Nanoparticles for the controlled release of curcumin, preparation method and use thereof

Abstract

The present invention relates to nanoparticles for the controlled release of curcumin comprising: a core formed by aggregation of gold nanoparticles where said aggregate is in the form of a nanostar; a first coating consisting of a mesoporous silica layer where the curcumin is encapsulated or embedded; a second coating covering the porous silica layer, where said second coating consists of octadecyltrimethoxysilane; a third coating covering the layer of octadecyltrimethoxysilane, where said third coating consists of a heat-sensitive compound. The present invention further relates to the preparation method for said nanoparticles and to the use thereof.

Description

[0001] DESCRIPTION

[0002] Nanoparticles for the controlled release of curcumin, preparation procedure and use thereof

[0003] The present invention relates to nanoparticles for the controlled release of an antibacterial agent such as curcumin by applying light in the near IR range, as well as to the process for preparing said nanoparticles and their use.

[0004] The present invention can be framed within the field of medicine, especially in the treatment of microbial infections.

[0005] BACKGROUND OF THE INVENTION

[0006] Due to the increasing antimicrobial resistance to conventional drug therapies, antimicrobial photodynamic therapy (aPDT) has been proposed as an alternative for the treatment of microbial infections (Hamblin, MR. “Antimicrobial photodynamic inactivation: a bright new technique to kill resistant microbes.” Curr Opin Microbiol, 2016 67-73). In aPDT, a compound called a photosensitizer (PS) interacts with light of a suitable wavelength, which, in the presence of oxygen, generates reactive oxygen species (ROS) that cause oxidative damage to microorganisms. Curcumin (CUR) is a naturally occurring PS successfully used in aPDT and has been evaluated in vitro against free microbial cells (planktonic cultures) and biofilms (Abrahamse, H, et al. “New photosensitizers for photodynamic therapy.” Biochem J, 2016, 347–64); as well as in vivo infection models (Dovigo, LN, et al.“Curcumin-mediated photodynamic inactivation of Candida albicans in a murine model of oral candidiasis.” Med Mycol, 2013, 243-251).

[0007] However, the clinical use of CUR as a SF remains limited due to its hydrophobicity, instability, and low bioavailability. Thus, its delivery and application require organic solvents such as dimethyl sulfoxide (DMSO), which exhibits cytotoxic effects (Sakima, VT, et al. “Antimicrobial Photodynamic Therapy Mediated by Curcumin-Loaded Polymeric Nanoparticles in a Murine Model of Oral Candidiasis.” Molecules, 2018, 23, 2075; Hanslick, JL, et al. “Dimethyl sulfoxide (DMSO) produces widespread apoptosis in the developing central nervous system.” Neurobiol Dis, 2009, 1-10;).

[0008] An alternative to improve these drawbacks is through drug delivery systems, such as polymeric nanoparticles, micelles, cyclodextrins, and mesoporous nanoparticles.

[0009] Among the potential nanomaterials, mesoporous silica nanoparticles (MSNs) have high loading capacity, chemical stability, and biocompatibility (Hernández-Montoto, A, et al. “Gold Nanostars Coated with Mesoporous Silica Are Effective and Nontoxic Photothermal Agents Capable of Gate Keeping and Laser- Induced Drug Release.” ACS Appl Mater Interfaces, 2018, 27644-27656; Aznar, E, et al. “pH- and Photo-Switched Release of Guest Molecules from Mesoporous Silica Supports.” J Am Chem Soc, 2009, 6833-6843). Studies on CUR were previously developed in MSN (Wang, J, et al. “Rational design of multifunctional dendritic mesoporous silica nanoparticles to load curcumin and enhance efficacy for breast cancer therapy.” ACS Appl Mater Interfaces, 2016, 26511-26523; Gangwar, RK, et al.” Curcumin conjugated silica nanoparticles for improving bioavailability and its anticancer applications.” J Agrie Food Chem, 2013, 9632–9637), but most of them were used against cancer cells. In a recent study, silica nanoparticles loaded with CUR (CUR-SNp) at a concentration of 1 mg / mL associated with 20 J / cm. 2Blue light irradiation promoted almost 6 logw decrease in planktonic cultures of Staphylococcus aureus and Pseudomonas aeruginosa (Mirzahosseinipour, M, et al. “Antimicrobial photodynamic and wound healing activity of curcumin encapsulated in silica nanoparticles. Photodiagnosis Photodyn Ther, 2020, 101639). Furthermore, these parameters of light dose and CUR loaded in nanoparticles were used against monospecies biofilms of S. aureus and P. aeruginosa, promoting a reduction in microbial colony counts of 3 log for S. aureus and 5 logw for P. aeruginosa compared to the untreated control group. In addition to the action of CUR as a SF, the compound transported in silica nanoparticles, compared to the free form of CUR, showed a greater healing effect after 24 hours. in contact with dermal fibroblasts in vitro.

[0010] Despite reported antimicrobial photodynamic efficacy of CUR in MSNs, the prolonged release of some systems could compromise the clinical use of CUR in nanocarriers. A possible alternative to improve the efficacy of aPDT could be the modification of the external surface of the MSN through the use of molecular gates (Garcia-Fernandez, A, et al. “New Advances in In Vivo Applications of Gated Mesoporous Silica as Drug Delivery Nanocarriers.” Small, 2020, 16 (3)), which offer a spatio-temporal release of the loaded compound, in order to control the amount of released FS and to avoid FS degradation before delivery to the target site. This concept has allowed the design of nanodevices for on-demand delivery that can be activated by chemical substances (such as redox molecules, selected anions and pH changes), physical substances (such as light, temperature and magnetic fields) and biochemical substances (such as enzymes, antibodies and DNA).

[0011] In this way, the present invention has the advantage of the controlled release of CUR from MSNs, which in turn are coated by a molecular gate that prevents the possible degradation of the FS until it is delivered to the site of action.

[0012] DESCRIPTION OF THE INVENTION

[0013] The present invention describes nanoparticles useful for the release of active compounds, such as curcumin, which allow the controlled spatio-temporal release of said compounds in the presence of near-infrared (NIR) irradiation.

[0014] In a first aspect, the present invention relates to nanoparticles for the controlled release of curcumin (hereinafter, the nanoparticles of the present invention), which comprise:

[0015] - a core formed by aggregation of gold nanoparticles where said aggregate has a nanostar shape,

[0016] - a first coating of the core, where said first coating is formed by a layer of mesoporous silica where the curcumin is encapsulated or embedded,

[0017] - a second coating formed by octadecyltrimethoxysilane, which is found covering the porous silica layer,

[0018] - a third coating covering the octadecyltrimethoxysilane layer, said third coating being formed by a heat-sensitive compound selected from 1-tetradecanol, poly(N-isopropylacrylamide), DNA and heneicosane, preferably heneicosane. This coating acts as a heat-sensitive molecular gate. The term “nanostar” refers to an aggregation of nanoparticles where the outer surface of the aggregate has conoidal structures ending in a point, giving rise to a nanometric-sized star-shaped structure. In a preferred embodiment, the nanostar-shaped core has an average largest dimension (maximum distance between two ends, or furthest tips, of the nanostar) of between 50 and 80 nm, as measured by transmission electron microscopy (TEM).

[0019] The term “mesoporous silica” is used in its ordinary meaning in the art, i.e., a silica material (also called silicon dioxide or SIO2) comprising mesopores, which are pores in the range of 2–50 nanometers (average pore diameter size). The technique used to measure average pore diameter is called porosimetry, determined by N2 adsorption–desorption isotherm.

[0020] The nanoparticles of the invention (core and three coatings) have an average diameter between 400 and 500 nm determined by dynamic light scattering (DLS). Said experiment is carried out by dispersing 1 mg of the nanoparticles in 1 mL of deionized water, at neutral pH (7.0) at a temperature of 25°C.

[0021] In a preferred embodiment, the nanostar-shaped core coated with a silica layer has an average diameter of between 100 and 150 nm, as measured by TEM.

[0022] In a preferred embodiment, the zeta potential of the nanoparticles of the present invention is negative and ranges between -19.2 and -23.8 mV. This value could reduce the interaction with microorganisms (AP Ribeiro, et al. Antimicrobial photodynamic therapy against pathogenic bacterial suspensions and biofilms using chloro-aluminum phthalocyanine encapsulated in nanoemulsions. Lasers Med Sci. 2015;30: 549-559).

[0023] The term “zeta potential (Z)” refers to a measure of the surface charge of nanoparticles. The zeta potential is determined for nanoparticles suspended in an electrolyte. In such a suspension, a distribution of electrolyte ions occurs around the nanoparticle surface. Determining the zeta potential makes it possible to estimate the surface charge and this technique can be used to verify the physical stability of nanoparticles (J. Jiang, G. Oberdórster, A. Eider, R. Gelein, P. Mercer, and P. Biswas, “Does nanoparticle activity depend upon size and crystal phase?,” Nanotoxicology, vol. 2, no. 1, pp. 33–42, 2008.). A large positive or negative zeta potential of nanoparticles indicates good physical stability of the nanoparticles, due to the electrostatic repulsion of the individual particles.

[0024] The Z potential was measured by dynamic light scattering (DLS). The measurement was performed on an aqueous suspension of particles (1 mg / mL) at neutral pH (pH=7) at 25°C in a Zetasizer Nano ZS (Malvern Instruments) using a transmission cuvette. The technique consists of subjecting the suspension to an electric field and measuring the particle displacement velocity under the effect of the electric field. The zeta potential is a magnitude of charge attraction or repulsion between particles, exhibited by any particle in suspension. In other words, it is the potential difference across the phase boundaries between solids and liquids. By applying an electric potential difference, a controlled electric field is applied by means of electrodes immersed in a suspended sample; this causes the charged particles to move through the electrode of opposite polarity.

[0025] In a second aspect, the invention relates to the process for preparing the nanoparticles described in the first aspect of the invention. Said process comprises the following steps: a) synthesizing gold nanoparticles (AuNsds), b) synthesizing gold nanostars (AuNSt) from the gold nanoparticles synthesized in step a), c) coating the gold nanostars obtained in b) with mesoporous silica, d) encapsulating curcumin in the mesoporous silica coating and coating the nanoparticles comprising the encapsulated curcumin with octadecyltrimethoxysilane, e) coating the nanoparticles obtained in d) with a thermosensitive compound selected from 1-tetradecanol, poly(N-isopropylacrylamide), deoxyribonucleic acid (DNA) and heneicosane, preferably heneicosane.

[0026] The AuNsds prepared in step a) have an average diameter size of between 20 and 40 nm, as measured by TEM. To this end, in a preferred embodiment, the AuNsds are prepared in step a) by contacting chloroauric acid HAuCl and sodium citrate in water under reflux for a time of between 15 and 20 minutes, preferably 15 min. The AuNp are then contacted with polyvinylpyrrolidone (PVP) in order to obtain spherical AuNPs and kept under stirring at room temperature (18-25 ° C) for a time between 16 and 20 h, preferably 18 h. The synthesized AuNsds are collected by centrifugation of the resulting reaction mixture. The most preferred proportions between reagents for this step are as follows:

[0027] 70 pL of HAuCl in 100 mL of distilled water

[0028] 15 mL of a 1% aqueous sodium citrate solution

[0029] 1 mL of aqueous solution of PVP at 500 mg / mL in 20 mL of a suspension of AuNsds.

[0030] In a preferred embodiment, the AuNSt are prepared in step b) by contacting PVP with an aqueous solution of HAuCl and then adding the AuNp obtained in step a), keeping the mixture under stirring at room temperature (18-25°C) for a period of between 20 and 26 hours, preferably 24 hours. After this time, the AuNSt are collected by centrifugation. The preferred proportions between reagents for this step are as follows:

[0031] 45 mL of a DMF solution containing 30% PVP, with 150 pL of an aqueous HAuCl solution

[0032] 5 mM of AuNsds are added in aqueous solution

[0033] In a preferred embodiment, to form the mesoporous silica coating in step c), an aqueous solution of cetyltrimethylammonium bromide (CTAB) is contacted with ethanol under an Argon (Ar) atmosphere, followed by the addition of ammonia and AuNSt obtained in the previous step. Subsequently, tetraethyl orthosilicate (TEOS) is added to the reaction mixture and the reaction mixture is maintained in an inert atmosphere for a time between 20 and 26 hours and a temperature between 23 and 28 ° C, preferably, 24 hours at 25 ° C. After this time, the silica-coated nanoparticles are centrifuged and washed with ethanol. Preferably, an extraction process is then carried out using an ethanolic solution of ammonium nitrate (NH4NO3) to eliminate the remaining CTAB molecules. The preferred proportions between reactants:

[0034] - To 50 mL of an aqueous solution of 6.6 mM CTAB are added 20 mL of ethanol, 50 pl of an aqueous solution of 32% ammonia and 5 mM AuSt, 40 pl of TEOS to the reaction. In a preferred embodiment, in order to include curcumin in the mesoporous silica layer, the nanoparticles obtained in step c) are suspended in acetonitrile (ACN) and curcumin is added to the reaction mixture which is kept under stirring for a time between 50 minutes to 1 hour at a temperature between 23 and 25 ° C, preferably, for 1 hour at 25 ° C under stirring. Subsequently, octadecylmethoxysilane (OCT) is added to the solution in order to form the OCT coating. The mixture with OCT is kept for 10 to 14 hours at a temperature of 23 to 28°C, preferably 12 hours at 25°C, under stirring. After this time, the particles are centrifuged, washed with an aqueous ACN solution, and dried under vacuum.The preferred proportions between reagents: a stock solution of CUR diluted in 1 mg / mL acetonitrile with 1 mg / mL of AuNst@mS¡02.

[0035] 20 pL of OCT in 2 mL of CUR solution: AuNst@mS¡02.

[0036] In a preferred embodiment, step e) is carried out by contacting the nanoparticles obtained in step d) with the thermosensitive compound, preferably using n-hexane as a solvent, and the mixture is subjected to ultrasound for a time between 40 s and 1 min, preferably 1 min and subsequent stirring in a vortex shaker for a time between 10 and 30 s, preferably 20 s. The step of ultrasound and stirring in a vortex shaker can be repeated several times. The preferred proportions between reactants: the nanoparticles loaded with CUR in 10 mL of n-hexane.

[0037] 1 mL of heneicosane or the thermostable compound in question (20 mg / mL diluted in n-hexane).

[0038] The present invention also relates to nanoparticles obtained by the process described above.

[0039] Another aspect of the invention relates to a composition comprising the nanoparticles described in the first aspect of the invention.

[0040] In a preferred embodiment, the composition is a pharmaceutical composition.

[0041] The pharmaceutical composition preferably includes at least one pharmaceutically acceptable excipient and / or vehicle. The "vehicle" or carrier is preferably an inert substance. The function of the vehicle is to facilitate the incorporation of other compounds, allow for improved dosage and administration, or provide consistency and shape to the pharmaceutical composition. Therefore, the vehicle is a substance used in the medication to dilute any of the components of the pharmaceutical composition of the present invention to a specific volume or weight; or even without diluting said components, it is capable of allowing for improved dosage and administration or providing consistency and shape to the medication. When the presentation form is liquid, the pharmaceutically acceptable vehicle is the diluent.

[0042] The term "excipient" refers to a substance that aids in the absorption of any of the components of the composition of the present invention, stabilizes said components, or aids in the preparation of the pharmaceutical composition by giving it consistency or providing flavors that make it more pleasant. Thus, excipients could have the function of keeping the components together, such as starches, sugars, or cellulose, sweetening function, coloring function, protecting the medication, such as isolating it from air and / or moisture, filling function of a pill, capsule, or any other form of presentation, such as dibasic calcium phosphate, or disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph.Therefore, the term "excipient" is defined as that substance, included in the dosage forms, which is added to the active ingredients or their combinations to enable their preparation and stability, modify their organoleptic properties, or determine the physical and chemical properties of the pharmaceutical composition and its bioavailability. The "pharmaceutically acceptable" excipient must allow the compounds in the pharmaceutical composition to function, that is, it must be compatible with said components.

[0043] Furthermore, as understood by those skilled in the art, the excipient and vehicle must be pharmacologically acceptable, that is, the excipient and vehicle must be permitted and evaluated so that they do not cause harm to the organisms to which they are administered.

[0044] Another aspect of the invention relates to the nanoparticles of the invention, or a composition comprising them, for use as a medicament. Another aspect of the invention relates to the nanoparticles of the invention, or a composition comprising them, for use in the controlled release of curcumin in the presence of near-infrared (NIR) light radiation of between 180-3000 nm wavelength, preferably between 600 and 900 nm, more preferably, 808 nm. In the presence of this radiation, the AuNSt transform the light into heat that is capable of melting the thermosensitive compound or changing the structure in the case of DNA, thus producing the release of the curcumin contained inside the pores.

[0045] A final aspect relates to the nanoparticles of the invention, or a composition comprising them, for use in antimicrobial photodynamic therapy (aPDT), preferably against Staphylococcus aureus and Pseudomonas aeruginosa. Curcumin acts as a photosensitizer (PS). Once the curcumin is released by NIR light irradiation, irradiation is carried out with a wavelength between 410 and 466 nm, preferably 450 nm (blue), which, in the presence of oxygen, results in the production of reactive oxygen species capable of causing microbial death.

[0046] Antimicrobial photodynamic therapy combines the use of a photosensitizing drug, light, and oxygen to eradicate pathogenic microorganisms.

[0047] The main advantage of this system is the controlled release of curcumin under NIR light irradiation (allowing the release of up to approximately 89% of the curcumin present in the nanoparticles in 30 min) and the possibility of using it in antimicrobial photodynamic therapy. Furthermore, the thermosensitive compound also acts as a protective barrier for curcumin until it reaches microbial cells, preventing its degradation.

[0048] The nanoparticles of the present invention have been shown to have advantages of controlled release under NIR irradiation and delivery of FS in microorganisms.

[0049] Throughout the description and claims, the word "comprise" and its variants are not intended to exclude other technical features, additives, components, or steps. For those skilled in the art, other objects, advantages, and features of the invention will be apparent in part from the description and in part from the practice of the invention. The following examples and figures are provided for illustrative purposes only and are not intended to be limiting of the present invention. BRIEF DESCRIPTION OF THE FIGURES

[0050] Fig. 1: TEM microscopy (a) and UV-visible absorption spectrum (b) of AuNsds

[0051] Fig. 2: TEM microscopy (a) and UV-visible absorption spectrum (b) of AuNSt

[0052] Fig. 3: TEM microscopy (a) and UV-visible absorption spectrum (b) of AuNSt@mSiC>2. Fig. 4: TEM microscopy (a) and UV-visible absorption spectrum (b) of AuNSt@mS¡O2@CUR@paraffin.

[0053] Fig. 5: Absorption spectrum of curcumin.

[0054] Fig. 6: Photorelease of CUR from AuNSt@mS¡O2@CUR@paraffin, scatter plot indicates percentage of CUR release as a function of irradiation time.

[0055] Fig. 7: Mean CFU / mL values ​​of planktonic cultures of (a) S. aureus and (b) P. aeruginosa. Error bars: standard deviation (n=12). Number of CFU / mL according to the groups evaluated.

[0056] EXAMPLES

[0057] The invention will then be illustrated by tests carried out by the inventors.

[0058] Materials and methods

[0059] Polyvinyl pyrrolidone (PVP-MW 10000), citrate, auric-lll-gold (HAuCl4), 1% sodium citrate, cetyltrimethylammonium bromide (CTAB), N,N-dimethylformamide (DMF), curcumin, tetraethyl orthosilicate (TECS), acetonitrile (ACN), octadecyltrimethoxysilane (OCT) were purchased from Sigma Aldrich.

[0060] For the characterization of nanoparticles, the following equipment has been used: UV-Visible spectrophotometer (JASCO V-630 Spectrophotometer, JASCO, Easton, USA), Fluorimeter (JASCO FP-8300, JASCO, Easton, USA), centrifuge (Eppendorf 5810 R) NIR Laser (developed by the engineering department of the Universitat Polytechnica of Valencia), Blue LED light at -450 nm with power of 30 mW / cm2 (Bulbs lighting. Co. Shenzhen, China).

[0061] Example 1 : The synthesis of photosensitive nanoparticles of the present invention

[0062] The synthesis process of the nanoparticles of the present invention includes five main steps. In the first step, gold nanoparticles (AuNp), also called nanoseeds, were synthesized by mixing 100 mL of deionized water with 70 pL of 30% chloroauric acid (HAuCU 1.444 M). The mixture was carried out in a double-necked, round-bottomed flask using a reflux system, which was heated to 100 °C, with magnetic stirring at 1200 rpm. After the mixture boiled, 15 mL of an aqueous sodium citrate solution (1%) was added through the side of the two-necked flask. The temperature (100 °C) and magnetic stirring were maintained for 15 minutes. After this time, magnetic stirring was maintained until the mixture cooled to room temperature.

[0063] Subsequently, 20 mL of AuNp and 1 mL of a 500 mg / mL aqueous solution of polyvinylpyrrolidone (PVP, M = 10,000 g / mol) were added to another round-bottom flask. The solution was stirred for 18 hours at room temperature. Finally, the AuNp were centrifuged at 10,000 rpm for 20 minutes at 25 °C, and the precipitate (pellet) (AuNp) was resuspended in 1.5 mL of 99% ethanol. The AuNp produced with a size of 31.1 nm (Figure 1a) was observed by transmission electron microscopy (TEM). Dynamic light scattering (DLS) showed AuNp with a size range of 48.1 to 75.57 nm. In the characterization of the UV-visible absorption spectrum, a maximum absorption was observed at 520 nm (Figure 1b).

[0064] In the second step, gold nanostars (AuNSt) were grown using a concentrated solution of 45 g of PVP diluted in 150 mL of dimethylformamide, in which 750 pL of an aqueous HAuCl solution (166 mM) were mixed. After 5 min, 50 pL of the previously synthesized AuNp at a concentration of 6.5 mM was added. The reaction was continued for 24 h without stirring and at room temperature. After this time, the AuNSt were washed 5 times by centrifugation (9,500 rpm for 20 min) and resuspension in deionized water. The AuNSt observed by TEM showed the formation of structures with sizes between 55.1 and 74 nm (Figure 2a). The size determined by DLS ranged between 124 and 175.5 nm. In the absorption spectrum, the nanostar band was located around 803 nm (Figure 2b).

[0065] In the third step, mesoporous silica nanoparticles (AuNSt@mSiC>2) were synthesized. 50 mL of an aqueous solution of cetyltrimethylammonium bromide (CTAB 6.6 mM) with 20 mL of ethanol were used in a double-necked, round-bottom flask. Argon (Ar) gas was bubbled into this solution for 1 h under magnetic stirring at 400 rpm and 25 °C. After this time, 50 pl of 32% ammonia and 3 mL of AuNSt suspension (5 mM) were added to the round-bottom flask. After 5 min, 40 pL of tetraethyl orthosilicate (TEOS) was added dropwise to the mixture. The reaction was maintained under an inert atmosphere for 24 h at 25 °C with magnetic stirring at 400 rpm. After this time, the nanoparticles were centrifuged (10 minutes, 9,500 rpm) and washed twice with ethanol (99%). Finally, an extraction process was performed using an ethanolic solution of ammonium nitrate (-NH4NO3- 10 mg / mL), and the remaining CTAB molecules were removed.The nanoparticles were washed 10 times using ethanolic NH4NO3 solution and dried under vacuum at room temperature. The size of AuNSt@mSiC>2 observed by TEM was 122 nm (Figure 3a). By DLS, the nanoparticle size ranged from 216.4 to 338.2 nm, and the polydispersity index (PDI) value ranged from 0.049 to 0.385 arbitrary units (au). The zeta potential of the formulation was evaluated before CTAB extraction (range +29.2 to +47.8 mV) and after CTAB extraction (range -25.7 to -32.9 mV). In addition, the maximum absorption spectrum was observed around 803 nm (Figure 3b).

[0066] In step 4, curcumin (CUR) was loaded onto the nanoparticles. 2 mg of AuNSt@mSiC>2 was suspended in 2 mL of acetonitrile (ACN). 1 mg of CUR was then added, and the mixture was kept for 1 h at 25°C under magnetic stirring at 400 rpm. Subsequently, 20 pL of octadecyltrimethoxysilane (OCT) was added to the solution. The mixture was kept for 12 h at 25°C under magnetic stirring at 400 rpm. After this time, the particles were centrifuged and washed twice with a 0.5% aqueous ACN solution. Finally, the particles were dried under vacuum.

[0067] In step 5, the heneicosane (also known as paraffin) gate was added to the system. To do this, the nanoparticles were diluted in 10 mL of n-hexane, followed by 1 mL of a heneicosane solution diluted in n-hexane (20 mg / mL). The nanoparticles were dispersed under ultrasound for 1 minute and agitated in a vortex for 20 seconds; this procedure was repeated for 30 minutes. TEM images showed that the nanoparticle size increased to 131 nm (Figure 4a).

[0068] This increase in size was also verified by DLS, which showed that the nanoparticle sizes ranged from 402 to 484.4 nm. The PDI values ​​observed at this stage of the synthesis ranged from 0.092 to 0.192, while the zeta potential ranged from -19.2 to -23.8 mV. Finally, the absorption spectrum was located around 803 nm (Figure 4b).

[0069] Example 2: Evaluation of the ability of the nanoparticles of the invention to load the CUR

[0070] The capacity of this formulation to load CUR was evaluated. To this end, a 500 mg / mL aqueous solution of AuNSt@mS¡O2@CUR@paraffin (synthesized in Example 1) was heated by magnetic stirring at 60 °C and 1200 rpm for 60 hours. After this time, the samples were centrifuged at 10,000 rpm for 5 minutes at 5 °C, the pellet (CUR) was solubilized in ACN, and its absorption was determined using a UV-visible spectrophotometer at 425 nm. The observed values ​​were between 0.218 and 0.265 au (Figures 5a and b), corresponding to 2.3 pg / mL and 2.7 pg / mL of CUR inside the mS¡O2 nanoparticles.

[0071] Example 3: Photorelease of CUR from nanoparticles

[0072] The photorelease of CUR from the nanoparticles synthesized in Example 1 was carried out using a laser light apparatus with a wavelength of 808 nm manufactured by the Molecular Research Institute of the Polytechnic University of Valencia. The nanoparticle samples (500 mg / mL) loaded with CUR were irradiated with the light apparatus for 0.5, 10, 15, 20, 25 and 30 minutes. After each irradiation period, the samples were centrifuged at 10,000 rpm for 5 minutes at 5°C, and the fluorescence of the supernatant was read at 498 nm. The non-irradiated samples remained in the dark as a control group. Figure 6 shows that the maximum CUR release from the nanoparticles was 89.1%.

[0073] Example 4: Use of the nanoparticles of the present invention as a photosensitizer (PS) in antimicrobial Photodynamic Therapy

[0074] The nanoparticles synthesized in Example 1 were used as photosensitizers (PS) in antimicrobial photodynamic therapy (aPDT) against planktonic cultures of Staphylococcus aureus and Pseudomonas aeruginosa. Each microorganism was grown in a specific culture medium (Müller Hinton Broth and Luria Bertoni broth for S. aureus and P. aeruginosa, respectively) and the inoculum was standardized at 4.72x10 7 (±3, 9x10 6 ) and 6.97x10 7 (±7, 52x10 6) colony forming units per milliliter (CFU / mL). For each microorganism in planktonic cultures, three different concentrations (500, 50 and 5 pg / mL) of AuNSt@mSiC>2@CUR@paraffin were evaluated, for which 100 pL of FS and 100 pL of microbial suspension were kept in the dark for 5 minutes. Then, each sample was irradiated with 808 nm laser light for 20 minutes, immediately, a second irradiation was performed, however, this second irradiation was performed using a blue light emitting diode device (-450 nm) (LED, power of 30 mW / cm 2 , Illumination Bulbs. Co. Shenzhen, China) for 20 minutes, corresponding to 36 J / cm 2 to generate reactive oxygen species responsible for microbial photoinactivation (aPDT groups).

[0075] Control samples included bacterial suspensions subjected to laser irradiation only with and without CUR-loaded nanoparticles (PS-L+ and PS+L+ groups, respectively). Another group was treated only with laser light without AuNSt@mS¡O2@CUR@paraffin (L+ group).

[0076] The effect of AuNSt@mS¡O2@CUR@paraffin was evaluated under blue LED irradiation (LED+PS+). The effect of LED light without any nanoparticles was also evaluated (LED+ group). Finally, the general bacterial growth control group received no treatment (control group).

[0077] Samples subjected to aPDT with 500 pg / mL of PS showed mean reductions of 3.15 and 2.18 log (CFU / mL) of S. aureus and P. aeruginosa, respectively, compared to untreated samples (control). Samples subjected to aPDT with 50 pg / mL of PS demonstrated a 1.5 log (CFU / mL) reduction for S. aureus, whereas no decrease in colony count was observed for P. aeruginosa. aPDT with 5 pg / mL of PS was not effective for either organism.

[0078] No decrease in microbial viability was observed in the groups treated solely with laser or LED, as well as in the samples treated with nanoparticles illuminated solely with laser or LED, as these groups showed CFU / mL values ​​similar to those of the control group (without any treatment). Figures 7a and 7b show the results for S. aureus and P. aeruginosa, respectively.

Claims

CLAIMS 1. Nanoparticles for the controlled release of curcumin comprising: - a core formed by aggregation of gold nanoparticles where said aggregate has a nanostar shape, - a first coating of the core, where said first coating is formed by a layer of mesoporous silica where the curcumin is encapsulated or embedded, -a second coating formed by a layer of octadecyltrimethoxysilane, which is covering the porous silica layer, - a third coating covering the octadecyltrimethoxysilane layer, wherein said third coating is formed by a heat-sensitive compound selected from 1-tetradecanol, poly(N-isopropylacrylamide), deoxyribonucleic acid (DNA) and heneicosane.

2. Nanoparticles, according to claim 1, whose average diameter is between 400 and 500 nm, determined by dynamic light scattering (DLS).

3. Nanoparticles according to any of the preceding claims, characterized in that they have a negative zeta potential between -19.2 and -23.8 mV.

4. Process for preparing the nanoparticles described in any of claims 1 to 3, comprising: a) synthesizing gold nanoparticles, b) synthesizing gold nanostars from the gold nanoparticles synthesized in step a), c) coating the gold nanostars obtained in step b) with a layer of mesoporous silica, d) encapsulating curcumin in the mesoporous silica layer of step c) and coating the layer comprising the encapsulated curcumin with octadecyltrimethoxysilane, e) coating the layer obtained in d) with a thermosensitive compound selected from 1-tetradecanol, poly(N-isopropylacrylamide), DNA and heneicosane.

5. Method according to claim 4, wherein the gold nanoparticles are prepared in step a) by contacting chloroauric acid (HAuCU) and sodium citrate in water under reflux for a time between 15 and 20 min; then the gold nanoparticles are Gold is brought into contact with polyvinylpyrrolidone (PVP) and kept under stirring at room temperature for a period of 16 to 20 hours.

6. Method according to claim 4 or 5, where the gold nanostars are prepared in step b) by contacting polyvinylpyrrolidone with an aqueous solution of HAuCU and subsequent addition of the gold nanoparticles obtained in step a), keeping the mixture under stirring at room temperature for a time between 20 and 26 hours.

7. Method according to any of claims 4 to 6, wherein, to prepare the mesoporous silica coating in step c), an aqueous solution of cetyltrimethylammonium bromide (CTAB) is contacted with ethanol under an Argon atmosphere, followed by the addition of ammonia and AuNSt obtained in step b), then tetraethyl orthosilicate (TEOS) is added to the reaction mixture and the reaction mixture is maintained in an inert atmosphere for a time between 20 and 26 hours and a temperature between 23 and 28°C.

8. Method according to any of claims 4 to 7, wherein the curcumin is encapsulated by contacting the nanoparticles obtained in step c) with curcumin using acetonitrile as a solvent and keeping the reaction mixture under stirring for a time between 50 minutes to 1 hour at a temperature between 23 and 25°C; subsequently, octadecyl methoxysilane (OCT) is added to the solution to form the OCT coating, the mixture with OCT is kept for a time between 10 and 14 hours and at a temperature of 23 to 28°C.

9. Method according to any of claims 4 to 8, wherein the coating of step e) is carried out by contacting the nanoparticles obtained in step d) with the thermosensitive compound, preferably using n-hexane as solvent, and the mixture is subjected to ultrasound for a time between 40 s and 1 min, and subsequent stirring in a vortex type stirrer for a time between 10 and 30 s.

10. Composition comprising the nanoparticles described in any of claims 1 to 3.

11. Nanoparticles described in any of claims 1 to 3, or a composition comprising them described in claim 10, for use as a medicine.

12. Nanoparticles described in any of claims 1 to 3, or a composition comprising them described in claim 10, for use in the treatment and / or prevention of a bacterial infection.

13. Nanoparticles or composition for use, according to claim 12, wherein the treatment and / or prevention is carried out by the controlled release of curcumin by irradiation with light in the near IR.

14. Nanoparticles described in any of claims 1 to 3, or a composition comprising them described in claim 10, for use in antimicrobial Photodynamic Therapy (aPDT).

15. Nanoparticles or composition for use thereof, according to any of claims 12 to 14, against Staphylococcus aureus or Pseudomonas aeruginosa infections.

16. Nanoparticles or composition for use according to claim 14 or 15, wherein the aPDT comprises irradiating the nanoparticles with near-IR wavelength light to release the curcumin and, once the curcumin is released, subsequent irradiation with wavelength light between 410 and 466 in the presence of oxygen to give rise to the production of reactive oxygen species capable of causing microbial death.

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