Gold nanoparticles and ecological method of production

Abstract

A method of preparing biocompatible and stable gold nanoparticles comprises preparing at least one flavonoid-rich plant extract, and mixing at least one of the plant extracts with an aqueous solution of at least one gold salt. The flavonoid-rich plant extract is an extract of Hubertia ambavilla or Hypericum lanceolatum. The gold nanoparticles may be used for medical and/or cosmetic purposes.

Claims

1. A method of preparing biocompatible and stable gold nanoparticles, comprising: preparing at least one total crude plant extract; and mixing at least one of the total crude plant extracts with an aqueous solution of at least one gold salt in a single step, wherein the total crude plant extract is an extract of Hubertia ambavilla or Hypericum lanceolatum.

2. The method of claim 1, wherein the total crude plant extract comprises flavonoids selected from among rutin, quercetin, hyperoside and isoquercetin or a combination of at least two thereof.

3. The method of claim 1, wherein the nanoparticles have a diameter of between 5 and 100 nm.

4. The method of claim 1, wherein the nanoparticles are anisotropic and flower-shaped, and the flower-shaped gold nanoparticles are obtained by mixing a total crude plant extract with an aqueous solution of at least one gold salt.

5. The method of claim 4, wherein a metal of the flower-shaped gold nanoparticles consists of gold.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a photograph taken by transmission electron microscopy of gold nanoparticles obtained according to the method of the disclosure based on gold salts reduced by a total crude extract of Hubertia ambavilla leaves. The nanoparticles obtained have a diameter of 40 nm and are anisotropic and flower-shaped. They are monodispersed.

(2) FIG. 2 is a photograph taken by transmission electron microscopy of gold nanoparticles obtained according to the method of the disclosure based on gold salts reduced by the flavonoids of Hubertia ambavilla flowers. The nanoparticles obtained have a diameter of 15 nm and are spherical. They are monodispersed.

(3) FIG. 3 is a photograph taken by transmission electron microscopy of gold nanoparticles obtained according to the method of the disclosure based on gold salts reduced by a total crude extract of Hypericum lanceolatum flowers. The nanoparticles obtained have a diameter of 40 nm and are anisotropic and flower-shaped. They are monodispersed.

(4) FIG. 4 shows the UV spectrum for the gold nanoparticles obtained using the method according to the disclosure.

(5) FIG. 5 shows the scan of the gold nanoparticles synthesized using the method according to the disclosure.

(6) FIG. 6 shows in Panel A an optical image (light field) of a glass slide with a colloidal nanoparticle applied (certain absorbent aggregates are circled). The aggregate indicated by an arrow in Panels A and B was targeted with the laser light (9.5 mW/μm.sup.2 for 5 seconds). In Panel B, a microbubble is formed at the nanoparticle level. The laser was subsequently focused on the position identified by X (no colloids); no bubble is formed in this case.

(7) FIG. 7 shows the Raman spectra obtained during focusing on a small aggregate of nanoparticles applied to glass and surrounded by water (λexc=660 nm, P=2 mW/μm.sup.2, integration time of 5 seconds). Represented as ordinate (y) is the intensity in pulses/minute and as abscissa (x) the wave number (cm.sup.−1).

(8) FIGS. 8 to 12 are photographs taken by TEM of sections of tissue from mice having received gold nanoparticles. The nanoparticles appear in the form of black dots.

(9) FIG. 8: Panel A, liver untreated with nanoparticles; Panel B, liver treated with nanoparticles (17 mg; 24 hours).

(10) FIG. 9: Panel A, liver untreated with nanoparticles (27 mg; 24 hours); Panel B, liver treated with nanoparticles (37 mg; 24 hours).

(11) FIG. 10: Panel A, lung untreated with nanoparticles; Panel B, lung treated with nanoparticles (7 mg; 24 hours).

(12) FIG. 11: Panel A, kidney untreated with nanoparticles (7 mg; 24 hours); Panel B, kidney treated with nanoparticles (27 mg; 24 hours).

(13) FIG. 12: kidney treated with nanoparticles (37 mg; 24 hours).

DETAILED DESCRIPTION

Examples

Example 1: Preparation of the Plant Extracts of Hubertia ambavilla and Hypericum lanceolatum

(14) Two plant species are used in the present disclosure. The first is Hubertia ambavilla, which is a bush endemic to Reunion Island. The second is Hypericum lanceolatum, which is an arborescent species of St. John's wort native to Reunion Island. Both of these plants are particularly rich in flavonoids, including rutin and quercetin in the case of Hypericum lanceolatum and isoquercetin and hyperoside in the case of Hubertia ambavilla.

(15) a) Preparation of a Total Crude Plant Extract

(16) Freshly harvested plants are washed in deionized water. Three grams are mixed with 50 mL of deionized water and the mixture is subsequently heated at 60° C. for 5 minutes, which releases the biological material by lysis of plant cells. The supernatant is subsequently cooled to ambient temperature, then on ice for 10 minutes. The cooled supernatant is subsequently filtered over a grade 2 pore size filter.

(17) When the starting material is Hubertia ambavilla, the extract obtained is green.

(18) When the starting material is Hypericum lanceolatum, the extract obtained is brown.

(19) No organic solvent is used in this preparation.

(20) b) Isolation of the Plant Flavonoids

(21) The extraction method used is a cold maceration method. The plants are crushed on a screen with a pore diameter of 10 mm then left to macerate under stirring at 150 rpm for 20 hours at ambient temperature. A mixture of equal parts of water and ethanol is added to the mixture in a solid/solvent ratio of 1:20 in order to obtain the best possible yield of phenolic compounds (N. Cujic et al.). After extraction, the macerates are filtered, dried under low pressure (maximum bath temperature: 45° C., pressure between 50 and 150 bars) and are subsequently freeze-dried for 48 hours.

(22) The extraction yield for Hubertia ambavilla under these conditions is nearly 50%.

Example 2: Preparation of Gold Nanoparticles by Mixing with Hubertia ambavilla and Hypericum lanceolatum Plant Extracts

(23) a) Preparation of the Gold Nanoparticles in Flower Form with the Total Crude Extracts

(24) 50 mL of an aqueous solution of 1 mM chloroauric acid (HAuCl.sub.4) is refluxed under vigorous stirring in a two-neck round-bottom flask topped by a reflux condenser, protected from light. When fine droplets appear on the walls, 20 mL of an aqueous solution of total crude plant extracts is very quickly added. The solution then rapidly turns midnight blue within 1 minute. The round-bottom flask is subsequently removed from the oil bath and the solution is maintained under vigorous stirring for an additional 15 minutes. The solution is finally maintained at 4° C. protected from light.

(25) The diameter of the nanoparticles thus obtained is measured as described in paragraph c) below.

(26) The nanoparticles obtained have a diameter measured by TEM of approximately 40 nm.

(27) b) Preparation of the Spherical Gold Nanoparticles with the Flavonoids

(28) 4 mL of an aqueous solution of the flavonoids is refluxed under vigorous stirring in a two-neck round-bottom flask topped by a reflux condenser, protected from light. When fine droplets appear on the walls, 4 mL of an aqueous solution of HAuCl.sub.4 is very quickly added. The solution then rapidly turns red-brown within 1 minute. The round-bottom flask is subsequently removed from the oil bath and the solution is maintained under vigorous stirring for an additional 15 minutes. The solution is finally maintained at 4° C. protected from light.

(29) The diameter of the nanoparticles thus obtained is measured as described in paragraph c) below.

(30) The nanoparticles obtained have a diameter measured by TEM of approximately 15 nm.

(31) A specific molar ratio between the reagents makes it possible to obtain spherical gold nanoparticles. This ratio is as follows:
n(flavonoids)/n(HAuCl.sub.4)=21
c) Characterization of the Nanoparticles Obtained Depending on the Method of Preparation

(32) Diameter of the Nanoparticles

(33) The diameter of the nanoparticles obtained according to the methods described in paragraphs a) and b) above is measured by transmission electron microscopy (TEM), by dynamic light scattering (DLS) and by atomic force microscopy (AFM).

(34) For the TEM analysis, the electronic microscopy images were recorded on a JEOL JEM 1011 microscope operating at an acceleration voltage of 100 kV.—carbon and dried ambient temperature. For the DLS analysis, the particle size and distribution were recorded on a DLS particle size analyzer (90 Plus Particle Size Analyzer, Brookhaven Instruments Corporation). For the AFM analysis, the samples were characterized by a Molecular Imaging PICOSCAN® II with a MAC extension for “acquisition mode.”

(35) The images obtained by TEM show two types of particle: spherical and individual particles 15 nm in diameter obtained with the totum flavonoid extracts and non-spherical, flower-shaped particles obtained with the total crude plant extracts. The latter flower-shaped particles, obtained with the total crude extract of Hubertia ambavilla or Hypericum lanceolatum have a diameter included between 40 nm-80 nm. It is observed that they are formed of well-individualized particles 15 nm in diameter. The smallest are synthesized with the total of the flavonoids.

(36) The differences observed between the two syntheses suggest that the crude extract is composed of polyphenols, which undergo multidirectional polymerization in space in solution during the chelation stage, resulting in the flower structure.

(37) The data obtained by DLS display two major groups of nanoparticles. The first group consists of individual particles that can polymerize to form the second group, dimers in solution. The results obtained by DLS show an average nanometric particle size greater than that obtained with the TEM and AFM methods. These differences are generally reported in the literature (Elia, 2014). This is due to the DLS method, which measures the hydrodynamic volume by considering the procession of biomolecules (polyphenols) surrounding the particles.

(38) Processing of the image obtained by AFM yields an average size of the flower-shaped nanoparticles of around 33 nm. The individual nanoparticles have an average size of approximately 15 nm.

(39) The sizes of the different nanoparticles measured using the different methods are summarized in Table 1.

(40) AuNP@EBHA=Gold nanoparticles (AuNP) obtained using a crude extract of Hubertia ambavilla. AuNP@EBHL=AuNP obtained using a crude extract of Hypericum lanceolatum.

(41) AuNP@F2HA=AuNP obtained using a totum of flavonoids of Hubertia ambavilla.

(42) TABLE-US-00001 TABLE 1 Nanoparticle size according to the measuring method used. AuNP@EBHA AuNP@EBHL AuNP@F2HA AFM Large band: 550-590 nm Large band: Narrow band λmax.sub.SPR = 550 nm 500-900 nm λmax.sub.SPR = λmax.sub.SPR = 670 nm 530 nm TEM Flower shaped Flower shaped Spherical Size: 30-80 nm Size: 30-80 nm Size: 10-15 nm DLS Average size: 109.7 nm Average size: 82.5 nm ND Polydispersity 0.19 Polydispersity 0.28

(43) Shapes of the Nanoparticles

(44) FIGS. 1 to 3 show that gold nanoparticles were indeed obtained using the method according to the disclosure. Furthermore, when a total crude plant extract is used, the nanoparticles are anisotropic and flower-shaped, whereas when a totum of flavonoids is used, the gold nanoparticles are spherical and smaller. The nanoparticles obtained using this method are monodispersed.

(45) The UV spectra of a Hypericum lanceolatum extract, of nanoparticles obtained by reaction of the gold salts with a Hypericum lanceolatum extract and nanoparticles obtained using the conventional method of Turkevich were compared. FIG. 4 shows the spectra obtained, in a, with the Hypericum lanceolatum extracts and, in b, the nanoparticles prepared by reaction with a.

(46) Hypericum lanceolatum extract and, in c, the nanoparticles prepared by reaction according to the method of Turkevich are also shown in FIG. 4. In spectrum b, a band appears at 568 nm confirming the presence of flower-shaped, anisotropic gold nanoparticles.

(47) Contrast of the Nanoparticles Studied by Scanner

(48) Feasibility of detection by scanner (CT scan) of the nanoparticles obtained using the method according to the disclosure was verified by tomography. To this end, three samples were scanned by computer-assisted tomography: Sample no. 1: a drop of gold nanoparticles prepared from citrates according to Turkevich, Sample no. 2: the supernatant (plant extract alone), Sample no. 3: a drop of gold nanoparticles prepared from a crude extract of Hypericum lanceolatum.

(49) The scan shows a drop of nanoparticles synthesized from gold salts and a Hypericum lanceolatum extract (sample no. 3). Following ultracentrifugation, samples no. 2 and no. 1 do not exhibit any significant differences in contrast. On the other hand, sample no. 3 shows a black deposit at the bottom of the ultracentrifugation tube displaying a marked contrast (FIG. 5). The radiological contrast of this sample no. 3 at 80 kVp is 621 UH, versus 25 only for the supernatant (sample no. 2) for a concentration of 6 mg Au/Kg, i.e., 15 times less than that of Boote et al. (data not shown).

(50) Boote et al. showed that a linear relationship existed between the gold nanoparticle concentration and the radiological contrast (expressed in HU). In the tests that they performed in vitro, the gold nanoparticles of 20 nm display a radiological contrast of approximately 10 HU at 80 kVp for a concentration of 90 mg Au/Kg. Experiments conducted on mice show a majority accumulation of gold nanoparticles in the liver, with a variation in contrast of between +22.3 HU (per mg Au absorbed/1 cm.sup.3) at 80 kVp and +26.7 HU (per mg Au absorbed/1 cm.sup.3) at 140 kVp in liver tissues. The data for the spleen show a variation in HU values of between +9.7 HU (mg Au absorbed/1 cm.sup.3) at 80 kVp and +10.1 HU (mg Au absorbed/1 cm.sup.3) at 140 kVp. Another team (Chanda et al.) showed a radiological contrast of approximately 45 HU at 80 kVp for a concentration of 0.016 M [Au] (i.e., approximately 1.54 g Au/Kg).

(51) Comparison of the results obtained by other teams (given above) with those obtained with the nanoparticles according to the disclosure indicate that the radiological contrast obtained with the nanoparticles obtained using the method that is the object of the disclosure is markedly greater than that previously described. The nanoparticles of the disclosure are, therefore, particularly suitable for imaging.

(52) Yield of Synthesis

(53) The yields of synthesis of the gold nanoparticles obtained using the method of the disclosure employing a total crude plant extract or totum flavonoid extract from the same plant are given in Table 2 below:

(54) TABLE-US-00002 TABLE 2 Yield of synthesis of gold nanoparticles with the different plant extracts. Hubertia ambavilla Hypericum lanceolatum Total crude extract  5 g/L 2.5 g/L Totum of flavonoids 25 g/L  30 g/L

(55) Synthesis with the total crude extract of Hubertia ambavilla shows a better yield than synthesis with the total crude extract of Hypericum lanceolatum.

(56) The yield of synthesis of gold nanoparticles is slightly greater with the totum of flavonoids of Hypericum lanceolatum than with that of Hubertia ambavilla.

(57) The yields of synthesis of gold nanoparticles are higher with flavonoid totums than with the total crude extracts.

(58) The yields obtained with the flavonoid totums were compared with the yields obtained with a single flavonoid. The totum of flavonoids of Hubertia ambavilla predominately contains hyperoside and a small amount of isoquercetin. By using the totum of flavonoids of Hubertia ambavilla in the method according to the disclosure, spherical, stable gold nanoparticles are obtained. Conversely, reaction of gold salts with isoquercetin alone or with hyperoside alone results in synthesis of unstable gold nanoparticles and a yield of 20 g/L, less than the yield obtained with the flavonoid totum. A synergistic action, therefore, exists between the hyperoside and the isoquercetin.

(59) The totum of flavonoids of Hypericum lanceolatum predominately contains rutin and a small amount of quercetin. By using the latter in the method according to the disclosure, spherical, stable gold nanoparticles are obtained. Conversely, the reaction between gold salts and rutin alone or quercetin alone does not allow synthesis of gold nanoparticles. A synergistic action, therefore, exists between the rutin and the quercetin.

Example 3: Study of the Photothermy of the Gold Nanoparticles In Vitro

(60) The ability of the nanoparticles to emit heat under infrared irradiation was studied in vitro. The AuNP used in this experiment were obtained by mixture with a totum of flavonoids of Hubertia ambavilla.

(61) It is known that gold nanoparticles offer a high potential for treating tumors by hyperthermia. Their efficacy, however, will depend on their size, shape and surface state.

(62) With the aim of assessing the laser irradiation power of the sample around the plasmon resonance of the nanoparticles used as well as the exposure time required to achieve a sufficient temperature for photothermal treatment of the cells, generation of microbubbles under continuous illumination of the waves was studied. According to Baffou et al. (J. Phys. Chem. C 2014, 118:4890), the local temperature required to initiate generation of bubbles is approximately 220° C.

(63) Results

(64) Small aggregates of nanoparticles were visualized by optical microscopy using an 80×, 0.75 NA lens. The smallest black spots observed in the bright field are likely to correspond to aggregates about 200 nm in size (FIG. 6, Panel A). It was not possible to locate individual nanoparticles owing to the diffraction limit. After having recorded a first optical image with the laser switched off, the laser light (exc=660 nm) was focused on the aggregate for a fixed period. A second optical image was subsequently recorded after cutting the laser light off. As shown in FIG. 6, Panel B, a microbubble was formed with an exposure of 5 seconds and an irradiation of 9.5 mW/μm.sup.2. The same experiment was repeated five times on other aggregates of similar size, always resulting in formation of microbubbles for exposure periods of between 2 to 5 seconds with the same laser irradiation. Greater irradiation results in larger bubbles.

(65) Negative tests were also performed when focusing the laser light away from the aggregate of nanoparticles; no bubble is formed (position X in FIG. 6, Panel A). The bubbles are generated at NP level and are due to plasmon absorption of the NP. Furthermore, irradiation of similar aggregates with 5 mW/μm.sup.2 for 30 seconds does not produce any microbubbles. The bubble formation threshold (T˜220° C.) is included between 5 and 9.5 mW/μm.sup.2 of laser irradiation. As the threshold occurs at high temperature, it could be assumed that the temperature and, therefore, the power necessary to induce cell death ought to be lower. Laser irradiation >6 mW/μm.sup.2 was selected for the photothermal measurements previously presented on the immobilized cells. A weak Raman signal (broad bands of amorphous carbon at −1300 and −1550 cm.sup.−1) can be detected from the NP aggregates using 2 mW/μm.sup.2 for 5 seconds (FIG. 7). The signal is still present after formation of the bubble (i.e., after heating), but with a much lower background.

(66) The plasmon band of the gold nanoparticles of the disclosure is located between 600 nm and 700 nm, offset by more than 100 nm in relation to the individual nanoparticles in solution. This offset may be the result of two phenomena. The first corresponds to the change of environment during nanoparticle internalization. In this case, the nanoparticle may be covered by proteins or other biological molecules, which will cause a redshift of the plasmon resonance. The second phenomenon corresponds to aggregation of the nanoparticles in the cell medium, confirming the observations by dark field microscopy. The plasmon resonances of the nanoparticles are close to the longest irradiation wavelengths (808 nm) considered less energetic and, therefore, less destructive to the living organism.

Example 4: Study of In Vivo Biodistribution of the Gold Nanoparticles

(67) Equipment and Methods

(68) Animal models: The mice used in this experimental protocol are male Swiss mice weighing 20 g to 30 g and 6 weeks of age obtained from R. Janvier.

(69) Synthesis of gold nanoparticles: The nanoparticles were synthesized ecologically according to the methods of the present disclosure and characterized by TEM, DLS and AFM. The gold nanoparticles (AuNP) were injected into the mice intravenously in order to conduct the biodistribution study. All the AuNP were administered following centrifugation; stabilized in an aqueous solution.

(70) Description of the experimental protocol: An injection of 200 μL of the different solutions was performed via the intravenous route. 200 μL was administered for a mouse weighing approximately 24 g.

(71) The following solutions were injected according to the protocols already described by Boote et al., 2010, and Chanda et al., 2014: NaCl (6 mice) Product 1 (AuNP@EBHL=AuNP obtained using a crude extract of Hypericum lanceolatum) Group A (6 mice)=one single injection of 7 mg/ml Group B (6 mice)=one single injection of 2 mg/ml Product 2 (AuNP@EBHA_A=AuNP obtained using a crude extract of Hubertia ambavilla) Group A (6 mice)=one single injection of 7 mg/ml Group B (6 mice)=one single injection of 2 mg/ml Product 3 (AuNP@F2HA obtained using a totum of flavonoids of Hubertia ambavilla) Group A (6 mice)=one single injection of 7 mg/ml Group B (6 mice)=one single injection of 2 mg/ml

(72) That is, a total of 42 mice.

(73) These mice were subsequently separated into three groups for each concentration of each product.

(74) The first group was killed after 6 hours (three mice per product and per concentration, i.e., a total of 27 mice) and the second group was killed after 24 hours.

(75) Finally, samples of the different organs were taken (Liver, Brain, Spleen, Lung and Heart) in order to study biodistribution of the different products.

(76) Results

(77) Following injection of the AuNP via the intravenous route, the organ extracts were analyzed by TEM in order to locate the nanoparticles.

(78) The nanoparticles were found accumulated in the following organs: lungs, kidneys and liver. The results corresponding to the mice injected with product 3 are illustrated in FIGS. 8 to 12. Similar biodistribution is obtained with products 1 and 2 (data not shown).

(79) These results show that the quantity of AuNP found in the different organs is proportional to the quantity administered.

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