers to electrons, known as Schottky barriers (12). It is believed that the presence of multi-domain, therefore, multi-domain walls, and Schottky barriers generate uncompensated charges that accumulate on domain walls. This phenomenon resulted in an electrical potential distributed by the structure of the present invention.
    [0257]
    [0258] Kelvin probe microscopy (KPM) is a modified version of Atomic Force Microscopy (AFM). The KPM consists of a non-destructive non-contact surface technique that allows two-dimensional profiles of the contact potential difference (VCPD) to be imagined, that is, the difference in the working functions of the tip and the sample. For a representative sample of the microparticles of the present invention (sample D), the contact potential difference voltage (CPD) reached values around -5 V at the top of the structure. Negative charge values were confirmed in aqueous solution of the microparticles of the present invention using zeta potential measurements (Figure 9b). As can be seen, the present invention shows a zeta potential value around -20 mV in the pH range of 6 to 10. This observation confirmed the existence of accumulation of negative charge along the structure of the present invention.
    [0259]
    [0260] The effects of the interactions between the particles of the present invention and the selected microorganisms are depicted in Figure 10. SEM micrographs show the damage caused by the flower-like particles of the invention in contact with E. coli bacteria (Figure 10a ) and S. aureus (Figure 10b) and the fungus A. niger (Figure 10c). It is believed that the contact is produced by electrostatic interactions between the charge of the microparticles of the present invention and the microorganisms. The main damages to the microorganisms (white arrows) were abrasions such as wrinkles, flattening or blisters, cracking and / or rupture of the membrane envelope. As a result, these abnormal textures lead to the "bored" membrane and ultimately to the death of microorganisms.
    [0261]
    [0262] The cathodoluminescence (CL) is an optical and electromagnetic phenomenon in which an electron beam impacts on a luminescent material that causes the emission of visible light. A cathodoluminescence detector attached to a scanning electron microscope (SEM), commonly called SEM-CL, is capable of producing high resolution digital cathodoluminescent images of luminescent materials. Figure 10d shows SEM-CL of the microstructure of the present invention (sample D) against the fungus A. niger. The areas more luminescent that were mostly terminal areas correspond to defects in the ZnO crystal. Defect areas accumulate more load. The areas with the highest load directly affect the microorganisms, in this case A. niger (dark areas). As can be seen, the interaction through a contact mechanism between the flower-like particles of the invention and the microorganism does not weaken its potential load. Therefore, the microparticles of the present invention maintained their activity for long periods of time and at very low concentrations or doses.
    [0263]
    [0264] Example 9. Cytotoxicity of the microparticles of the present invention.
    [0265]
    [0266] After observing excellent antimicrobial behavior of the ZnO microparticles of the present invention against bacteria and fungi, it is important to know their cytotoxicity against human cells. For this study, the human cells used are the HeLa tumor epithelial cell line (cervical adenocarcinoma). HeLa cells were grown in Dulbecco-modified Eagle's medium (DMEM) supplemented with 10% (v / v) fetal bovine serum (FBS), 50 units / ml penicillin and 50 ^ g / ml streptomycin. Cell cultures were performed in a 5% CO2 atmosphere at 37 ° C and maintained in a Steri-Cult 2000 incubator (Hucoa-Erloss, Madrid, Spain). Cells were seeded in 24 multiwell plates (Falcon, St. Louis, MO, USA). Experiments were performed with 60-70% confluence cells.
    [0267]
    [0268] Prior to the addition of ZnO, the nanoZnO commercial references and the microparticles of the present invention (heat treated at 500 ° C) were sterilized by dry heat at 110 ° C for 24 h. A suspension of ZnO particles (references and present invention) was prepared in phosphate buffered saline (PBS). After this, the different ZnO concentrations (0.1 mg / ml, 0.2 mg / ml and 0.5 mg / ml) were prepared in DMEM from the suspension in PBS. The cells were incubated for 24 h with the appropriate concentration of ZnO. After incubation, the cells were washed three times with PBS and incubated again with DMEM for 24 h. Finally, cell viability was determined after treatments by (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, called the MTT assay. The MTT assay is a colorimetric method for measuring the mitochondrial enzyme activity Enzymes reduce the yellow MTT dye to its insoluble formazan, which has a purple color, so the amount of formazan formed is proportional to the number of living cells To determine cell survival, the band Absorption of 540 nm is measured in each test.The results were expressed as a percentage compared to control cells (Figure 11).
    [0269]
    [0270] As shown in Figure 11, the ZnO particle size has an important function in cytotoxicity. The nanoZnO induces a drastic reduction in the population of living cells by increasing the concentrations of ZnO nanoparticles in the culture medium, reaching values of 40% cell survival at a concentration of ZnO nanoparticles of 0.5 mg / ml. In contrast, the ZnO microparticles of the present invention heat treated at 500 ° C maintain a low cytotoxicity of HeLa cells at high concentrations (0.5 mg / ml), the cell survival being> 88% at all concentrations.
    [0271]
    [0272] Surprisingly, the particles of the present invention show high antimicrobial activity against fungi and bacteria without showing cytotoxicity to human cells. The organized hexagonal stack design of the particles of the present invention is considered a determining factor. Without pretending to be limited by theory, it is believed that the stack organized in star-type morphology combines the beneficial properties of ZnO nanoparticles, such as high antimicrobial activity, and the beneficial properties of ZnO microparticles, such as a Low cytotoxicity in cells. Therefore, the particles of the present invention are considered potentially non-cytotoxic to human cells, allowing the use of the ZnO microparticles of the present invention in applications such as cosmetic and medical applications.
    权利要求:
    Claims (14)
    [1]
    1. Zinc oxide microparticles comprising platelets, in which platelets,
    - they are prisms with hexagonal base,
    - have an edge length between 30 and 200 nm, and
    - they are stacked in direct contact with each other along the stacking axis c, by means of their crystalline planes [000T] or [0001], and
    - they are rotated by a non-zero angle with respect to their adjacent platelets along said stacking axis c;
    wherein said zinc oxide microparticles are characterized by a specific surface area that is less than or equal to 4 m2 / g.
    [2]
    2. The zinc oxide microparticles according to claim 1, wherein the specific surface area is less than or equal to 2 m2 / g.
    [3]
    3. The zinc oxide microparticles according to any one of claims 1 to 2, characterized in that they are flower-shaped microparticle structures.
    [4]
    4. Method for preparing the zinc oxide microparticles according to any one of claims 1 to 3, comprising the steps of:
    i) adding urea to an aqueous zinc salt solution, where the zinc salt solution has a concentration between 3 and 10 M.
    ii) heating the solution resulting from step (i),
    iii) isolate the product resulting from step (ii), and
    iv) subject the product isolated from step (iii) to an annealing treatment.
    [5]
    5. Method according to claim 4, wherein the salt solution of step (ii) is heated at a temperature between 80 ° C and 140 ° C for a period of time between 1 and 3 hours.
    [6]
    6. Method according to any of claims 4 to 5, wherein step (iii) comprises:
    - cooling the solution resulting from step (ii) to room temperature, and adding a hot aqueous solvent to obtain a precipitated product, and
    - isolate the precipitated product.
    [7]
    7. Method according to any of claims 4 to 6, wherein the annealing treatment of step (iv) is carried out at a temperature between 350 ° C and 700 ° C.
    [8]
    8. Method according to any of claims 4 to 7, wherein the annealing treatment of step (iv) is carried out for a period of time between 1 and 24 hours.
    [9]
    9. Cosmetic product, paint, ink, paper, cardboard, textiles, food, agricultural product, home care, air conditioning, animal care, personal and work hygiene, contact lenses, chromatography material, medical equipment, product dermatological, lacquer, coating and / or plastic product containing the zinc oxide microparticles according to any one of claims 1 to 3.
    [10]
    10. Biocidal composition comprising the zinc oxide microparticles according to any one of claims 1 to 3 and optionally a vehicle.
    [11]
    11. Use of the biocidal composition according to claim 10 for the elimination, inhibition of growth or inhibition of the progeny of microorganisms, excluding any treatment or diagnostic methods on the human or animal body.
    [12]
    12. Use of the biocidal composition according to claim 11, wherein the microorganisms are selected from bacteria and fungi.
    [13]
    13. The zinc oxide microparticles according to any one of claims 1 to 3 or a pharmaceutical composition comprising the same for use in the treatment or prevention of an infection.
    [14]
    14. The zinc oxide microparticles or pharmaceutical composition for use according to claim 13, wherein the infection is an infection of the skin, mucous membranes, nails, skin lesions and / or mucous membranes.
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    同族专利:
    公开号 | 公开日
    ES2724825B2|2020-03-16|
    EP3326975A1|2018-05-30|
    WO2018099945A1|2018-06-07|
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    申请号 | 申请日 | 专利标题
    EP16382571.4A|EP3326975A1|2016-11-29|2016-11-29|Zinc oxide microparticles, preparation method, and use thereof|
    PCT/EP2017/080766|WO2018099945A1|2016-11-29|2017-11-29|Zinc oxide microparticles, preparation method, and use thereof|
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