Functionalized porous silicon nanoparticles and use thereof in photodynamic therapy

Abstract

Disclosed are nanovectors of formula (I) that can be used simultaneously for the targeting, imaging and treatment, by photodynamic therapy, of cancer cells, and to biodegradable silicon nanoparticles containing a variety of photosensitizing molecules, in particular porphyrins, capable of targeting diseased cells and inducing cell death by excitation in the near-infrared region (>600 nm) in monophotonic and biphotonic modes. In formula (I), (AA) is a porous silicon nanoparticle.

Claims

1. Nanoparticles corresponding to formula (I): ##STR00016## in which: ##STR00017## represents a porous silicon nanoparticle, x represents 0 or 1, M represents a transition metal atom, X represents a halide or an anion of a pharmaceutically acceptable carboxylic acid, R represents a urea (NHCONH) or a thiourea (NHCSNH), W represents a C1-C12 alkanediyl group, R represents: ##STR00018## Z.sup.+ represents a pharmaceutically acceptable organic or mineral cation, Y.sup. represents COO.sup. or SO.sub.3.sup., A.sup. represents a halide or an anion of a pharmaceutically acceptable carboxylic acid, and R1 represents a C1 to C10 alkyl group.

2. The nanoparticles according to claim 1 corresponding to formula (Ia): ##STR00019## wherein Cbg represents a specific targeting molecule for neoplastic tissues.

3. The nanoparticles according to claim 2, the size of which is from 20 to 200 nm.

4. The nanoparticles according to claim 2, in which x represents 0.

5. The nanoparticles according to claim 2, in which: X represents a group selected from the group consisting of: Cl.sup., Br.sup., I.sup., acetate, propionate, butyrate, ascorbate, benzoate, cinnamate, citrate, fumarate, glycolate, malonate, tartrate, malate, maleate, mandelate, and tosylate, W represents a (CH.sub.2).sub.3 group, R is: ##STR00020## and Cbg is selected from the group consisting of folic acid, peptides, carbohydrates and antibodies.

6. A method for producing nanoparticles according to claim 2, said method comprising the steps of: (i) providing porous silicon nanoparticles, (ii) functionalizing the porous silicon nanoparticles with groups comprising at least one C1-C12 NH.sub.2 function or at least one C1-C12 isocyanate or C1-C12 isothiocyanate, (iii) providing and grafting a porphyrin-type photosensitizing molecule corresponding to formula (II), ##STR00021## in which: x represents 0 or 1, M represents a transition metal atom, X represents a halide or an anion of a pharmaceutically acceptable carboxylic acid, Q represents a group selected from the group consisting of NH.sub.2, NCO, and NCS, R represents: ##STR00022## Z.sup.+ represents a pharmaceutically acceptable organic or mineral cation, Y.sup. represents COO.sup. or SO.sub.3.sup., A.sup. represents a halide or an anion of a pharmaceutically acceptable carboxylic acid, and R1 represents a C1 to C10 alkyl group, and optionally (iv) grafting with at least one targeting molecule.

7. The nanoparticles according to claim 1, the size of which is from 20 to 200 nm.

8. The nanoparticles according to claim 7, in which x represents 0.

9. The nanoparticles according to claim 7, in which: X represents a group selected from the group consisting of: Cl.sup., Br.sup., I.sup., acetate, propionate, butyrate, ascorbate, benzoate, cinnamate, citrate, fumarate, glycolate, malonate, tartrate, malate, maleate, mandelate, and tosylate, W represents a (CH.sub.2).sub.3 group, R is: ##STR00023##

10. The nanoparticles according to claim 1, in which x represents 0.

11. The nanoparticles according to claim 10, in which: X represents a group selected from the group consisting of: Cl.sup., Br.sup., I.sup., acetate, propionate, butyrate, ascorbate, benzoate, cinnamate, citrate, fumarate, glycolate, malonate, tartrate, malate, maleate, mandelate, and tosylate, W represents a (CH.sub.2).sub.3 group, R is: ##STR00024##

12. The nanoparticles according to claim 1, in which: X represents a group selected from the group consisting of: Cl.sup., Br.sup., I.sup., acetate, propionate, butyrate, ascorbate, benzoate, cinnamate, citrate, fumarate, glycolate, malonate, tartrate, malate, maleate, mandelate, and tosylate, W represents a (CH.sub.2).sub.3 group, and R is: ##STR00025##

13. The nanoparticles according to claim 12, wherein the nanoparticles have a structure according to one of: ##STR00026##

14. A method for producing nanoparticles according to claim 1, said method comprising the steps of: (i) providing porous silicon nanoparticles, (ii) functionalizing the porous silicon nanoparticles with groups comprising at least one C1-C12 NH.sub.2 function or at least one C1-C12 isocyanate or C1-C12 isothiocyanate, and (iii) providing and grafting a porphyrin-type photosensitizing molecule corresponding to formula (II), ##STR00027## in which: x represents 0 or 1, M represents a transition metal atom, X represents a halide or an anion of a pharmaceutically acceptable carboxylic acid, Q represents a group selected from the group consisting of NH.sub.2, NCO, and NCS, R represents: ##STR00028## Z.sup.+ represents a pharmaceutically acceptable organic or mineral cation, Y.sup. represents COO.sup. or SO.sub.3.sup., A.sup. represents a halide or an anion of a pharmaceutically acceptable carboxylic acid, and R1 represents a C1 to C10 alkyl group.

15. The method according to claim 14, which comprises the steps of: (i) aelectrochemical etching of monocrystalline silicon plates in a hydrofluoric (HF) ethanol solution, bremoval of the porous film and treatment by ultrasound, (ii) acontrolled oxidation followed by silanization so as to produce SiOH groups and SiO.sub.2 species on the surface of the porous silicon nanoparticles, and treatment by an aminoalkylsilanyl group, and/or bhydrosilylation with a C1-C12 allylamine, C1-C12 allyl isocyanate or C1-C12 allyl isothiocyanate, and (iii) grafting the porous nanoparticles of step (ii) with a porphyrin-type photosensitizing molecule corresponding to formula (II).

16. A medicinal composition comprising nanoparticles according to claim 1 in a pharmaceutically acceptable support.

17. A cosmetic composition comprising nanoparticles according to claim 1 in a cosmetically acceptable support.

18. A kit for the detection, treatment, monitoring, prevention, and delay of the appearance and/or recurrence of a pathology selected from the group consisting of cancers, tumors, and cell proliferation diseases, comprising: nanoparticles according to claim 1, and means that allow an irradiation in the infrared.

Description

FIGURES

(1) FIG. 1: Diagrammatic representation of the preparation of silicon nanoparticles by electrochemical anodization of crystalline silicon, electrodissolution, and ultrasonic fracturing.

(2) FIG. 2: SEM imaging photograph of a porous silicon film, precursor of the nanoparticles, on the surface (2A) and in cross section (2B).

(3) FIG. 3: SEM (3A) and TEM (3B) imaging photograph of the porous silicon nanoparticles prepared by ultrasonic fracturing and shown in FIG. 1.

(4) FIG. 4: Graph showing the light scattering intensity in kcps as a function of the size of the nanoparticles (hydrodynamic diameter measured in nm).

(5) FIG. 5: Graph showing the nitrogen adsorption and desorption isotherm for the porous silicon nanoparticles etched at 200 mA/cm.sup.2. The adsorbed quantity (volume adsorbed in mL STP/g) is shown as a function of the relative pressure P/P.sub.0.

(6) FIG. 6: Diagrammatic representation of two synthesis routes that make it possible to graft functional groups onto porous silicon nanoparticles.

(7) FIG. 7: Diagrammatic representation of the coupling of an amine porphyrin on a porous silicon nanoparticle functionalized by an allyl isocyanate group.

(8) FIG. 8: Diagrammatic representation of the coupling of a porphyrin functionalized by an isocyanate on a porous silicon nanoparticle functionalized by an allylamine group.

(9) FIG. 9: Diagrammatic representation of the coupling of a mannose group on a porous silicon nanoparticle via a squarate group.

(10) FIG. 10A: Graph showing the FTIR spectrum (wavenumber in cm.sup.1) of the porous silicon nanoparticles not grafted (solid) and hydrosilylated with allylamine (dotted). FIG. 10B: Graph showing the FTIR spectrum (wavenumber in cm.sup.1) of porous silicon nanoparticles at different stages of the coupling of a mannose-squarate: after hydrosilylation by an allylamine (solid), after coupling with mannose (dotted).

(11) FIG. 11: Graph showing the UV-VIS spectrum of a solution obtained after dissolution, in KOH, of the porous silicon nanoparticles grafted with porphyrin-NCS, with 74 g porphyrin/mg nanoparticles, i.e. a loading of 7.4% by mass (absorbance Log I/I0 as a function of the wavelength in nm).

(12) FIG. 12: Graph showing the FTIR spectrum (wavenumber in cm.sup.1) of porous silicon nanoparticles at different stages of the coupling of a porphyrin-NCS: after hydrosilylation by an allylamine (solid), after coupling with porphyrin-NCS (dotted).

(13) FIG. 13: Graph showing the FTIR spectrum (wavenumber in cm.sup.1) of porous silicon nanoparticles grafted with a porphyrin-NH.sub.2 by the allyl isocyanate route.

(14) FIG. 14A: Graph showing the FTIR spectrum (wavenumber in cm.sup.1) of porous silicon nanoparticles at different stages of the simultaneous coupling of a mannose-ketone and a porphyrin-NCS: (a) porous silicon grafted with semicarbazide, (b) after hydrosilylation by an allylamine, (c) after coupling with porphyrin-NCS.

(15) FIG. 14B: Graph showing the UV-VIS spectrum of a solution obtained after dissolution, in KOH, of the porous silicon nanoparticles grafted with porphyrin-NCS and with semicarbazide, with 10 g porphyrin/mg nanoparticles (absorbance Log I/I0 as a function of the wavelength in nm).

(16) FIG. 15: Graph showing the produced quantity of singlet oxygen as a function of the wavelength of irradiation for nanoparticles without porphyrin (intensity expressed in arbitrary units: phosphorescence of the singlet oxygen as a function of the wavelength in nm).

(17) FIG. 16: Graph showing the UV-VIS spectrum of the porous silicon nanoparticles grafted with a porphyrin and with mannose (absorbance in Log I/I0 as a function of the wavelength in nm).

(18) FIG. 17: Graph showing the produced quantity of singlet oxygen as a function of the wavelength of irradiation for porous silicon nanoparticles grafted with a porphyrin and with mannose (intensity expressed in arbitrary units: phosphorescence of the singlet oxygen as a function of the wavelength in nm).

(19) FIG. 18: Confocal microscopy images of living MCF-7 breast cancer cells. The cells were incubated for 5 h with 20 pg ml1-pSiNP Porph-NH.sub.2 nanoparticles, 0.25 g ml1 Porph-NH.sub.2, or a vehicle (ethanol) and co-coloured with a membrane marker (cell mask). The cell membranes were visualized in green at 561 nm, the nanoparticles and the porphyrins were visualized at 633 nm and the images have been merged. The photos represent at least 3 independent experiments. The intracellular aggregates of pSiNP-Porph-NH.sub.2 are identified by solid arrows and the aggregates of the cell membrane, which look yellow in the merged image, are labelled with dotted arrows. The scale bars are at 5 m.

(20) Left-hand column: membrane, middle column: compounds, right-hand column: merged.

(21) First row: control, second row: porphyrin-NH.sub.2, third row: porous silicon nanoparticles grafted with porphyrin-NH.sub.2.

(22) FIG. 19: Graph showing the % of living MCF7 cells as a function of the treatment which has been administered to them. Efficacy of the in vitro PDT in biphotonic excitation mode on the MCF-7 cells incubated with: control culture medium containing 4% ethanol (EtOH, control experiment), free porphyrin-NCS (porph-NCS), non-grafted porous silicon nanoparticles (Si), porous silicon nanoparticles grafted with mannose (Si-m), porous silicon nanoparticles grafted with porphyrin-NCS (Si-p) and porous silicon nanoparticles grafted with porphyrin and with mannose (Si-m-p). Irradiation at 750 nm, 3 scans of 1.57 s. The living cells are counted by an MTS assay 2 days after irradiation.

(23) FIG. 20: Diagrammatic representation of the preparation of the nanoparticles grafted with a porphyrin.

(24) FIG. 21: X-ray diffractograms of the porous silicon nanoparticles: FIG. 21A at wide angles, FIG. 21B at narrow angles. The relative intensity (u.a.) is shown as a function of 2 (in degrees)

(25) FIG. 22A: Diagrammatic representation of the coupling of a semicarbazide group on a porous silicon nanoparticle by hydrosilylation.

(26) FIG. 22B: Diagrammatic representation of the coupling of a mannose group on a porous silicon nanoparticle via a semicarbazone function.

(27) FIG. 23: Curves plotting ln(A) as a function of the illumination time (minutes) for methylene blue (diamond), porphyrin-NCS (triangle) and porous silicon nanoparticles grafted with porphyrin-NCS (square).

(28) FIG. 24: Curves plotting the absorbance at 411 nm ln(A) as a function of the illumination time (minutes) for DPBF on its own (square), and for porous silicon nanoparticles grafted with porphyrin-NH.sub.2+DPBF (diamond).

(29) FIG. 25: Kinetics of the release of porphyrin-NCS from the porous silicon nanoparticles grafted with porphyrin-NCS incubated at 37 C. in different media. Diamond: in PBS at pH=7.4, square: in DMEM culture medium with 10% FBS, triangle: in DMEM culture medium without serum.

(30) FIG. 26: Release kinetics of the porphyrin-NH.sub.2 from the nanoparticles grafted with porphyrin-NH.sub.2, incubated at 37 C. in PBS (pH=7.4). The stability of the porous silicon nanoparticles grafted with porphyrin-NH.sub.2 in PBS was monitored by spectroscopic determination of the quantity of porphyrin occurring in the solution as a function of the time (hours). The data show that 50% of the porphyrin was released at the end of 5 h, and that 89% of the porphyrin was released after 48 h of incubation. Under the experiment conditions used in this study, this degradation time is compatible with the use of the porous silicon nanoparticles grafted with porphyrin-NH.sub.2 for imaging and PDT.

(31) FIG. 27: Quantification of the porphyrin-NH.sub.2 contained in the porous silicon nanoparticles (black) and of the free porphyrin (grey) internalized in the living MCF-7 breast cancer cells after 5 h of incubation. The internalization was monitored by a spectroscopic determination of the quantity of porphyrin in the cell lysates and compared with a series of dilutions. The quantity of porphyrin internalized from the nanoparticles is in the range 1055 ng.Math.ml1 porphyrins, whereas the free porphyrin internalized corresponds to 355 ng.Math.ml1.

(32) FIG. 28: Semi-quantitative analysis of nanoparticles grafted with porphyrin-NH.sub.2 (pSiNP-Porph-NH.sub.2) and of the free porphyrin (Porph-NH.sub.2) internalized in the living MCF-7 breast cancer cells after 5 h of incubation. An average intensity inside the cells was determined from five distinct zones and expressed as greyness per m.sup.2. It was noted that the internalized nanoparticles represent 45% of the total of the nanoparticles detected.

(33) FIG. 29: Confocal microscopy images, in monophotonic excitation mode at 405 nm, of MCF-7 cells incubated with control medium, of non-grafted pSiNP (porous silicon nanoparticles), of free porphyrin-NCS, of pSiNP-porphyrin-NCS and of pSiNP-mannose-porphyrin-NCS. On the left: autofluorescence of the cells (emission collected between 540 nm and 620 nm), in the middle: nanoparticles and porphyrin (emission collected between 620 nm and 700 nm), on the right: superimposition of two fluorescence images. a) control, b) pSiNP c) free porphyrin-NCS d) pSiNP-porphyrin-NCS e) pSiNP-mannose-porphyrin-NCS.

(34) FIGS. 30A and 30B: Multiphoton confocal microscopy imaging of MCF-7 cells incubated with the different types of porous silicon nanoparticles. The images were taken with a spectral detector during a biphotonic excitation at 750 nm. On the left: fluorescence of the membrane marker (Cell Mask Orange), in the middle: fluorescence emitted between 620 and 700 nm, on the right: superimposition of the two fluorescence images. a) ethanol b) pSiNP (porous silicon nanoparticles) c) free porphyrin-NCS d) pSiNP+free porphyrin e) pSiNP-porphyrin-NCS f) pSiNP-mannose g) pSiNP-mannose+free mannose h) pSiNP-mannose-porphyrin-NCS i) pSiNP-mannose-porphyrin-NCS+free mannose.

(35) FIG. 31. Efficacy of the in vitro PDT in monophotonic excitation mode, as a percentage of living cells, with free porphyrin-NH.sub.2 at 0.25 g/mL, non-grafted pSiNP (porous silicon nanoparticles) at 20 g/mL and pSiNP-porphyrin-NH.sub.2 at 20 g/mL, with or without irradiation. Irradiation at 650 nm (14 J/cm.sup.2) for 40 min. The living cells are counted by an MTT assay 2 days after irradiation.

(36) FIG. 32. Efficacy of the in vitro PDT in monophotonic excitation mode, as a percentage of living cells, on the MCF-7 cells incubated with: control culture medium containing 4% ethanol (EtOH, control experiment), free porphyrin-NCS (porph-NCS), non-grafted porous silicon nanoparticles (Si), porous silicon nanoparticles grafted with mannose (Si-m), porous silicon nanoparticles grafted with porphyrin-NCS (Si-p) and porous silicon nanoparticles grafted with porphyrin and with mannose (Si-m-p). Irradiation at 650 nm, 7 mW/cm.sup.2, 40 min. The living cells are counted by an MTS assay 2 days after irradiation.

(37) FIG. 33. Dotted: normalized UV-VIS absorption spectrum of the porphyrin-NCS. Solid: normalized emission spectrum of the porous silicon nanoparticles in water (excitation at 250 nm).

EXPERIMENTAL PART

(38) 1. Synthesis of the Biodegradable Porous Silicon Nanoparticles and Chemical Functionalization

(39) This part describes the preparation of the nanostructured components/nanoparticles and the development of novel grafting chemistries for attaching porphyrins and targeting agents, in order to study their effectiveness in targeting, penetrating and delivering the porphyrin-type therapeutic agents to in vivo cancer or endothelial cells.

(40) 1.1. Synthesis and Characterization of the Porous Silicon Nanostructures/Nanoparticles

(41) The porous silicon nanoparticles are prepared by electrochemical etching of monocrystalline silicon wafers in a hydrofluoric acid (HF) electrolyte solution in ethanol, followed by detachment of the porous layer by electrodissolution and an ultrasonic fracturing in accordance with a published procedure (FIG. 1), (Sailor, M. J. et al., Adv. Funct. Mater. 2009, 19 (20), 3195-3208; Bley, R. A. et al. Chem. Mat. 1996, 8 (8), 1881-1888).

(42) Monocrystalline silicon of the p++ type (doped with boron, resistivity 0.8-1.2 m.Math.cm, orientation, <100>) is etched electrochemically in a solution of hydrofluoric acid (48%)/ethanol at 3:1 by volume (FIG. 1, first step). The etching is carried out in a Teflon cell, using a circular platinum counter electrode. A constant current of 200 mA/cm.sup.2 is applied for 150 seconds, and the sample is rinsed three times with ethanol. The porous layer is then electrodissolved in a solution of HF in ethanol at 3.3% by applying a constant current of 4 mA/cm for 250 seconds (step 2 of FIG. 1). After rinsing three times with ethanol, the porous layer is deposited in ethanol, in a glass flask. After having been degassed under a nitrogen flow for 20 minutes, the sample is sonicated in an ultrasonic bath for 16 hours (steps 3 and 4 of FIG. 1). The obtained dispersion of particles in ethanol is filtered (0.2-m nylon syringe filter) and centrifuged at 14000 rpm for 30 minutes. Nanoparticles with a size distribution centred on 115 nm are obtained (FIG. 4). The size and the texture of the nanoparticles are characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), light scattering (DLS) and nitrogen adsorption (BET). The nanoparticles are also characterized by X-ray diffraction.

(43) FIG. 2 shows the SEM image of a surface and of a cross section of a porous Si film.

(44) FIG. 3 shows the SEM and TEM images of porous Si nanoparticles prepared by ultrasonic fracturing of the porous film shown in FIG. 2.

(45) FIG. 4 shows the light scattering curve for the porous Si nanoparticles shown in FIG. 3.

(46) FIG. 5 shows the nitrogen adsorption/desorption isotherm curve for the porous Si nanoparticles shown in FIG. 3.

(47) FIGS. 21A and 21B show the X-ray powder diffractograms at wide angles and at narrow angles for the porous Si nanoparticles shown in FIG. 3.

(48) 1.2. Modification of the Surface of the Porous Silicon Nanostructures/Nanoparticles.

(49) Hydrophilic synthetic porphyrins are anchored covalently onto the surface of the porous silicon nanostructures/nanoparticles by novel grafting chemistries. Targeting species are also grafted onto the surface of the porous silicon nanostructures/nanoparticles. Upstream of the grafting of the targeting species and the porphyrins, the porous silicon nanostructures/nanoparticles are chemically modified by functional ligands.

(50) 1.2.1. Chemical Modification of the Porous Silicon Nanostructures/Nanoparticles by Functional Ligands:

(51) The freshly etched porous silicon nanostructures/nanoparticles, having SiH bonds on their surface, are very hydrophobic. Two surface modifications are developed.

(52) 1.2.1.1. Controlled Oxidation (Thermal, by Ozone, Dimethyl Sulphoxide, Aqueous Borate) and Silanization:

(53) SiOH and SiO.sub.2 species are produced on the surface of the nanostructures/nanoparticles. The chemistry of silanization on oxidized surfaces of porous silicon nanostructures/nanoparticles has been developed for the grafting of functional carbon chains such as aminopropyltriethoxysilane, incorporating amine groups (as shown in FIG. 6A), then used for the grafting of porphyrins and targeting species.

(54) After oxidation of the nanoparticles, they are redispersed in aminopropyltriethoxysilane. The silanization reaction is carried out at 150 C. under a nitrogen flow for 2 hours. The nanoparticles are then rinsed 4 times with ethanol and twice with diethyl ether, before being dried under a nitrogen stream.

(55) 1.2.1.2. Hydrosilylation:

(56) Functional groups are grafted covalently onto the surface of the porous Si nanostructures/nanoparticles via SiC bonds according to published procedures (Buriak, J. M., Chem. Rev. 2002, 102 (5), 1272-1308). Hydrosilylation is a chemical treatment known to strengthen the chemical stability of porous silicon in aqueous medium (15). Functional groups of the amine, isocyanate and semicarbazide type are anchored by hydrosilylation with allylamine, allyl isocyanate, and allylsemicarbazide derivatives as shown in FIG. 6B and in FIG. 22A.

(57) 1.2.2. Grafting of the Porphyrins

(58) Amine and isocyanate porphyrins are grafted onto the different types of ligands (amino and isocyanato) previously anchored to the surface of these porous Si nanoparticles.

(59) 1.2.2.1. Grafting of Porphyrin-NH.sub.2 onto the Porous Si Nanoparticles Hydrosilylated with Allyl Isocyanate.

(60) The nanoparticles are dispersed in allyl isocyanate. The hydrosilylation is carried out at 90 C. under a nitrogen flow for 3 hours. The particles are then rinsed three times in tetrahydrofuran (THF) and redispersed in 4 ml THF.

(61) 500 L porphyrin-NH.sub.2 at 1 mg/mL in dimethylformamide (DMF) is added to 200 L ethanol. The reaction takes place at 80 C. under nitrogen reflux for 4 hours. The particles are then rinsed twice with ethanol, twice with water, twice with ethanol, and twice with diethyl ether before being dried under a nitrogen stream.

(62) This grafting procedure shown in FIG. 7 has never been described before for porous Si nanoparticles.

(63) 1.2.2.2. Grafting of Porphyrin-NCS onto the Porous Si Nanoparticles Hydrosilylated with Allylamine (Shown in FIG. 8):

(64) The nanoparticles are dispersed in allylamine. This solution is then degassed by three consecutive freeze-pump-thaw cycles.

(65) The hydrosilylation is carried out at 70 C. under a nitrogen flow for 2 hours and the nanoparticles are rinsed four times in ethanol. The particles are redispersed in 2 ml ethanol. 2.5 mL of a solution of H.sub.2O/ethanol at 1:1 (by volume) is then added to 500 L of a solution of porphyrin-NCS at 1 mg/mL in ethanol.

(66) The reaction takes place at ambient temperature accompanied by stirring for 18 hours. The particles are then rinsed four times with ethanol and twice with diethyl ether before being dried under a nitrogen stream.

(67) 1.2.3. Anchoring of the Targeting Species

(68) Molecules of the carbohydrate (mannose) type are coupled to the surface of the porous Si nanoparticles according to a procedure developed previously for mesoporous silica nanoparticles and shown in FIG. 9, and according to another procedure shown in FIG. 22B. It has been shown here that squarate and semicarbazide groups allow the coupling of carbohydrate-type molecules under mild conditions. The expected role of the carbohydrate species is the vectorization of the nanoparticles to the cancer cells.

(69) 1.2.3.1 Anchoring of a Mannose-Squarate

(70) In a typical reaction, the porous Si nanoparticles are dispersed in allylamine. This suspension is then degassed by three consecutive freeze-pump-thaw cycles.

(71) The hydrosilylation is carried out at 70 C. under nitrogen reflux for 2 hours and the nanoparticles are rinsed four times in ethanol. The particles are redispersed in 2.5 ml ethanol. 2.5 mL of a solution of mannose-squarate at 6 mg/mL in H.sub.2O/ethanol at 1:1 (by volume) is then added to 250 L triethylamine.

(72) The reaction takes place at ambient temperature accompanied by stirring for 18 hours. The nanoparticles are then rinsed four times with diethyl ether before being dried under a nitrogen stream.

(73) 1.2.3.2 Anchoring of a Mannose-Ketone Hydrosilylation of the porous silicon nanoparticles with semicarbazide: The porous silicon nanoparticles dispersed in ethanol are first centrifuged at 22000 g for 30 min, then rinsed twice with THF, with centrifugation at 22000 g for 30 min each time. The nanoparticles are then redispersed in 3 mL of a solution of tert-butyl-2-[(allylamino)carbonyl]hydrazine-carboxylate, at 10.sup.1 M in THF. The hydrosilylation reaction then takes place at 85 C. for 3 h accompanied by stirring and under a nitrogen flow. The particles are then centrifuged and rinsed twice with THF, once with ethanol and once with dichloromethane.

(74) Deprotection of the Semicarbazide Function

(75) In order to remove the Boc group protecting the semicarbazide function, the porous silicon nanoparticles are centrifuged at 22000 g for 30 min, to then be redispersed in 5 mL of a solution of trifluoroacetic acid (TFA) at 40% by volume in dichloromethane. This dispersion is stirred for 4 h at ambient temperature, then the nanoparticles are rinsed twice with CH.sub.2Cl.sub.2, once with ethanol, once with water, once with ethanol and once with CH.sub.2Cl.sub.2.

(76) Grafting of Mannose-Ketone

(77) Following the deprotection of the semicarbazide, the nanoparticles are redispersed in 2.5 mL ethanol. 2.5 mL of a solution of mannose-ketone at 1.610.sup.2 M in a 1:1 water/ethanol mixture and 250 L triethylamine were then added dropwise. The reaction takes place accompanied by stirring and at ambient temperature for 18 h. After the reaction, 4 to 5 washings are carried out with distilled water then two washings with absolute ethanol and two other washings with diethyl ether. Finally, the grafted particles are dried under nitrogen.

(78) 1.2.4. Simultaneous Grafting of Mannose-Squarate and of Porphyrin-NCS:

(79) The porous Si nanoparticles are dispersed in allylamine. This suspension is then degassed by three consecutive freeze-pump-thaw cycles. The hydrosilylation is carried out at 70 C. under nitrogen reflux for 2 hours and the nanoparticles are rinsed four times in ethanol. The particles are redispersed in 2 ml ethanol. 2.5 mL of a solution of mannose-squarate at 6 mg/mL in H.sub.2O/ethanol at 1:1 (by volume) is then added to 250 L triethylamine, and to 500 L of a solution of porphyrin-NCS at 1 mg/mL in ethanol. The reaction takes place at ambient temperature accompanied by stirring for 18 hours. The nanoparticles are then rinsed four times with ethanol and twice with diethyl ether before being dried under a nitrogen stream.

(80) 1.2.5. Simultaneous Grafting of a Mannose-Ketone and of Porphyrin-NCS: Hydrosilylation of the porous silicon nanoparticles with semicarbazide (as above) Hydrosilylation of the porous silicon nanoparticles with allylamine (as above) Grafting of porphyrin-NCS (as above) Grafting of mannose-ketone (as above)

(81) Notes:

(82) 1/Galactose can be substituted for mannose, following the same experimental conditions.

(83) 2/If the nanoparticle formulations have poor circulation properties, PEG (polyethylene glycol) chains can be used to bond the carbohydrate species to the nanoparticles via squarate or amide couplings.

(84) Chitosan and serum albumin can be used as another alternative surface chemistry for improving the biocompatibility of the nanoparticles. It will be possible to adjust the molecular weight and chain length of the PEGs in order to maximize the biocompatibility, the cell penetration, and in order to control the degradation kinetics of the porous silicon nanostructures.

(85) 1.2.6. Characterization

(86) The chemically modified nanoparticles are characterized by infrared vibrational spectroscopy (FTIR), elemental analysis, and XPS for the grafting of the mannose, and by UV-VIS absorption spectroscopy and FTIR for the grafting of the different porphyrins to the surface of the porous Si nanoparticles. For the UV-VIS absorption spectroscopy the porous Si nanoparticles are dissolved in a solution of KOH before the absorbance of porphyrin is measured (FIGS. 11 and 14B).

(87) 1.2.6.1. Grafting of Mannose

(88) The grafting of mannose-squarate onto the allylamine-functionalized nanoparticles is carried out by nucleophilic addition of the amine followed by the removal of the ethoxy group of the squarate. The electrophilicity of the squarate is increased by the presence of hydrogen bonds with water, a cosolvent of the reaction. The triethylamine catalyzes the reaction by trapping the protons released during the reaction. The FTIR spectrum of the mannose-functionalized nanoparticles is shown in FIG. 10B.
In this FTIR spectrum, the band appearing at 1654 cm.sup.1 is attributed to the angular deformation vibration of the NH bond of the amines that have reacted. The vibration band at 1630 cm.sup.1 is always present in the form of a shoulder in the band centred on 1654 cm.sup.1. The presence of this band, attributed to the angular deformation of the NH bond of the primary amine, means that the primary amine is always partially present on the surface of the porous silicon nanoparticles.

(89) The results of the elemental analyses after grafting of the mannose show, below, the percentage of elements present: 5.25% C 0.72% N 4.18% O

(90) These values are consistent with the presence of mannose on the surface of the porous Si particles.

(91) Quantification by XPS

(92) Experimental: The XPS analyses were carried out on a Thermo Electron ESCALAB 250 device from the analysis technical support centre of Montpellier 2 University. The excitation source is the Al K line (1486.6 eV). The analyzed surface has a diameter of 400 m, the photoelectron spectra are calibrated by binding energy relative to the energy of the CC component of the carbon C1s at 284.8 eV, and the charge is compensated by an electron beam.

(93) TABLE-US-00001 TABLE 1 Table showing the quantifications obtained by XPS for the porous silicon nanoparticles grafted with mannose-squarate (on allylamine). Name Peak BE Height Counts FWHM eV Area (P) CPS .Math. eV Area (N) At. % Si2p 103.22 10204.45 1.78 19956.09 0.56 30.98 C1s 284.89 3324.17 2.73 9935.02 0.23 12.76 N1s 401.07 400.97 3.81 1551.23 0.02 1.12 O1s 532.53 65969.95 1.62 118334.22 0.96 53.42 F1s 688.97 2728.87 1.83 5593.67 0.03 1.72

(94) 1.2.6.2. Grafting of the Porphyrins

(95) The UV-VIS spectrum of a solution obtained after dissolution, in KOH, of the porous silicon nanoparticles grafted with porphyrin-NCS is shown in FIG. 11. 74 g porphyrin/mg nanoparticles, i.e. a loading of 7.4% by mass, is obtained.

(96) The FTIR spectrum of the porous silicon nanoparticles grafted with a porphyrin-NCS is shown in FIG. 12. Three intense bands are observed between 2850 and 2960 cm.sup.1, corresponding to the stretching vibrations of the CH bonds. This is due to the numerous CH bonds provided by porphyrin. Between 1460 cm.sup.1 and 1600 cm.sup.1, the vibrational spectrum is complex. These bands are characteristic of the aromatic CC bonds present thanks to the porphyrin. The intense band observed at 1642 cm.sup.1 could be characteristic of the thiourea bond or of the NH angular deformation vibration of the amines that have not reacted. Finally, the band observed at 797 cm.sup.1 is always attributed to the stretching vibration of the SiC bond, whereas the bands at 882 cm.sup.1 and between 1000 and 1250 cm.sup.1 (stretching vibrations of the SiO bonds) indicate a partially oxidized surface.
The FTIR spectrum of the porous silicon nanoparticles grafted with a porphyrin-NH.sub.2 is shown in FIG. 13. In particular, two bands characteristic of the urea bond are observed at 1620 cm.sup.1 and 1542 cm.sup.1, attributed respectively to the stretching vibration of the CO bond and the stretching vibration of the NH bond, which indicates the success of the coupling between the nanoparticles functionalized with isocyanate and porphyrin-NH.sub.2.
The quantification of the grafted porphyrin-NH.sub.2 carried out by dissolution of the nanoparticles in a basic KOH solution indicates that a quantity of 13.3 g porphyrin per mg nanoparticles is obtained. This quantity is sufficient for PDT applications.

(97) 2. Production of .sup.1O.sub.2 (oxygen) and of ROS (reactive oxygen species). The measurements consist of irradiating a photosensitizer in the presence of a probe molecule, diphenylisobenzofuran (DPBF), the oxidation of which by .sup.1O.sub.2 manifests itself as a decrease in the absorption spectrum. It is a comparative method: from knowing the quantum yield of .sup.1O.sub.2 generation of a reference molecule (such as rose bengal or methylene blue), that of the functionalized porous silicon nanoparticles is deduced by spectrophotometric monitoring.

(98) Experimental: 2 mL of a solution of DPBF at 0.08 mM and of nanoparticles (or photosensitizing molecule) at a known concentration is placed in a quartz vessel. The vessel is stirred, closed and irradiated for 15 min. The irradiation is carried out with an optical fibre 0.63 cm in diameter. The light is filtered through an anti-IR filter so as not to heat up the sample, and a band-pass filter between 410 and 490 nm (Figure IV.1). The absorbance of the solution is measured every minute for the first 5 minutes, then every 5 minutes.

(99) No production of singlet oxygen is observed for the porous Si nanoparticles that do not contain porphyrin (FIG. 15). A production of singlet oxygen is observed for the porous Si nanoparticles functionalized with porphyrin-NCS (FIG. 23), for the porous Si nanoparticles functionalized with porphyrin-NH.sub.2 (FIG. 24) and for the porous Si nanoparticles functionalized with porphyrin-NCS of mannose (FIG. 17).

(100) 3. Release Kinetics of the Porphyrins (FIGS. 25, 26)

(101) Experimental: The release of porphyrin as a function of time was studied in PBS (phosphate buffer saline) at pH=7.2, in DMEM F12 culture medium without serum and in DMEM F12 culture medium at 10% FBS (fetal bovine serum). A known quantity of grafted nanoparticles (approximately 500 g) is dispersed in 3 mL of medium and incubated at 37 C. on a stir plate at 100 rpm. At given times, 500 L of each dispersion is removed and 500 L of the study medium is added so as to keep the volume constant. The 500 L of dispersion removed is centrifuged at 22000 g for 30 min. The supernatant is kept away from light at 4 C. until analysis, the nanoparticle pellet is redispersed in the study medium.

(102) The removed samples are analyzed by UV-visible absorption spectroscopy in order to determine the porphyrin concentration of each sample. The presence of mannose on the surface of the porous silicon nanoparticles does not influence their degradation kinetics. The porous silicon nanoparticles are completely degraded after 24 h of incubation in biological medium, they are biodegradable.

(103) 4. Targeting of Tumours and Homing

(104) 4.1 Imaging in Monophotonic Excitation Mode with the Porous Silicon Nanoparticles Grafted with Porphyrin-NH.sub.2 (FIG. 18)

(105) Experimental: The MCF-7 cells are seeded in glass-bottomed wells at a density of 10.sup.6 cells/cm.sup.2. After 24 hours they are rinsed once then incubated in 1 mL of culture medium containing the porous silicon nanoparticles at a concentration of 20 g/mL, and/or the free porphyrin at an equivalent concentration for 3 or 5 h depending on the experiments. Fifteen minutes before the end of the incubation, the cell membranes are labelled with Cell Mask Orange (Invitrogen) at a final concentration of 5 g/mL (for the experiment with the nanoparticles grafted with porphyrin-NH.sub.2 only). They are then rinsed with white DMEM culture medium (without phenol red). The microscopy photos are taken on an LSM 780 LIVE confocal microscope. The fluorescence excitation is carried out at 405 nm or at 633 nm depending on the samples. The emission is collected between 620 and 700 nm.

(106) FIG. 18 also shows that free porphyrin-NH.sub.2 is not detected inside the cell, whereas the nanoparticles grafted to porphyrin-NH.sub.2 are internalized and appear luminescent. This experiment shows that the use of porous silicon nanoparticles is an effective means for internalizing porphyrin-NH.sub.2 in the cells. The quantity of porphyrin internalized from the nanoparticles is in the range 1055 ng.Math.ml1 porphyrins, whereas the free porphyrin internalized corresponds to 355 ng.Math.ml1 (FIG. 27 and FIG. 28).

(107) 4.2 Imaging in Monophotonic Excitation Mode with the Porous Silicon Nanoparticles Grafted with Porphyrin-NCS (FIG. 29)

(108) When the MCF-7 cells are incubated with the porous silicon nanoparticles grafted with porphyrin-NCS, distinct points of fluorescence are observed on the surface or in the cells. This result confirms the possibility of internalizing porphyrin in MCF-7 cells by means of porous silicon nanoparticles, even without a targeting agent. This internalization is possible through the action of clathrins and the formation of invagination vesicles. When the MCF-7 cells are incubated with the nanoparticles grafted with porphyrin and mannose, intense fluorescence aggregates are observed inside the cells. The porous silicon nanoparticles grafted with porphyrin-NCS and mannose are therefore present to a considerably greater extent than the porous silicon nanoparticles grafted with porphyrin-NCS without mannose: the mannose here plays its role of targeting agent with great effectiveness. It is thought that the mannose on the surface of the nanoparticles ensures the endocytosis thereof by the cells.

(109) Similar results can be obtained by functionalizing the porous Si with galactose species.

(110) 4.3 Imaging in Biphotonic Excitation Mode with the Porous Silicon Nanoparticles Grafted with Porphyrin-NCS (FIG. 30A and FIG. 30B)

(111) Experimental: the MCF-7 cells were incubated for 5 h with the different types of nanoparticles: pSiNP, pSiNP-porphyrin-NCS, pSiNP-mannose, pSiNP-mannose-porphyrin-NCS at 40 g/mL. The MCF-7 cells were also incubated for 5 h with free porphyrin-NCS at 3.2 g/mL, with pSiNP and free porphyrin, as well as with pSiNP-mannose and pSiNP-mannose-porphyrin-NCS with free mannose (at a concentration of 10 mM) in order to study the reversion of the targeting by mannose. The cells were then rinsed, then imaged after excitation with a biphotonic pulsed laser at 750 nm. A membrane dye was added so as to identify the cells. The development was carried out in confocal mode.

(112) When the cells are incubated with the porous silicon nanoparticles, a few fluorescent zones are observed, indicating that these nanoparticles can be internalized, even if it appears that this only happens in quite small proportions. Moreover, the observation of the fluorescence here indicates that porous silicon can absorb light in biphotonic mode.

(113) By comparison, when the MCF-7 cells are incubated with free porphyrin-NCS, no fluorescence is observed after biphotonic excitation at 750 nm. This means that the porphyrin-NCS present on/in the cells after rinsing, which could be distinguished by imaging in monophotonic excitation mode (at 650 nm), is not excited in biphotonic light at 750 nm.

(114) When the cells are incubated with the mannose-porphyrin-NCS porous silicon nanoparticles, the fluorescence observed is much greater than with the other types of nanoparticles. The fluorescence is also intracellular, which indicates an internalization of the nanoparticles which is clearly improved by the presence of the mannose on their surface. When the same experiment is carried out in the presence of an excess of free mannose in the culture medium, a blockage of the internalization of the nanoparticles is observed which is characterized by a fluorescence that is more membranous than intracellular.

(115) The fluorescence observed in biphotonic excitation mode for each type of nanoparticles can be classified according to the following scale: free porphyrin-NCS<pSiNPpSiNP-mannosepSiNP+free porphyrin-NCS<pSinp-porphyrin-NCS<pSiNP-mannose-porphyrin-NCS.

(116) The results obtained here show that, as in monophotonic imaging mode, the presence of the mannose allows a better internalization of the porous silicon nanoparticles. The combined presence of porphyrin-NCS and mannose on the surface of the porous silicon nanoparticles makes it possible to optimize their internalization in the cells and their visualization by biphotonic imaging. A joint action mechanism between silicon and the grafted porphyrin-NCS, of the energy-transfer type, is envisaged for the emission of the fluorescence of the pSiNP grafted with porphyrin.

(117) 4.4 Photodynamic Efficacy in Monophotonic Excitation Mode

(118) Experimental: The MCF-7 cells are seeded in a 96-well plate, at a density of 10.sup.6 cells/cm.sup.2. After 24 h, the cells are incubated with the porous silicon nanoparticles at a concentration of 20 g/mL or 40 g/mL for the nanoparticles grafted respectively with porphyrin-NH.sub.2 and porphyrin-NCS, or with free porphyrin at an equivalent concentration for 5 h. The cells are then rinsed with PBS, then kept in 100 L of culture medium, and finally irradiated for 40 min with a laser at 650 nm with a power of 7 mW/cm.sup.2 (i.e. 16.8 J/cm.sup.2). After incubation for 48 h, an MTS assay (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium) is carried out in order to quantify the living cells in the experiment with the porous silicon nanoparticles grafted with porphyrin-NCS. In the case of the nanoparticles grafted with porphyrin-NH.sub.2, an MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is carried out in order to quantify the living cells. In the case of the MTT assay, the cells are incubated for 4 h in culture medium supplemented with 0.5 mg/mL MTT. The medium is then removed and the purple MTT crystals are dissolved in an ethanol/DMSO (1:1) solution. The absorbance of the solution is read at 540 nm. In the case of the MTS assay, the cells are incubated for 2 h in culture medium supplemented with 0.5 mg/mL MTS. The absorbance of the solution is then read directly at 490 nm

(119) 4.4.1 Porphyrin-NH.sub.2 (FIG. 31)

(120) Irradiation of the cancer cells alone does not induce any toxicity. The porous silicon nanoparticles (pSiNP) are not cytotoxic, 99% and 100% of the cells remain alive after incubation for 5 h with or without irradiation respectively. The fact that the pSiNP are not cytotoxic after irradiation at 650 nm was expected, taking into account the fact that the pSiNP do not absorb light at this wavelength.

(121) Porphyrin-NH.sub.2 induces 8% cytotoxicity in the absence of irradiation. After irradiation, a slightly higher cell death rate (16%) is observed. This quite low cytotoxicity of the free porphyrin after irradiation was expected, because the imaging and quantification experiments showed that the free porphyrin-NH.sub.2 was poorly internalized by the cells. By comparison, the cells incubated with the pSiNP-porphyrin-NH.sub.2 and not irradiated have a cell death of 19%. In this case, the observed toxicity in the dark is attributed to the release of a small quantity of cytotoxic porphyrin-NH.sub.2 inside the cell, after the internalization and the poor metabolization of the nanoparticles. Finally, the pSiNP-porphyrin-NH.sub.2 induce a cell death of 42% after irradiation at 650 nm. The toxicity of the porphyrin-NH.sub.2 confined in the porous silicon nanoparticles is therefore significantly increased compared with the free porphyrin-NH.sub.2.

(122) 4.4.2 Porphyrin-NCS (FIG. 32)

(123) The pSiNP and the pSiNP-mannose, irradiated at 650 nm, are not toxic for the cells. 99% and 104% of the MCF-7 cells respectively survive after incubation for 5 hours with pSiNP functionalized or not functionalized with mannose and after irradiation. In a parallel experiment, the toxicity of the pSiNP at different concentrations without irradiation was studied on the same cells. At 40 g/mL, the porous silicon nanoparticles are of very low toxicity without irradiation for the cells.

(124) After incubation of the free porphyrin-NCS and irradiation, a cell death of 22% is observed. Sufficient porphyrin-NCS adheres to the membranes and/or is internalized by the cells, as was seen in the confocal microscopy images, to generate a not insignificant effect in monophotonic PDT.

(125) By comparison, the cells treated with the pSiNP-porphyrin-NCS show a cell death of 28%. The effect of the porphyrin-NCS vectorized by the nanoparticles here is not significantly increased compared with the effect of the free porphyrin. By contrast, during the treatment by PDT of the MCF-7 cells with the pSiNP-mannose-porphyrin-NCS, the cell survival rate falls to 58%. The increase in cytotoxicity and in the efficacy of the PDT observed here is attributed to a more effective targeting of the cells thanks to the presence of the mannose, as well as to a greater internalization or anchoring to the surface of the cells than for the pSiNP-porphyrin-NCS, in accordance with the imaging experiments.

(126) 4.5 Photodynamic Effectiveness in Biphotonic Excitation Mode

(127) A particularly useful approach consists of replacing the conventional one-photon excitation in the visible range with a two-photon excitation in the near-infrared range, because this makes it possible to limit the side effects due to light in the tissues treated by photodynamic therapy. The advantage of a two-photon excitation is also that this makes it possible to treat cancers in deeper tissues.

(128) One of the inventors has shown previously that porous silicon nanoparticles can be excited by an excitation in 2-photon mode in the near infrared (780 nm). Here, in an experiment on 2-photon PDT, it is shown that the porous Si nanoparticles containing a polycationic porphyrin excited in 2-photon mode (750 nm) can kill cancer cells after endocytosis of the nanoparticles (FIG. 19).

(129) Experimental: The MCF-7 cells are seeded in a 384-well plate, at a density of 10.sup.6 cells/cm.sup.2. After 24 h, the cells are incubated with the porous silicon nanoparticles at a concentration of 40 g/mL, or with free porphyrin at a concentration of 3.2 g/mL for 5 h. The cells are then rinsed (this rinsing step not having been carried out for certain control experiments), then irradiated with a biphotonic laser at 750 nm. 3 scans of 1.57 s are carried out. After incubation for 48 h, an MTS assay is carried out in order to quantify the living cells.

(130) The cells incubated in the control culture medium alone and irradiated do not display any cell death, which indicates that neither the culture medium containing ethanol nor the biphotonic irradiation are toxic for the cells. Nor is any cell death observed when the MCF-7 cells are incubated with free porphyrin-NCS, rinsed, then irradiated. Contrary to what was observed under monophotonic irradiation, the porphyrin-NCS remaining on the cells after rinsing is not sensitive to biphotonic excitation and therefore is not toxic for the cells.

(131) When the MCF-7 cells are treated with the non-functionalized pSiNP, a cell death of 15% is observed after rinsing and biphotonic irradiation. This result indicates that the pSiNP, which are capable of absorbing biphotonic light, generate .sup.1O.sub.2 or other ROS and kill the cells. However, the effect observed here is quite weak. This result can be explained by two reasons: it is known that the quantum yield of .sup.1O.sub.2 generation of the porous silicon is quite low on the one hand; on the other hand it has been observed in the imaging experiments that the internalization of the pSiNP was not very high. By comparison, it can be observed that the nanoparticles, when they are functionalized with mannose (pSiNP-mannose), kill 31% of cells after irradiation. The presence of mannose grafted to the surface of the nanoparticles seems to allow a better internalization in the cells. After incubation of the cellules with the pSiNP-porphyrin-NCS, rinsing and biphotonic irradiation, a cell death of 44% is observed.

(132) The hypothesis being made here is based on the possibility of an energy transfer between the pSiNP, which are sensitive to biphotonic excitation, and the porphyrin-NCS.

(133) This result suggests a mechanism of FRET (fluorescence resonance energy transfer) in 2-photon excitation mode from the porous Si nanoparticles to the porphyrins and, as a result, the generation of singlet oxygen and reactive oxygen species under 2-photon irradiation.

(134) The conditions needed for such a transfer, defined by the Forster-Dexter theory, are that the two systems, donor and acceptor, silicon and porphyrin respectively in this case, be close enough to each other in space (the distance between the two must not exceed 10 nm), and that the emission spectrum of the donor and the absorption spectrum of the acceptor at least partially overlap. In the case in point, the proximity of the silicon and the porphyrin is ensured by the covalent grafting. Using Chem3D software, the distance between the silicon and the porphyrin was estimated at 7.8 , which is much less than the limit distance that allows the energy transfer. Moreover, the overlap of the emission spectrum of the donor and the absorption spectrum of the acceptor is shown in FIG. 33.

(135) Finally, when the MCF-7 cells are incubated with the pSiNP-mannose-porphyrin-NCS, the cell death rate observed is 30%. This result is surprising. In fact, there was expected to be a maximum cell death rate for these nanoparticles because, on the one hand, the quantity of grafted porphyrin is greater than on the nanoparticles grafted only with porphyrin and, on the other hand, the presence of mannose-squarate on the surface of the nanoparticles improves the internalization of the nanoparticles by the cells. However, these nanoparticles have a lesser effect in 2-photon PDT than the nanoparticles grafted with porphyrin. Given the hypothesis of the energy transfer between the pSiNP and the porphyrin-NCS, this is explained by the absorption of some of the transmitted energy by the mannose. In fact, the mannose, in order to be grafted to the surface of the pSiNP, has a phenyl group and a squarate group. The aromaticity and the conjugation of the squarate are suspected of being at the origin of a deactivation of the excited state of the porous silicon, and explains this absorption of the energy. Another type of bond for the mannose, such as a ketone, described previously can be used for the grafting of the mannose.

(136) Of course the present invention is not limited to the examples and embodiment described and shown, but is capable of numerous variants accessible to a person skilled in the art.

(137) This application was filed first in a foreign country without the knowledge or consent of the Regents of the University of California, and this was done in error and without the intention to deceive.