THERANOSTIC SYSTEM FOR DIRECTED DIFFUSION OF THERAPEUTIC AND IMAGING AGENTS TO CANCER CELLS

Abstract

The invention relates to a multifunctional system stable in a physiological medium, which includes in the same platform an anti-carcinogenic molecule, an imaging agent and a directing molecule that interacts specifically with cancer-cell membrane receptors, the system allowing pathological tissue imaging and pharmacological action to be carried out jointly with high specificity. The intratumoral administration of the system facilitates selective diffusion to cancer cells and minimises the disadvantages of chemotherapy.

Claims

1. A theranostic system comprising: nanoparticles of a porous material wherein said porous material has a crystalline structure that corresponds to a covalent organic framework to which the following elements are bound through terminal amino groups present in the crystalline structure thereof: at least one therapeutic molecule, preferably selected from a small molecule of biological or synthetic origin the molecular weight of which is less than 3,000 Da, a macromolecule, an antibody, a peptide, a protein, an enzyme, a nucleic acid or an organic polymer an imaging agent, preferably selected from a fluorochrome, a paramagnetic element, and a radionucleus and an anti-FOLH1 monoclonal antibody that interacts specifically with FOLH1 receptors, wherein the nanoparticles have a diameter greater than 20 nm.

2-5. (canceled)

6. The theranostic system according to claim 1, wherein the crystalline structure also has further comprises terminal methoxy groups.

7. (canceled)

8. The theranostic system according to claim 1, wherein the small therapeutic molecule is an anti-tumor agent, selected from camptothecin, doxorubicin, docetaxel, paclitaxel, and cabazitaxel, or a combination thereof, and preferably the concentration of the therapeutic molecule ranges between 0.1 and 40% by weight.

9. (canceled)

10. The theranostic system according to claim 8, wherein the therapeutic molecule is bound to the nanoparticles by covalent bond, preferably through a linker that facilitates the covalent bond thereof to the terminal amino group through a bio-hydrolysable bond selected from an ester, amide, carbamate or carbonate.

11. (canceled)

12. The theranostic system according to claim 10, wherein the therapeutic molecule is docetaxel (DTX), and the linker is succinic acid.

13. (canceled)

14. The theranostic system according to claim 1, wherein the imaging agent is bound to the nanoparticles by covalent bond, preferably through a linker that facilitates the covalent bond thereof to a terminal amino group through an amide or carbamate bond.

15. (canceled)

16. The theranostic system according to claim 1, wherein the fluorochrome is selected from fluorescein, rhodamine, coumarin, and cyanine, or a combination thereof, and preferably the concentration of the fluorochrome can range between 0.01 and 10% by weight.

17. (canceled)

18. The theranostic system according to claim 1, wherein the paramagnetic element is selected from Gd (III), Fe (III), Mn (II), and .sup.19F, or a combination thereof, and preferably the concentration of the paramagnetic element can range between 0.01 and 10% by weight.

19. (canceled)

20. The theranostic system according to claim 1, wherein the radionucleus is selected from a β.sup.+ emitter such as .sup.18F, .sup.64Cu, .sup.68Ga, .sup.89Zr, .sup.99Tc, .sup.177Lu, an Auger electron emitter, such as .sup.111In, .sup.125I, an α emitter such as .sup.211At, .sup.212Bi, .sup.213Bi, .sup.225Ac, and .sup.227Th or a combination thereof, and preferably the concentration of the radionucleus can range between 0.0001 and 1% by weight.

21. (canceled)

22. The theranostic system according to claim 1, wherein the anti-FOLH1 monoclonal antibody is selected 175 Canal Street from an anti-FOLH1 monoclonal antibody of synthetic, semi-synthetic or natural origin, preferably selected from the list consisting of: J591, J415, D2B, 107-1A4, GCP-05, 2G7, YPSMA-1, YPSMA-2, GCP-02, GCP-04, 3E6, 24.4E6, clone C803N and 7E11-05.3, and more preferably that the amount of antibody linked to the nanoparticles can range between 0.0008 and 0.16 μmol/g.

23. (canceled)

24. The theranostic system according to claim 22, wherein the anti-FOLH1 monoclonal antibody is bound to the nanoparticles by covalent bond, preferably through a linker of an amide or carbamate bond.

25-26. (canceled)

27. The theranostic system according to claim 126, wherein the linker is 11-aminoundecanoic acid.

28. (canceled)

29. The theranostic system according to claim 1, further comprising a PEG molecule that is bound to the nanoparticle by covalent bond to a terminal amino group, preferably the PEG molecule has a molecular weight between 200 and 5,000 Da, and/or preferably the concentration of PEG can range between 0.05 and 10% by weight.

30-31. (canceled)

32. A theranostic system according to claim 1 for use as a medicament.

33. The theranostic system according to claim 1, for use in the treatment of prostate, breast, lung, colorectal, and kidney cancer, preferably wherein the route of administration is selected from intra-tumoral or parenteral, preferably performed by intra-tumor injection on the prostate gland.

34-38. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0057] FIG. 1 is a schematic illustration of the functionalisation sequence of nanoparticles of the COF-5 porous material with terminal amino and methoxy groups by covalent anchoring of the therapeutic molecule Suc-DTX, the imaging agent [.sup.18F]FDG, the spacer chain 11-aminoundecanoic acid, and the anti-FOLH1 antibody (PROTEIN DATA BANK ID FOLH1: 1Z8L).

[0058] FIG. 2 is a schematic illustration of the functionalisation sequence of nanoparticles of the COF-5 porous material with terminal amino and methoxy groups by covalent anchoring of the B rhodamine imaging agent, the spacer chain 11-aminoundecanoic acid, and the anti-FOLH1 antibody (PROTEIN DATA BANK ID FOLH1: 1Z8L).

[0059] FIG. 3 Image obtained in the transmission electron microscope (TEM) showing the morphology of nanoparticles of the COF-25 material of Example 3.

[0060] FIG. 4 Representation of the powder diffraction patterns obtained for nanoparticles of the COF-0 (COF-5) and COF-25 materials obtained in Example 3, and presented in Table 1.

[0061] FIG. 5 DTX release curve from the theranostic system of Example 4 in phosphate buffered saline (PBS, 1×, pH 7.4). Experimental conditions: T=37° C.; stirring=1500 rpm; concentration=5 mg/ml.

EXAMPLES

[0062] The invention is illustrated below by means of tests carried out by the inventors which reveal the effectiveness of the product of the invention.

[0063] Materials

Example 1: Suc-DTX Synthesis

[0064] 200 mg of DTX (0.24 mmol, I), 50 mg of succinic anhydride (0.500 mmol), and 1 mg of 4-(dimethylamino)pyridine (DMAP, 0.008 mmol) are introduced in a 50 ml flask. An anhydrous mixture of 12 ml (11:1) of dichloromethane (DCM) and dimethylformamide (DMF) is then injected. The solution is kept under stirring for 24 h at room temperature and under an inert atmosphere. After this time, the solvent is evaporated under vacuum (50 torr), a white solid being obtained, which is redissolved in 30 ml of ethyl acetate. The organic phase is then washed with aqueous hydrochloric acid solution (1%, w/v) (3×30 ml), and afterwards with ultrapure water (3×30 ml). The organic phase is separated, dried over anhydrous magnesium sulphate, and the solvent is evaporated under vacuum (50 torr), a white solid being obtained. Said white residue is dissolved in the minimum amount of solvent and purified by means of a chromatographic column, using DCM and methanol (100%.fwdarw.96:4%, v/v) as mobile phase.

[0065] The fraction containing the desired product, the solvent is evaporated under vacuum (50 torr), and 168 mg (75%) of a white solid (Suc-DTX, II) are obtained.

[0066] .sup.1H-RMN (300 MHz, dmso-d.sub.6) 0.97 (s, 6H); 1.38 (s, 9H); 1.51 (s, 3H); 1.68 (s, 3H); 2.23 (s, 3H); 2.51 (2H, below solvent signal); 2.61 (d, 2H, J=6.3 Hz); 3.61 (m, 2H); 4.03 (m, 3H); 4.44 (s, 1H); 5.00 (m, 5H); 5.39 (d, 1H); 5.77 (m, 1H); 7.19 (t, 1H); 7.39 (m, 4H); 7.69 (m, 3H); 7.87 (d, 1H); 7.98 (d, 2H); 12.83 (s.sub.a, 1H); .sup.13C-RMN (75 MHz, dmso-d.sub.6) 9.74; 13.64; 20.72; 22.43; 26.40; 28.47; 28.62; 28.72; 30.73; 34.90; 36.56; 42.82; 45.94; 51.32; 55.08; 56.92; 70.70; 71.04; 73.69; 74.76; 75.33; 76.77; 78.46; 80.25; 83.71; 127.42; 128.01; 128.50; 128.64; 129.52; 130.00; 133.34; 135.96; 136.75; 137.39; 155.16; 162.27; 165.24; 168.82; 169.50; 171.45; 172.55; 172.97; 209.28. Q-TOF MS (ESI, m/z) [M-Na].sup.− calculated for C.sub.47H.sub.57NO.sub.17Na, 930.3524; found 930.3502.

##STR00001##

Example 2: 2-amino-5-methoxy-benzene-1,4-diboronic Acid Synthesis

[0067] 2.96 g (11.13 mmol) of 1,4-dibromo-5-methoxybenzene are dissolved in anhydrous tetrahydrofuran (100 ml) and the temperature of the mixture is lowered to −78° C. Next, 14.8 ml of n-BuLi (23.65 mmol, 1.6 M in hexane) are added drop by drop for 30 minutes. Once the n-BuLi has been added, stirring is maintained for 5 hours under refrigeration. After this time, 5.4 ml of trimethyl borate (48.30 mmol) are slowly added and magnetic stirring of the mixture is maintained at −78° C. It is left stirring overnight, allowing it to reach room temperature. After this time, acetic acid is added and the mixture is stirred for 2 hours at room temperature. The solvent is evaporated under vacuum, the suspension is filtered and washed with water. The compound is recrystallised in a water/acetonitrile mixture (100 ml, 50:50 v/v), filtered and the solid obtained is dried under vacuum (8 torr) for 24 hours. 1.02 g (47%) of 5-methoxy-1,4-benzenediboronic acid (MDMB) are obtained.

[0068] 30 ml of 90% nitric acid are cooled at 0° C. for 15 minutes. Next, 100 mg of urea (1.67 mmol) are added and the temperature of the mixture is lowered to −10° C. Subsequently, 5.91 g (30.1 mmol) of 2-methoxy-1,4-benzenediboronic acid (MBDB) is added in small portions with strong stirring for 1 hour. After completing the addition of the MBDB, the magnetic stirring of the mixture is maintained at −10° C. for an additional 15 minutes. After this time, the dark red solution is poured onto an ice bath containing a small amount of water. The suspension is filtered, washed with water, and the solid obtained is dried under vacuum (8 torr) for 24 hours. 4.36 g (60%) of a yellow solid (2-nitro-5-methoxy-1,4-benzenediboronic acid (NO.sub.2-MBDB)) are obtained.

[0069] .sup.1H-RMN (300 MHz, dmso-d.sub.6) 4.13 (s, 3H); 7.53 (s, 1H); 8.54 (s, 1H). .sup.13C-RMN (75 MHz, dmso-d6) 56.2; 115.28; 124.80; 125.46; 135.40; 139.34; 168.48. Q-TOF MS (ESI, m/z) [MH].sup.− calculated for C.sub.7H.sub.10B.sub.2NO.sub.7, 241.05; found 240.75.

[0070] In a second reaction step, to a solution of 570 mg of NO.sub.2-MBDB in a mixture of 10 ml of methanol and 0.2 ml of 36.5% hydrochloric acid, 150 mg of palladium supported on carbon (Pd/C, 10% wt Pd) are added. The treatment is carried out in a hydrogenation reactor (H.sub.2, 10 bar) at room temperature and with stirring for 1 hour. After this time, the suspension is filtered over Celita® to separate the palladium supported on carbon. The fraction containing the desired product, the solvent is evaporated under vacuum (50 torr), and 580 mg (99.4%) of a white solid are obtained (2-amino-5-methoxy-1,4-benzenediboronic acid (NMBB)).

[0071] .sup.1H-RMN (300 MHz, dmso-d.sub.6) 3.83 (s, 3H); 7.46 (s, 1H); 7.61 (s, 1H); .sup.13C-RMN (75 MHz, dmso-d.sub.6) 56.24; 115.11; 116.35; 125.30; 128.03; 128.60; 143.60; 152.38. Q-TOF MS (ESI, m/z) [MH].sup.− calculated for C.sub.7H.sub.12B.sub.2NO.sub.5, 211.07; found 210.76.

##STR00002##

Example 3: Preparation of Nanoparticles of the COF-25 Porous Material with Terminal Methoxy and Amino Groups

[0072] For the synthesis of COF-5 derivatives with terminal amino groups, the sequential replacement of MBDB by NMBB was carried out during the condensation of the covalent organic framework. The material obtained is designated by the nomenclature CF-x, wherein x represents the degree of replacement: percentage of NMBB over the total bi-coordinated ligand (MBDB+NMBB).

[0073] Initially, 243 mg of HHTP (0.749 mmol) are added to a solution of MBDB (221 mg, 1.128 mmol) and CH.sub.3OH (0.47 ml, 11.7 mmol) in 75 ml of an anhydrous 1,4-dioxane:mesitylene (4:1 v/v) mixture. The resulting suspension is subjected to sonication for 1 minute and filtered (0.45 μm PTFE) to introduce it into a Schlenk tube. Next, 187 ml of anhydrous acetonitrile and a further 112 ml of the anhydrous 1,4-dioxane/mesitylene (4:1, v/v) mixture are injected to obtain nanoparticles with a 2 mM concentration of HHTP in a 50% (v/v) acetonitrile. The mixture is purged with N.sub.2 and 2 cycles of N.sub.2/vacuum are carried out to remove any traces of moisture. The reaction mixture is left under magnetic stirring at 90° C. for 20 hours. After this time, the suspension is filtered, washed with anhydrous toluene, and the solid obtained is dried under vacuum (8 torr) for 24 hours. 206 mg of nanoparticles of the COF-25 porous material are collected.

[0074] The sequential replacement of MBDB by NMBB led to obtaining the different COF-x materials, the composition of which is summarised in Table 1.

[0075] FIG. 3 shows an image of the nanoparticles of the COF-25 material obtained in this example. Likewise, FIG. 4 shows the X-ray diffractograms of the COF-0 materials (it does not incorporate terminal amino groups) and COF-25 materials obtained in this example.

TABLE-US-00001 TABLE 1 Initial composition used for the preparation of nanoparticles of the COF-5 material and COF-x derivatives stable in physiological environment. Composition of the synthesis gel x Molar ratio HHTP HHTP MBDB MBDB NMBB NMBB COF-x [%] (MBDB:HHTP:NMBB) (mg) (mmol) (mg) (mmol) (mg) (mmol) COF-0 0 3.00:2:0.00 243 0.75 187 0.96 — — COF-25 25 2.53:2:0.86 243 0.75 186 0.95 68 0.32 COF-40 40 1.91:2:1.29 243 0.75 140 0.72 102 0.48 COF-60 57 1.54:2:2.06 243 0.75 113 0.58 163 0.77 COF-70 71 1.27:2:3.11 243 0.75 93 0.48 246 1.17 COF-80 78 1.04:2:3.71 243 0.75 76 0.39 293 1.39

Example 4: Preparation of Nanoparticles of the COF-25 Porous Material with Terminal Methoxy and Amino Groups on which Suc-DTX is Bound by Means of Covalent Bond

[0076] 200 mg of the COF-25 porous material obtained in Example 3 are dried (100° C., vacuum, 24 hours). Next, they are suspended in 20 ml of anhydrous DCM, 200 mg of the prodrug Suc-DTX (0.22 mmol, prepared according to Example 1) are added, 84 mg 1-oxide (bis(dimethylamino)methylene)-3H-[1,2,3]triazolo[4,5-bipiri-dine hexafluorophosphate (V) (HATU, 0.22 mmol) and 115 μl N,N′-diisopropylethylamine (DIPEA, 0.66 mmol). The reaction mixture is left stirring at room temperature under an argon atmosphere for 48 h. After this time, the suspension is filtered, washed with anhydrous DCM and the grey solid obtained is dried under vacuum (8 torr) for 24 hours. 224 mg of nanoparticles of the CF-25 porous material are collected the surface of which is covered with terminal amine groups to which Suc-DTX molecules are bound by amide bond (COF-25-DTX, FIG. 1).

Example 5: Preparation of Nanoparticles of the COF-25 Porous Material with Terminal Methoxy Groups and Terminal Amino Groups on which Suc-DTX and a PEG Molecule are Bound by Covalent Bond

[0077] 200 mg of the COF-25-DTX porous material obtained in Example 4 are dried (80° C., vacuum, 24 hours), and suspended in 30 ml of anhydrous DCM, and 250 μl of N,N′-diisopropylethylamine (DIPEA, 0.46 mmol) are added under argon. Next, 150 mg of 2,5,8,11-tetraoxatetradecane-14-oic acid succinimidyl ester (PEGS) is added and the mixture is stirred at room temperature for 16 h. After removing the solvent on a rotary evaporator, the solid is re-dispersed by stirring in 100 ml of ethanol. This suspension is filtered and washed with ethanol. Finally, the solid is lyophilised (−55° C., 16 h). 268 mg of nanoparticles of the COF-25-DTX@PEG porous material are collected the surface of which is covered with terminal amine groups to which Suc-DTX and PEG.sub.3 molecules are bound by amide bond.

Example 6: Preparation of Nanoparticles of the COF-25 Porous Material with Terminal Methoxy Groups and Terminal Amino Groups on which Suc-DTX, the Acid-11-aminoundecanoic Linker and the Anti-FOLH1 Directing Molecule (Clone C803N, Creative Diagnostics; PROTEIN DATA BANK ID FOLH1: 1Z8L) are Bound by Covalent Bond

[0078] 100 mg of COF-25-DTX nanoparticles obtained according to Example 4 are dried (60° C., vacuum, 24 hours), and suspended in 10 ml of anhydrous DCM. Next, 61 mg of 11-aminoundecanoic acid (AU, 0.30 mmol), 114 mg of HATU (0.30 mmol) and 157 μl of DIPEA (0.90 mmol) are added, and it is left stirring at room temperature and under argon atmosphere for 48 hours. After this time, the suspension is filtered, washed with anhydrous DCM, and the solid obtained is dried under vacuum (8 torr) for 24 hours. 115 mg of nanoparticles of COF-25 porous material are obtained the surface of which is covered with terminal amine groups to which Suc-DTX and 11-aminoundecanoic acid molecules are bound, respectively, by amide bond (COF-25-DTX/AU, FIG. 1).

[0079] 10 μl of anti-FOLH1 antibody (clone C803N, Creative Diagnostics, 5 mg/ml) are suspended in 100 μl of commercial 2-(N-morpholino) ethanesulphonic acid monohydrate buffer (MES, 0.1 M, pH 6.0). Next, 100 μl of a solution of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC, 8 mg/ml) and 100 μl of a solution of N-hydroxysuccinimide (NHS, 20 mg/mL) are added, and left stirring at room temperature for 30 minutes. After this time, 1 mg of COF-25-DTX/AU nanoparticles obtained according to the previous step are dispersed in 1 ml of PBS (1×, pH 8.5) and added to the previously prepared antibody solution. It is left stirring for 2 h at room temperature. At the end of the reaction period, the sample is centrifuged (16100 g), the residue is washed with PBS (1 x, pH 7.4), lyophilised at −55° C. for 16 hours and stored at 4° C. until use thereof (COF-25-DTX/aFOLH1, FIG. 1).

[0080] The amount of antibody bound by amide bond to the terminal amino group of the AU spacer chain is determined by the Bradford method. In this assay, the amount of protein (antibody) not conjugated to the particles is quantified by visible spectrophotometry (□=595 nm) through the binding that is established between a dye (Coomassie blue G-250) and the protein under study.

Example 7: Preparation of Nanoparticles of the COF-5 Porous Material with Terminal Methoxy Groups and Terminal Amino Groups on which Suc-DTX, the Imaging Agent [.SUP.18.F]FDG, the 11-Aminoundecanoic Acid Linker, and the Anti-FOLH1 Directing Molecule (Clone C803N, Creative Diagnostics; (PROTEIN DATA BANK ID FOLH1: 1Z8L) are Bound by Covalent Bond

[0081] A freshly obtained aliquot of [.sup.18F]-fluorodeoxyglucose [.sup.18F]FDG (˜3 mCi) is dried at 110° C. and vacuum. 1 ml of a suspension in sterile COF-25-DTX/aFOLH1 nanoparticles (1 mg/ml) Milli Q® water obtained in Example 6 is added on the dry [.sup.18F]FDG. The resulting suspension is stirred at room temperature for 15 min in a sterile environment. After this time, it is centrifuged (16100 g) and washed several times with sterile Milli Q® water. The final mixture contains 1 mg/ml of COF-25-DTX/aFOLH1/[.sup.18F] in sterile Milli Q® water and is ready for intratumour or parenteral administration.

Example 8: Preparation of Nanoparticles of the COF-25 Porous Material with Terminal Methoxy Groups and Terminal Amino Groups on which the Imaging Agent Rhodamine B is Bound by Covalent Bond

[0082] 200 mg of the COF-25 porous material obtained in Example 3 are dried (100° C., vacuum, 24 hours). It is subsequently suspended in 10 ml of anhydrous DCM. Next, 3.0 mg of rhodamine B (0.006 mmol), 11 mg of HATU (0.030 mmol) and 16 μl of DIPEA (0.090 mmol) are added, and it is left stirring at room temperature and under an argon atmosphere for 48 hours. After this time, the suspension is filtered, washed with anhydrous DCM, and the solid obtained is dried under vacuum (8 torr) for 24 hours. 233 mg of nanoparticles of the COF-25 porous material are collected the surface of which is covered with terminal amine groups to which rhodamine B molecules are bound by amide bond (COF-25-RhB, FIG. 2).

Example 9: Preparation of Nanoparticles of the COF-25 Porous Material with Terminal Methoxy Groups and Terminal Amino Groups on which the Imaging Agent Rhodamine B, the 11-Aminoundecanoic Acid Linker, and the Anti-FOLH1 Directing Molecule (Clone C803N, Creative Diagnostics; (PROTEIN DATA BANK ID FOLH1: 1Z8L) are Bound by Covalent Bond

[0083] 100 mg of COF-25-RhB nanoparticles obtained according to Example 8 are dried (60° C., vacuum, 24 hours), and suspended in 10 ml of anhydrous DCM. Next, 61 mg of 11-aminoundecanoic acid (AU, 0.30 mmol), 114 mg of HATU (0.30 mmol) and 157 μl of DIPEA (0.90 mmol) are added, and it is left stirring at room temperature and under argon atmosphere for 48 hours. After this time, the suspension is filtered, washed with anhydrous DCM, and the solid obtained is dried under vacuum (8 torr) for 24 hours. 112 mg of nanoparticles of the COF-25 porous material are obtained the surface of which is covered with terminal amine groups to which rhodamine B and 11-aminoundecanoic acid molecules are bound, respectively, by amide bond (COF-25-RhB/AU, FIG. 2).

[0084] 10 μl of anti-FOLH1 antibody (clone C803N, Creative Diagnostics, 5 mg/ml) is suspended in 100 μl MES (0.1 M, pH 6.0). Next, 100 μl of a solution of EDC (8 mg/ml) and 100 μl of a solution of NHS (20 mg/ml) are added, and left stirring at room temperature for 30 minutes. After this time, 1 mg of COF-25-RhB/AU nanoparticles obtained according to the previous step are dispersed in 1 ml of PBS (1×, pH 8.5) and added to the previously prepared antibody solution. It is left stirring for 2 h at room temperature. At the end of the reaction period, the sample is centrifuged (16100 g), the residue is washed with PBS (1×, pH 7.4), lyophilised at −55° C. for 16 hours and stored at 4° C. until use (COF-25-RhB/aFOLH1, FIG. 2).

Example 10: DTX Release in Phosphate Buffered Saline

[0085] A 5 mg/ml suspension of nanoparticles obtained according to Example 4 is prepared in 1 ml of PBS in a 2 ml eppendorf tube and incubated at 37° C. for the different times established. The samples incubated at the different times are centrifuged, the residue is washed with methanol (2×1 ml) and the supernatant is lyophilised at −55° C. for 24 h. The residue obtained is reconstituted with 1 ml of a methanol solution. 5 μl of said solution are injected into an HPLC equipment. The DTX peak area is integrated and compared to a calibration curve. All measurements were made in triplicate, calculating the mean±standard deviation (SD) in each case.

[0086] It is verified that the material presents great stability in PBS, with a release of around 10% of the total load in the first hour of incubation, characterised as a mixture of free DTX and Suc-DTX (approximately 20% of the total). This initial release is related to prodrug molecules adsorbed inside the pores. Subsequently, the release of DTX is low until at least 6 h of exposure (15% of the total load).

[0087] FIG. 5 shows the release curve in PBS of DTX from the COF-25-DTX theranostic system.

Example 11: Determination of the IC.SUB.50 .by Means of MTT Assay

[0088] The in vitro cytotoxic activity of the theranostic system was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) bromide test. In these assays, the proliferation of living cells is determined via mitochondrial dehydrogenase activity: viable mitochondria reduce MTT by generating MTT-formazan crystals that are quantifiable by visible spectrophotometry.

[0089] Two types of human prostate adenocarcinoma cell lines were used for these assays: LNCaP (with membrane receptors FOLH1, PROTEIN DATA BANK ID FOLH1: 1Z8L) and PC3 (without FOLH1 receptors).

[0090] The cells are seeded in 96-well plates at a rate of 10,000 cells/well in both cases, 24 h before incorporating the nanoparticles. Aliquots of 3 mg ml-.sup.1 of DTX in DMSO are prepared and kept at −20° C. When required, an aliquot is thawed, and working aliquots (100 μg/ml by dilution in enriched RPMI medium) are prepared. Alternatively, aliquots of 100 μg/mL DTX are prepared in enriched RPMI medium and kept at −20° C. until required.

[0091] A suspension of nanoparticles obtained according to Example 4 is prepared in RPMI medium enriched with 10% foetal bovine serum and 1% penicillin/streptomycin at 37° C., in a humid air (95%) and CO.sub.2 (5%) atmosphere, with an equivalent concentration of 100 μg/ml of DTX and treated with ultrasound to improve the dispersion of the nanoparticles. The suspension is divided into aliquots that are kept at −20° C. until required.

[0092] Alternatively, a suspension of nanoparticles obtained according to Example 6 is prepared in RPMI medium enriched with 10% foetal bovine serum and 1% penicillin/streptomycin at 37° C., in a humid air (95%) and CO.sub.2 (5%) atmosphere, with an equivalent concentration of 100 μg/ml DTX and treated with ultrasound under identical conditions.

[0093] The cells are treated with DTX or with nanoparticles obtained according to Example 4, in a range of DTX doses between 0.005 μg/ml and 100 μg/ml for 72 hours. Alternatively, the cells are treated with nanoparticles obtained according to Example 6 for 72 hours. At the end of the incubation period, 10 μl (5 mg/ml) of the MTT solution are added to each well and 4 hours later the formazan crystals are dissolved with isopropanol, and the absorbance at 570 nm is measured. The percentage of cell survival relative to untreated cells is determined and the values of IC.sub.50 from the curve DTX concentration-(%) live cells are calculated. The values of IC.sub.50 are listed in Table 2.

TABLE-US-00002 TABLE 2 Values of IC.sub.50 (mean ± SD, in μg/ml) for free DTX, COF-25-DTX from Example 4 and COF-25-DTX/aFOLH1 from Example 6 in PC3 and LNCaP cells (n = number of experiments). Cell line DTX COF-25-DTX COF-25-DTX/aFOLH1 n LNCaP 0.80 ± 0.20 0.14 ± 0.02 0.02 ± 0.01 3 PC3 — — — 3