RELEASE OF SUBSTANCES IN SENESCENT CELLS

Abstract

The invention relates to nanodevices for the controlled release of substances which comprises a support coated in oligosaccharides, wherein said oligosaccharides comprise at least 3 units of monosaccharides, and wherein at least one of the monosaccharides is galactose. These nanodevices release their load specifically in senescent cells. The invention also encompasses the method of obtainment and its uses.

Claims

1. A nanodevice for the controlled release of a substance, wherein the nanodevice comprises a support coated in oligosaccharides comprising at least 3 units of monosaccharides, and at least one of the monosaccharides is galactose.

2. The nanodevice of claim 1, wherein the oligosaccharides comprise between 3 to 10 units of monosaccharides.

3. The nanodevice of claim 1, wherein at least 50% of the monosaccharides are galactose.

4. The nanodevice of claim 1, wherein the diameter of the nanodevice is between 10 nm and 250 nm.

5. The nanodevice of claim 1, wherein the support comprises mesoporous silica.

6. The nanodevice of claim 1, wherein the substance is selected from an indicator, a compound for the reactivation of telomerase, and a cytotoxic compound.

7. The nanodevice of claim 1, wherein the substance is selected from GSE 24-2 peptide, TAT2 peptide, cisplatin, oxaliplatin, carboplatin, doxorubicin, camptothecin, etoposide, 5-fluorouracil, gemcitabine, cytosine, arabinoside, 6-mercaptopurine, taxane, vincristine, vinblastine, adriamycin methotrexate, and pemetrexed.

8. The nanodevice of claim 1, formulated as a pharmaceutical composition.

9. The nanodevice of claim 8, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

10. The nanodevice of claim 8, wherein the pharmaceutical composition is in the form of a tablet, capsule, powder, granule, solution, suppository, syrup, cream, lotion, gel, or powder.

11. A method of making a nanodevice for the controlled release of a substance comprising a support coated in oligosaccharides, wherein the method comprises the steps of: a) functionalizing the oligosaccharides with a silane group; b) suspending the support in a solution of the substance to be released; c) adding an excess of the oligosaccharides of a) to the solution of b) to form a mixture, wherein said oligosaccharides comprise at least 3 units of monosaccharides, and wherein at least one of the monosaccharides is galactose.

12. The method of claim 11, wherein the suspending of b) occurs between 1 and 48 hours.

13. The method of claim 11, wherein solution of b) comprises methanol, ethanol, propanol, isopropanol, acetone, or acetonitrile.

14. The method of claim 11, wherein the proportion of oligosaccharide functionalized in a) to the support is 1:5 by mass.

15. The method of claim 11, further comprising d) allowing the mixture of c) to stir for between 0.5 hours to 20 hours.

16. A method of treating a disease characterized by overexpression of -galactosidase in a subject, wherein the method comprises administering to the subject a nanodevice for the controlled release of a substance, and the nanodevice comprises a support coated in oligosaccharides comprising at least 3 units of monosaccharides, wherein at least one of the monosaccharides is galactose.

17. The method of claim 16, wherein the overexpression of -galactosidase occurs in a senescent cell.

18. The method of claim 17, wherein the disease is selected from Wiedemann-Rautenstrauch syndrome of neonatal progeria, Werner syndrome of adult progeria, Hutchinson-Gilford syndrome, Rothmund Thompson syndrome, Mulvill-Smith syndrome, Cockayne syndrome, Dyskeratosis Congenita, idiopathic pulmonary fibrosis, aplastic anemia, Type 2 diabetes, and degeneration of cartilage.

19. The method of claim 17, wherein the subject is a human.

20. A method of detecting a senescent cell in a subject, the method comprising administering to the subject a nanodevice for the controlled release of a substance, and the nanodevice comprises a support coated in oligosaccharides comprising at least 3 units of monosaccharides, wherein at least one of the monosaccharides is galactose.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0069] FIG. 1. Synthesis of the derived galactooligosaccharide (I).

[0070] FIG. 2. X-ray diffractogram of the mesoporous silica and Transmission Electron Microscopy (TEM) images of the nanodevices of the invention loaded with Rhodamine-B (S1) and calcined MCM-41 support; I: Intensity, 2d: 2 degrees, S1: nanodevices loaded with Rhodamine coated with galactooligosaccharides (I), MCM-41 c: calcined mesoporous silica nanoparticles, MCM-41 m: non-calcined mesoporous silica nanoparticles, nm: nanometres.

[0071] FIG. 3. Graph of release of the Rhodamine-B in the presence of -gal; B: Rhodamine B; G: galactooligosaccharide.

[0072] FIG. 4. Release profiles of Rhodamine-B of S1 in the absence and presence of -gal enzyme in water at pH 7.5 and room temperature; P: presence of -gal; A: absence of -gal; A.U.: Fluorescence intensity in %; T: time (hours).

[0073] FIG. 5. % Cell viability in the presence of the nanoparticles of the invention. A: % Viability of the yeast cells in the presence of the indicated quantities of S1; B: % Viability of the H460 cells after 48 hours of treatment with the indicated quantities of S1; % V: % Viability.

EXAMPLES

Example 1 Synthesis of the Silane Derivative of the Oligosaccharide

[0074] The galactooligosaccharide (I) (FIG. 1, n=3-6) was obtained as a syrup with a pH of 3.8. This syrup was diluted in water and the pH was increased up to 7 with NaHCO.sub.3. Then, the solution was lyophilised obtaining a white solid. Afterwards, 5 grammes of the solid were dissolved in 100 ml of anhydrous ethanol, and a solution of 3-aminopropyltriethoxysilane (2, 5.85 ml, 25 mmol) was added. The reaction mixture was stirred for 24 h at room temperature. The solvent was evaporated under reduced pressure to obtain a white solid (I). FIG. 1.

[0075] .sup.1H NMR (300 MHz, D.sub.2O): 0.42 (t, 2H, CH.sub.2Si), 1.02 (t, 9H, CH.sub.3CH.sub.2OSi), 1.53 (m, 2H, CH.sub.2CH.sub.2Si), 2.74 (t, 2H, NHCH.sub.2CH.sub.2CH.sub.2Si), 3.20-3.77 (m, nH, galactooligosaccharide, CH.sub.3CH.sub.2OSi), 5.13 (d, 1H, OCHO) ppm, .sup.13C NMR (300 MHz, D.sub.2O): 9.62 (CH.sub.2Si), 16.65 (CH.sub.3CH.sub.2OSi), 21.74 (CH.sub.2CH.sub.2Si), 41.96 (NHCH.sub.2CH.sub.2CH.sub.2Si), 57.25 (CH.sub.3CH.sub.2OSi), 61.84 (HOCH.sub.2CH), 72.67-78.11 (HOCH), 89.57 (OCHCH), 94.84 (OCHNH), 100.25 (OCHO) ppm.

Example 2 Synthesis of the Mesoporous Support

[0076] The mesoporous support that was used was mesoporous silica, specifically MCM-41.

[0077] N-cetyltrimethylammoniumbromide (CTABr, 2.00 g, 5.48 mmol) was dissolved in 960 ml of deionised water. NaOH (ac) (2.00 M, 7.00 ml) was added to the CTABr solution. Next the temperature was adjusted to 95 C. Tetraethyl orthosilicate (TEOS, 10.00 ml, 5.14*10.sup.2 mol) was then added drop by drop to the previous solution. The mixture was left to stir for 2.5 h and a white precipitate was obtained. The solid product was centrifuged and washed with deionised water until neutral pH and dried at 60 C. for 13 h. To prepare the final porous product MCM-41, the dry solid product was calcined at 550 C. in an oxidising atmosphere for 5 h to remove the surfactant, also known as the structure directing agent.

[0078] The X-ray diffractogram (XRD) of the MCM-41 material (FIG. 2) shows four small angle reflections typical of the hexagonal geometry that can be indexed as Bragg peaks (100), (110), (200) and (210). A significant displacement in the reflection (100) can be clearly appreciated and a widening of the peaks (110) and (200) in the XRD in powder of the calcined MCM-41 sample. This corresponds to a unit cell contraction of about 6-8 due to the condensation of silanols during the calcination stage. In spite of this clear partial loss of order, observation of the reflections (100) and (200) indicates that certain relative mesoporous symmetry is preserved after calcination. The Na.sub.2 adsorption desorption isotherms of the mesoporous nanoparticles shows a typical type IV curve with a specific surface of 999.6 m.sup.2g.sup.1, and a pore volume of 1.17 cm.sup.3g.sup.1. From the XRD, porosimetry, and Transmission Electron Microscopy (TEM) studies the a.sub.0 parameter of the hexagonal cell (4.43 nm), pore diameter (2.45 nm) and wall thickness value (1.98 nm) were calculated.

Example 3. Synthesis of the Nanodevices of the Invention Loaded with Rhodamine-B (Fluorescent Substance)

[0079] 50 mg of MCM-41 and 19 mg of Rhodamine-B (0.04 mmol) were suspended in 10 ml of ethanol in a round bottom flask in an inert atmosphere. The mixture was left stirring for 24 h at room temperature with the objective of achieving maximum load in the pores of MCM-41. Then, an excess of (I), silane derivative of the galactooligosaccharide, (100 mg in 10 ml of ethanol) were added and the final mixture was left stirring for 5.5 h at room temperature. Finally, the nanodevices of the invention loaded with Rhodamine-B (S1) were filtered and washed with water.

[0080] The reflections (110) and (200) were partially lost, certainly because of a reduction in the contrast due to the fact that the empty space of the pores was filled with the Rhodamine-B. Also, the preservation of the mesoporous structure in the final functionalised solids was confirmed by TEM.

[0081] To determine the content of Rhodamine-B in the final hybrid material, 30 mg of S1 was suspended in 75 ml of water at pH 7.5 in the presence of 7500 ppm of -galactosidase. The mixture was left stirring at room temperature for 72 h to achieve the full release of the Rhodamine-B. Afterwards, the suspension was centrifuged and the supernatant was isolated and dried at reduced pressure. The crude obtained was dissolved in acetone and filtered. The solution obtained was dried and the residue was dissolved in 4 ml of ethanol. The final content of Rhodamine-B was determined by means of a calibration curve based on the increase in the absorption band centred in 550 nm with the concentration, obtaining 0.14 g/g SiO.sub.2 in weight.

[0082] The content of the galactooligosaccharide (I) in S1 was calculated through thermogravimetric analysis, 0.28 g/g SiO.sub.2 in weight.

Example 4. Load Release Studies

[0083] 10 mg of S1 were suspended in 18.75 ml of water at pH 7.5 and next 6.25 ml of a lactozyme enzyme solution (-D-galactosidase) were added (0.3 g of enzyme in 10 ml of water at pH 7.5). The release of the Rhodamine-B of the pores to the aqueous solution was monitored through the emission spectrum of Rhodamine-B centred in 580 nm.

Example 5. Experiments of Load Release in S. cerevisiae

Strains and Culture Conditions

[0084] All the strains of yeast used in this study have the genetic base of BY4741 (MAT leu20, his30, met150, ura30). The -Gar.sup.oe strain was generated by transforming the WT BY4741 strain with expression vector -gal p477 (URA3). Standard methods were used for the culture and manipulation of the yeasts. The minimum medium (SD) contained 2% glucose, 0.67% of yeast nitrogen base and the amino acid supplements appropriate for maintaining the selection of URA3. The yeast cultures were maintained at 30 C.

Fluorescence Microscope and Cell Viability Studies

[0085] The BY4741(WT, wild type) and -Gar.sup.oe yeasts were cultured in SD medium overnight at 28 C. stirring continuously in a density of 10.sup.8 cells/ml. Then, aliquotes of 1 ml of this suspension were centrifuged and re-suspended in 100 l of SD medium at pH 3.7 containing 5 mg/ml of S1 nanoparticles loaded with Rhodamine-B and were incubated for 6 h at 40 C. The intracellular release was monitored by fluorescent microscopy using a NIKON ECLIPSE 600 microscope equipped with a fluorescent lamp NIKON Y-FL, .sub.ex=540 nm, .sub.em=650 nm. After the incubation period, approximately 300 cells were seeded in a YPD plate and were incubated for 72 h. Finally, the formation of colonies was quantified and cell viability was calculated. The experiments were repeated twice containing triplicates. The data is expressed as (meanS.D., standard deviation). The incubation with S1 nanoparticles did not affect cell viability throughout the experiment (table 1, FIG. 5 A)

TABLE-US-00001 TABLE 1 % Viability of -Gal.sup.oe yeast incubated with S1. S1 (mg/ml) % Viability S.D. 0 100 2 1 100 5 2.5 98 5 5 97 6 10 96 10

[0086] Fluorescence was only detected in the -Gal.sup.oe east cells with great -gal activity. In these conditions the percentage of internalisation in -Gal.sup.oe, i.e., of cells stained with the Rhodamine B colouring is 85% with an S.E. of 5% (p<0.01) for 5 mg/ml of S1.

TABLE-US-00002 yeast -Galactosidase Activity (enzymatic units) S.D. WT 2.0000 1.0000 -Galoe 32.0000 2.0000

Example 6. Experiments of Load Release in Human Cells

Cell Culture

[0087] The reagents for the tissue culture were obtained from GIBCO/Invitrogen Corporation/Life Technologies Life Sciences. Dermal fibroblasts from two patients with X-linked Dyskeratosis congenita (X-DC) (X-DC1774 and X-DC4646) and control fibroblasts with a high number of passages (DC1787) were obtained from the Coriell Cell Repository. The X-DC1774 and X-DC4646 fibroblasts belong to males with mutations c.109_11delCTT, c.385A>T and c.5C>T DKCI respectively. The DC17871 control fibroblasts belong to a woman carrier of mutation c.109_11delCTT DKCI. The H460 cells (non-small cell lung cancer cells expressing telomerase) were obtained from ATCC and were maintained in RMPM supplemented with 10% foetal bovine serum (FBS) and the primary human cells were maintained in MEM (Minimum Essential Medium, GIBCO) supplemented with 15% FBS at 37 C. and 5% of CO.sub.2. The culture medium was supplemented with gentamicin (56 g/ml and L-glutamine (2 mM).

Analysis of Senescence by Fluorescent Microscopy In Vivo.

[0088] Control and X-DC (1*10.sup.4 cells) fibroblasts were placed on 6 plates of wells and were fixed after 4 days to test the activity of the -galactosidase SA--gal (Senescence Detection Kit, BioVision, USA). The location of the load release of the nanoparticles was visualised using fluorescent microscopy. To this end, the cells were cultured in ibiTreat (Ibidi) microscopy chambers of eight wells with plates of 1 and were treated with the mesoporous particles S1. The fluorescence was visualised by means of in vivo microscopy during 48 h in a Microscope Observer Z1. Image acquisition and processing was carried out as follows: type, numerical aperture and magnification of the objective lenses: APOCHROMAT Plan by Zeiss 20, Temperature 37 C. and 5% CO.sub.2. Camera: Cascade 1k. Acquisition software: Axiovision 4.8.

[0089] The percentage of senescent cells (blue) was calculated in the images by bright field microcopy at 100 magnification (Nikon Eclipse TS100 Microscopy, USA). The following values were obtained:

TABLE-US-00003 TABLE 2 % of senescent cells. Cells % of senescent cells DC1787 35.5 X-DC1774 55.0 X-DC4646 65.00 H460 0.3

Cell Viability Assays

[0090] The viability of H460 and DC1787 in the presence of S1 was determined using a crystal violet staining method. The cells were cultured in 24-well plates, treated with different quantities of mesoporous silica particles and 48 h after treatment were fixed with 1% glutaraldehyde, were washed with PBS and the remaining cells were stained with 0.1% crystal violet. The colorimetric assay using 595 nm Elisa was used to estimate the number of cells per well. The experiments were repeated 3 times in quadruplicate.

[0091] The assays showed that the nanoparticles of the invention are not toxic in human cells and in yeasts (FIG. 5 B).

Example 7. Control Experiment

[0092] To verify that the lack of Rhodamine-B stain in the H460 cells was not an artefact, nanoparticles loaded with Rhodamine-B were prepared and coated in hydrolysed starch (S2). These nanoparticles are similar to S1 but contain an oligosaccharide capable of hydrolysing by the amylase in lysosomes. The S2 nanoparticles were synthesised following a similar method to the one described, using mesoporous silica as support, Rhodamine B as load and hydrolysed starch (Glucidex 47) as coating.

[0093] When S2 was used in the internalisation studies under similar conditions to those described above, a clear staining was detected in all cell lines used, indicating that the release of S1 was clearly dependent on the presence of -galactosidase.