Mesoporous organosilica nanoparticles, production method thereof and uses of same

Abstract

The present invention relates to mesoporous organosilica nanoparticles, the method of preparation thereof, and uses of the same in treatment by means of photodynamic therapy or in imaging.

Claims

1. Mesoporous organosilica nanoparticles comprising: (i) a porphyrin derivative being a compound having the formula A ##STR00013## in which: either R.sub.1, R.sub.2, R.sub.3, and R.sub.4 all correspond to ##STR00014## where X is an oxygen atom or a sulfur atom; or R.sub.1, R.sub.2, R.sub.3, and R.sub.4 all correspond to ##STR00015## (ii) a compound having the formula I:
(EtO).sub.3Si(CH.sub.2).sub.nSi(OEt).sub.3, in which n represents an integer selected from 1 to 10, wherein the porphyrin derivative aggregates into a J aggregate and wherein the nanoparticles optionally encapsulate at least one hydrophilic and/or hydrophobic anticancer compound.

2. The nanoparticles according to claim 1, wherein the hydrophilic anticancer compound is selected from the group consisting of gemcitabine, gemcitabine monophosphate, 5-fluorouracil, cytarabine, topotecane, irinotecane, and oxalylplatin; wherein the hydrophobic anticancer compound is selected from the group consisting of doxorubicin, paclitaxel, and camptothecin.

3. The nanoparticles according to claim 1, formed by the elements comprising: (i) a porphyrin derivative having the formula here below: ##STR00016## and (ii) a compound of formula I, as defined according to claim 1.

4. The nanoparticles according to claim 1, comprising: (i) a porphyrin derivative having the formula here below: ##STR00017## and (ii) a compound having the formula I as defined in claim 1, wherein the nanoparticles encapsulate gemcitabine or gemcitabine monophosphate.

5. The nanoparticles according to claim 1, comprising: (i) a porphyrin derivative having the formula here below: ##STR00018## (ii) a compound having the formula I as defined according to claim 1, and (iii) a compound having the formula II
(EtO).sub.3Si(CH.sub.2).sub.3—(SS).sub.m—(CH.sub.2).sub.3—Si(OEt).sub.3 in which m is an integer that is equal to 2 or 4.

6. The nanoparticles according to claim 1, comprising: (i) a porphyrin derivative having the formula: ##STR00019## (ii) a compound having the formula I as defined according to claim 1, and (iii) a compound having the formula II
(EtO).sub.3Si(CH.sub.2).sub.3—(SS).sub.m—(CH.sub.2).sub.3—Si(OEt).sub.3 in which m is an integer that is equal to 2 or 4, wherein the nanoparticles encapsulate gemcitabine or gemcitabine monophosphate.

7. The nanoparticles according to claim 1, wherein the molar ratio between the porphyrin derivative and the compound having the formula I is between 2:98 and 20:80.

8. The nanoparticles according to claim 1, wherein the load of hydrophilic or hydrophobic anticancer compound as defined in claim 1 is from 2% to 100% by weight relative to the initial weight of the nanoparticles prior to the encapsulation of the said anticancer compound.

9. The nanoparticles according to claim 1, whereof the diameter of particles is from 20 to 400 nm and the specific area is from 100 to 1500 m.sup.2/g.

10. A method of treating cancers, tumors, cell proliferative disorders and diseases, or skin conditions and diseases comprising administering to a subject the nanoparticles according to claim 1.

11. A method of detecting or monitoring a cancer, a tumor, a cell proliferative disorder, a cell proliferative disease, a skin condition or a skin disease in a subject comprising: administering to the subject the nanoparticles according to claim 1 as a luminescent agent or a fluorescent agent; irradiating the nanoparticles with mono-photon or bi-photon irradiation; and detecting visible light or fluorescence emitted from the nanoparticles in order to detect or monitor the cancer, tumor, cell proliferative disorder, cell proliferative disease, skin condition or skin disease in the subject.

12. A method of photosensitizing a cell comprising administering to the cell the nanoparticles according to claim 1 as a photosensitizing agent.

13. A pharmaceutical composition comprising the nanoparticles as claimed in claim 1 and a pharmaceutically acceptable carrier.

14. A nanoparticle preparation method for preparing the nanoparticles according to claim 1, the said method comprises the steps of: (a) reacting in a basic aqueous solution at a temperature of 50° C. to 90° C. in the presence of a surfactant, the compounds comprising: (i) a porphyrin being a compound having the formula A ##STR00020## in which: either R.sub.1, R.sub.2, R.sub.3, and R.sub.4 all correspond to ##STR00021## where X is an oxygen atom or a sulfur atom; or R.sub.1, R.sub.2, R.sub.3, and R.sub.4 all correspond to ##STR00022## (ii) a compound having the formula I here below:
(EtO).sub.3Si(CH.sub.2).sub.nSi(OEt).sub.3, in which n represents an integer selected from 1 to 10, and (iii) optionally a compound having the formula II
(EtO).sub.3Si(CH.sub.2).sub.3—(SS).sub.m—(CH.sub.2).sub.3—Si(OEt).sub.3, in which m is an integer that is equal to 2 or 4, and (b) recovering the nanoparticles formed in the preceding step, and optionally: (c) reacting in a solvent, the nanoparticles obtained in the step (b) with at least one hydrophilic and/or hydrophobic anticancer compound in order to encapsulate the latter, and (d) recovering the nanoparticles obtained at the end of the step (c).

15. Mesoporous nanoparticles obtained by the method according to claim 14.

16. A detection kit for the detection of a pathology selected from the group consisting of cancers, tumors and cell proliferative disorders and diseases, the kit comprising: the nanoparticles according to claim 1 or a medicine composition comprising the same; and a light source configured to provide LED, blue to near IR laser irradiation.

17. The nanoparticles according to claim 1, further comprising: (iii) a compound having the formula II
(EtO).sub.3Si(CH.sub.2).sub.3—(SS).sub.m—(CH.sub.2).sub.3—Si(OEt).sub.3 in which m is an integer that is equal to 2 or 4.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Graph representing the UV-VIS spectrum of a solution of CM238 nanoparticles obtained after being put in suspension in ethanol.

(2) FIG. 2: Two-photon confocal microscopy imaging of MCF-7 cancer cells surviving after 20 hours of incubation with CM238 nanoparticles. At left: fluorescence of the membrane marker; in the middle: fluorescence emitted by the living cells at 750 nm; at right: superposition of the two images of fluorescence illustrated in the figures on the left and in the middle.

(3) FIG. 3: Percentage of surviving MCF-7 cells before (black bar) and after (white bar) excitation at 800 nm by 3 scans of 1.57 s at maximum power by making use of a microscope and a two-photon laser, after 20 hours of incubation in a control culture medium (“Control”) or with 80 μg/ml of CM238 nanoparticles.

(4) FIG. 4: Profile of the release, in water, of the encapsulated gemcitabine, from the “CM238+gemcitabine” Nanoparticles. The arrow signifies the addition of HCl. The abscissa or x-axis represents the time in minutes. The ordinate or y-axis represents the gemcitabine released.

(5) FIG. 5: Cytotoxicity of the “CM238+gemcitabine” nanoparticles on MCF-7 cells in the absence of excitation. The abscissa axis represents the concentration of nanoparticles expressed in μg/mL. The ordinate axis represents the percentage of surviving cells as determined by the MTT (3-(4, 5-dimethytthiazolyl-2)-2, 5-diphenytetrazolium bromide) assay.

(6) FIG. 6: Percentage of MCF-7 cells surviving before (light gray bar) and after (dark gray bar) excitation at 800 nm by 3 scans at maximum power by making use of two-photon microscopy, after 20 hours of incubation in a control culture medium (“Control”) or with 40 μg/ml of “CM238+gemcitabine” nanoparticles.

(7) FIG. 7: Graph representing the UV-VIS spectrum of a solution of CM 240 nanoparticles obtained after its dispersion in ethanol.

(8) FIG. 8: Percentage of MCF-7 cells surviving before (light gray bar) and after excitation (dark gray bar) at 800 nm by 3 scans at maximum power using two-photon microscopy, after 20 hours of incubation in a control culture medium (“Control”) or with 40 μg/ml or 80 μg/ml of CM240 nanoparticles.

(9) FIG. 9: Two-photon confocal microscopy imaging of surviving MCF-7 cancer cells after 20 hours of incubation in a control culture medium (top) or with 80 μg/ml of CM240 nanoparticles (bottom). Column A: fluorescence of the membrane marker; Column B: fluorescence emitted by the CM240 nanoparticles in the living cells at 800 nm; Column C: superposition of the two fluorescence images illustrated in the figures of columns A and B.

(10) FIG. 10: Cytotoxicity of the CM240 nanoparticles on MCF-7 cells in the absence of excitation. The abscissa axis represents the concentration of nanoparticles expressed in μg/ml. The ordinate axis represents the percentage of surviving cells as determined by the MTT assay.

(11) FIG. 11: Percentage of MCF-7 cells surviving before (white bar) and after (black bar) one-photon laser excitation with 10 mW power at 405 nm for a period of 10 min after 20 hours of incubation in a control culture medium (“Control”) or with 80 μg/ml of CM238 nanoparticles.

(12) FIG. 12: Detection of ROS (Reactive Oxygen Species) from MCF-7 cells incubated for a period of 24 hours with 80 μg/ml of CM 238 nanoparticles.

(13) FIG. 13: Detection of ROS from MCF-7 cells incubated for a period of 24 hours with 80 μg/ml of CM 240 nanoparticles.

(14) FIG. 14: Profile of the release, in water, of the encapsulated gemcitabine, from the “CM240-b+gemcitabine” nanoparticles. The arrow signifies the addition of HCl. The abscissa axis represents the time in minutes. The ordinate axis represents the gemcitabine released.

(15) FIG. 15: Graph representing the UV-VIS spectrum of a solution of PMOS1 nanoparticles obtained after being put in suspension in ethanol.

(16) FIG. 16: Two-photon confocal microscopy imaging of MCF-7 cancer cells surviving after 20 hours of incubation in a control culture medium (top) or with 80 μg/ml of PMOS1 nanoparticles (bottom). Column A: fluorescence of the nuclei; Column B: fluorescence of the membrane marker; Column C: fluorescence emitted by the PMOS1 nanoparticles in the living cells at 850 nm; Column D: superposition of the two fluorescence images illustrated in the figures of columns A, B and C.

(17) FIG. 17: Percentage of MCF-7 cells surviving before (white bar) and after (black bar) one-photon laser excitation with 10 mW power at 405 nm for a period of 10 min after 20 hours of incubation in a control culture medium (“Control”) or with 80 μg/ml of PMOS1 nanoparticles.

(18) FIG. 18: Profile of the release, in water, of the encapsulated gemcitabine, from the “PMOS1+gemcitabine” nanoparticles. The arrow signifies the addition of HCl. The abscissa axis represents the time in minutes. The ordinate axis represents the gemcitabine released.

DESCRIPTION

Examples

(19) 1. Materials and Methods

(20) 1.1. Preparation of the CM238 Nanoparticles

(21) 250 mg of cetyitrimethylammonium bromide and 875 μL of NaOH (2M) are introduced into 120 mL of ultrapure water. The mixture is agitated at 750 revolutions per minute (rpm) for a period of 50 minutes at 80° C. The porphyrin having the formula A1a (in 1 ml of absolute ethanol) is introduced simultaneously in reaction with bis(triethoxysilyl)ethane (10/90 by weight). The reaction is maintained for a period of 1 hour 45 minutes at 80° C. Then the CM 238 nanoparticles obtained are centrifuged. The surfactant is extracted with an ethanolic solution of ammonium nitrate (6 g/L). The nanoparticles are put in suspension in this solution (50 ml) for a period of 30 minutes under ultrasound at 50° C., and centrifuged at 20000 rpm for a period of 20 minutes. The protocol is repeated three times. Three washes with ethanol are then carried out. The nanoparticles are dried under vacuum.

(22) 1.2 Preparation of the CM240 Nanoparticles

(23) 2M NaOH (875 μl) and the cetyitrimethylammonium bromide (250 mg) are mixed in 120 ml of water at 80° C. for a period of 120 minutes. The porphyrin having the formula A1a (1.40×10.sup.−2 mmol, 23.8 mg diluted in 1 mL of EtOH), bis(triethoxysilyl)ethane (1.78 mmol) and bis triethoxysilylpropyl disulfide (1.3 mmol) (ratio 1/55/44 in moles) are then added. The reaction is maintained for a period of 2 hours at 80° C., at 750 rpm. The nanoparticles are thereafter centrifuged at 20,000 rpm for a period of 15 minutes. The surfactant is extracted with an ethanoic solution of ammonium nitrate (6 g/L). The nanoparticles are put in suspension in this solution (50 ml) for a period of 30 minutes under ultrasound at 50° C., and centrifuged. The protocol is repeated three times. The nanoparticles are dried under vacuum.

(24) 1.3 Preparation of the CM240-b Nanoparticles

(25) 2M NaOH (875 μl) and the cetyitrimethylammonium bromide (250 mg) are mixed in 120 ml of water at 80° C. for a period of 120 minutes. The porphyrin having the formula A1a (1.40×10−2 mmol, 23.8 mg diluted in 1 mL of EtOH), bis(triethoxysilyl)ethane (1.78 mmol) and bis triethoxysilylpropyl disulfide (0.3 mmol) (ratio 1/83/16 in moles) are then added. The reaction is maintained for a period of 2 hours at 80° C. at 750 rpm. The nanoparticles are thereafter centrifuged at 20,000 rpm for a period of 15 minutes. The surfactant is extracted with an ethanolic solution of ammonium nitrate (6 g/L). The nanoparticles are put in suspension in this solution (50 ml) for a period of 30 minutes under ultrasound at 50° C., and centrifuged. The protocol is repeated three times. The nanoparticles are dried under vacuum.

(26) 1.4. Preparation of the PMOS1 Nanoparticles

(27) 2M NaOH (437 μl) and cetyttrimethylammonium bromide (125 mg) are mixed in 60 ml of water at 80° C. for a period of 120 minutes. The porphyrin having the formula B (1.3×10 −2 mmol, 12 mg diluted in 1 mL of EtOH) and bis(triethoxysilyl)ethane (0.89 mmol) are then added. The reaction is maintained for a period of 2 hours at 80° C. at 750 rpm. The nanoparticles are thereafter centrifuged at 20,000 rpm for a period of 15 minutes. The surfactant is extracted with an ethanoic solution of ammonium nitrate (6 g/L). The nanoparticles are put in suspension in this solution (50 ml) for a period of 30 minutes under ultrasound at 50° C., and centrifuged. The protocol is repeated three times. The nanoparticles are dried under vacuum.

(28) 1.5. Encapsulation of Gemcitabine

(29) 1.6 mg of CM238 nanoparticles are put in suspension with 1.9 mg of gemcitabine in 2 mL of water (pH=7.4) for a period of 24 hours. The nanoparticles are thereafter centrifuged and washed 4 times with water and dried under vacuum. The supernatants are collected in order to determine the quantity of medicament encapsulated in the nanoparticles.

(30) 1.6. UV-Vis Spectrum of the Nanoparticles

(31) 1 mg of nanoparticles are dispersed in 1 mL of EtOH. The UV-Vis spectrum of the nanoparticles is observed by using a UV-Vis spectrometer.

(32) 1.7. Two-Photon Confocal Microscopy Imaging of Cells

(33) The MCF-7 breast cancer cells are incubated for a period of 20 hours with the nanoparticles. 15 minutes prior to imaging, the membranes of the cells are stained with a dye. The nanoparticles are observed at 750 nm with a two-photon confocal microscope and a low power laser (5% of the total power (3 W) delivered by the Chameleon femtosecond pulsed laser).

(34) 1.8. Release Kinetics of the Encapsulated Gemcitabine

(35) The “NPs+gemcitabine” nanoparticles are introduced at the bottom of a UV tank and thereafter followed by addition of an aqueous solution at pH 7.4 without agitation. The concentration of gemcitabine released into the solution is measured after a period of 10, 20, 30 minutes. HCl is added into the solution after a period of 50 minutes. The concentration of gemcitabine released into the solution is then measured at 60, 90, 120, 130 and 140 minutes.

(36) 1.9. Cytotoxicity of the Nanoparticles

(37) The MCF-7 cancer cells are incubated for a period of 20 hours with different concentrations of the nanoparticles. The cytotoxicity of the nanoparticles is measured without or after excitation. The excitation is carried out by means of Zeiss LSM 780 confocal microscope (×10 lens) at 800 nm.

(38) The quantification of living cells is obtained by means of the MTT (3-(4, 5-dimethytthiazolyl-2)-2, 5-diphenytetrazolium bromide) assay (cell survival test) after 48 hours of irradiation. The MTT assay is carried out in accordance with a conventional protocol (Mosmann, Journal of Immunological Methods, 1983, 65 (1-2): 55-63).

(39) 1.10 Determination of ROS Production

(40) The generation of ROS (Reactive Oxygen Species) is examined in cells by using the DCFDA (dichlorodihydrofluorescein diacetate) kit. In contact with ROS species, the non-fluorescent DCFDA is oxidised into fluorescent dihydrofluorescein (DCF).

(41) Prior to the two-photon irradiation, the DCFDA was therefore incubated for a period of 45 minutes with the cells after endocytosis of the nanoparticles. The experiment reveals no significant fluorescence without irradiation while a high fluorescent signal is detected during the irradiation of the cells, which confirmed the production of ROS with two photon excitation (TPE). The intensity of the fluorescence is proportional to the quantity of ROS generated (detection at 535 nm).

(42) 2. Results

(43) 2.1. Analysis of the CM238 Nanoparticles

(44) The CM 238 nanoparticles obtained have a very high specific surface area (832 m.sup.2 g.sup.−1) and a pore size of 3 nm. They are monodisperse with a diameter of 200-250 nm. They disperse in water or ethanol. The porphyrins in these nanoparticles aggregate into J aggregates with a shift of the UV-Vis spectrum towards red (red-shifted) (FIG. 1).

(45) These particles may be used for the two-photon imaging of cancer cells. The MCF-7 breast cancer cells are incubated for a period of 20 hours with the CM238 nanoparticles and then observed at 750 nm with a confocal microscope and low power laser (5% of the total power (3 W) delivered by the Chameleon femtosecond pulsed laser). This experiment shows that the nanoparticles are internalised within the MCF-7 cells (FIG. 2).

(46) After 20 hours of incubation of the MCF-7 cancer cells with 80 μg/ml of CM238 nanoparticles, the cells are irradiated with 3 scans of 1.57 s at 800 nm by two-photon confocal microscopy. About 27% of the cancer cells are destroyed after irradiation (FIG. 3). Under the same conditions of incubation, the cells are irradiated for a period of 10 minutes by means of a one-photon laser at 405 nm and a power measuring 10 mW. 71% of the cells are destroyed (FIG. 11).

(47) The production of ROS is consistent with two-photon irradiation and is proportional to the intensity of fluorescence (FIG. 12).

(48) 2.2. Analysis of the “CM238+Gemcitabine” Nanoparticles

(49) The CM238 basic nanoparticles, the matrix of which is formed by a porphyrin derivative having the formula A1a and bis(triethoxysilyl)ethane, are obtained according to the method described in section 1.1.

(50) The gemcitabine is encapsulated within the CM238 nanoparticles according to the method described in section 1.2.

(51) The gemcitabine load by weight relative to the weight of the basic nanoparticles is 50%. This signifies that gemcitabine is encapsulated effectively within the basic nanoparticles.

(52) The delivery of gemcitabine is sensitive to pH. At pH 7.4, the nanoparticles in suspension do not release gemcitabine, whereas at pH 5.5 (pH of the cancer cells), there is a significant delivery of gemcitabine (FIG. 4).

(53) The cytotoxicity of the “CM238+gemcitabine” nanoparticles at various different concentrations without excitation was tested on MCF-7 cell cultures. After three days of incubation, up to 40% of the cells are destroyed (FIG. 5). These results show that gemcitabine is delivered efficiently in cancer cells.

(54) When the “CM238+gemcitabine” nanoparticles are incubated at a concentration of 40 μg.Math.mL.sup.−1 for a period of 20 hours with the MCF-7 breast cancer cells, after irradiation at 800 nm with 3 scans at maximum power, 62% of the cells are destroyed due to dual treatment with gemcitabine and photodynamic therapy (FIG. 6).

(55) 2.3. Analysis of the CM240 Nanoparticles

(56) The CM 240 nanoparticles obtained have a very high specific surface area (950 m.sup.2 g.sup.−1) and a pore size of 2.2 nm. They are monodisperse. They disperse in water or ethanol. The porphyrins in these nanoparticles aggregate into J aggregates with a shift of the UV-Vis spectrum towards red (red-shifted) (FIG. 7).

(57) At 80 μg/ml, after 20 hours of incubation with MCF-7 cancer cells, the CM240 nanoparticles after excitation at 800 nm by means of two-photon confocal microscopy using the Zeiss LSM 780 microscope (×10 lens), are able to destroy 53% of the cancer cells (FIG. 8). On the other hand, at 40 μg/ml, the CM240 nanoparticles do not show a significant cytotoxicity.

(58) The surviving cells after 20 hours of incubation with 80 μg/ml of CM240 nanoparticles are observed by means of Zeiss LSM 780 two-photon confocal microscope (×63 lens). It is observed that the nanoparticles have entered into the cells (FIG. 9).

(59) Furthermore, when MCF-7 cells are treated with increasing concentrations of CM240, in the absence of excitation, the CM240 nanoparticles are not toxic to the cells (FIG. 10).

(60) The production of ROS is consistent with two-photon irradiation and is proportional to the intensity of fluorescence (FIG. 13).

(61) 2.4. Analysis of the “CM240-b+Gemcitabine” Nanoparticles

(62) The basic CM240-b nanoparticles whose matrix is formed by a porphyrin derivative having the formula A1a and bis(triethoxysilyl)ethane as well as bis triethoxysilylpropyl disulfide are obtained according to the method described in section 1.3.

(63) The gemcitabine is encapsulated within the CM240-b nanoparticles according to the method described in section 1.5. The gemcitabine load by weight relative to the weight of the basic nanoparticles is 98%. This signifies that gemcitabine is encapsulated effectively within the basic nanoparticles.

(64) The delivery of gemcitabine is sensitive to pH. At pH 7.4, as well as at pH 5.5 (pH of the cancer cells), the nanoparticles in suspension do not release gemcitabine (FIG. 14).

(65) 2.5. Analysis of the PMOS1 Nanoparticles

(66) The PMOS1 nanoparticles obtained have a very high specific surface area (892 m2 g-1) and a pore size of 3 nm. They are monodisperse with an average diameter of 305 nm. They disperse in water or ethanol.

(67) The porphyrins in these nanoparticles show no shift in the UV-Vis spectrum as compared to the porphyrin derivative thereof having the formula B (FIG. 15). These particles may thus be used for one-photon imaging of cancer cells. The MCF-7 breast cancer cells are incubated for a period of 20 hours with the PMOS1 nanoparticles and then observed at 850 nm.

(68) This experiment shows that the nanoparticles are internalised within the MCF-7 cells (FIG. 16).

(69) After 20 hours of incubation of the MCF-7 cancer cells with 80 μg/ml of PMOS1 nanoparticles, the cells are irradiated at 405 nm by means of a one-photon laser with a power measuring 10 mW. About 80% of the cancer cells are destroyed after irradiation (FIG. 17).

(70) 2.6. Analysis of the “PMOs1+Gemcitabine” Nanoparticles

(71) The PMOS1 basic nanoparticles whose matrix is formed by a porphyrin derivative having the formula B and bis(triethoxysilyl)ethane are obtained according to the method described in section 1.4.

(72) The gemcitabine is encapsulated within the PMOS1 nanoparticles according to the method described in section 1.5. The gemcitabine load by weight relative to the weight of the basic nanoparticles is 72%. This signifies that gemcitabine is encapsulated effectively within the basic nanoparticles.

(73) The delivery of gemcitabine is sensitive to pH. At pH 7.4, the nanoparticles in suspension do not release gemcitabine, and for the most part not even at pH 5.5 (pH of the cancer cells), where only 0.5% is released (FIG. 18).