A COMPOSITION FOR CONTROLLED RELEASE OF A BIOMOLECULE, METHOD OF PREPARATION AND USES THEREOF

Abstract

The present disclosure relates to the modulation of cell activity by sequential release of several biomolecule from a given nanocarrier using a given wavelength. The present disclosure describes a composition for controlled release of biomolecules comprising: a gold nanoparticle; at least two different biomolecules; at least two different oligonucleotides having different melting points, binding said biomolecules to the surface of the gold nanoparticle; wherein the binding is obtainable via hybridization of complementary oligonucleotides; wherein each of the oligonucleotides is a photo-active such that the respective biomolecule is releasable by photo-activation. The composition of the present disclosure may be use in medicine, namely in the treatment of cancer, aging diseases, and accelerated aging syndromes, infectious diseases such as AIDS, epigenetic diseases such as polycystic ovary syndrome, or cardiac diseases, such as cardiac regeneration.

Claims

1. A composition for controlled release of biomolecules comprising: a gold nanoparticle; at least two different biomolecules; at least two different oligonucleotides having different melting points, binding said biomolecules to the surface of the gold nanoparticle; wherein the binding is obtainable via hybridization of complementary oligonucleotides; wherein each of the oligonucleotides is a photo-active such that the respective biomolecule is releasable by photo-activation.

2. The composition according to claim 1 wherein the biomolecule is selected from a list consisting of: a peptide, a protein a micro RNA, a mRNA, and mixtures thereof.

3. The composition according to the previous claim 1 wherein the oligonucleotide sequences comprise between 13-30 base pairs, preferably 13-16 base pairs.

4. The composition according to the previous claims wherein the melting point of a oligonucleotide sequence of the plurality of oligonucleotides sequences varies between 40-90 C., preferably between 50-70 C.

5. The composition according to the previous claims wherein a first oligonucleotide sequence of the plurality of oligonucleotides sequences has a melting point of 50-55 C., and a second oligonucleotide sequence of the plurality of oligonucleotides sequences has a melting point of 65-70 C.

6. The composition according to the previous claims wherein the plurality of oligonucleotides sequences further comprise a poly thymine spacer.

7. The composition according to the previous claims wherein the gold nanoparticle is a nanorod.

8. The composition according to the previous claims comprising at least two oligonucleotides, wherein each oligonucleotide is a sequence 95% identical to: SEQ. ID. 1; SEQ. ID. 2; SEQ. ID. 3; SEQ. ID. 4; and mixtures thereof.

9. The composition according to the previous claims comprising at least two oligonucleotides, wherein each oligonucleotide is a sequence identical to: SEQ. ID 1, SEQ. ID 2, SEQ. ID 3, SEQ. ID 4, or mixtures thereof.

10. The composition according to the previous claims further comprising a cell transfection enhancer.

11. The composition according to the previous claim wherein the cell transfection enhancer is a penetrating peptide, in particular cecropin melitin.

12. The composition according to the previous claims wherein the density of oligonucleotide per gold nanoparticle is between 10-400.

13. The composition according to the previous claims wherein the composition is activated with light wavelengths between 600-1000 nm.

14. The composition according to the previous claims wherein the gold nanoparticle has a size between 10-45 nm, preferably a size between 15-30 nm.

15. The composition according to the previous claims the gold nanoparticle has aspect ratio of 1.5-10, preferably between 3-3.4.

16. The composition according to any one of the previous claims, wherein the composition is a topic formulation or an injectable formulation.

17. The composition according to the previous claims for the use in medicine or veterinary.

18. The composition according to the previous claims for the use in regenerative medicine.

19. The composition according to the previous claims for the use in cell reprograming, in particular adult stem cells, and/or embryonic stem cells.

20. The composition according to the previous claims for the use in the treatment of diseases that respond positively to cell reprograming.

21. The composition according to the previous claims for the use in the treatment of retinal diseases, neurodegenerative diseases, or viral infections.

22. The composition according to the previous claims for the use in the treatment of cancer, aging diseases, and accelerated aging syndromes, infectious diseases such as AIDS, epigenetic diseases such as polycystic ovary syndrome, or cardiac diseases, such as cardiac regeneration.

23. Use of the composition describe in any of the previous claims as a coating of medical devices wherein the composition further comprises an adhesive to immobilize the nanoparticles on top of the medical device.

24. Use of the composition describe in any of the previous claims wherein the medical device is a patch, a catheter, a stent, a contact lens or a pacemaker.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0079] The following figures provide preferred embodiments for the present disclosure and should not be seen as limiting the scope of the disclosure.

[0080] FIG. 1: Schematic representation of AuNR-DNA-protein. A DNA-protein conjugate is bound to the AuNR surface through hybridization with a complementary DNA strand attached to the AuNR via thiol gold chemistry. Light induced sequential release of different proteins from the AuNR upon the application of different stimuli.

[0081] FIG. 2: Light induced release profiles of DL-BSA attached to gold nanorods. a) Effect of the laser power in the amount of protein released b) Percentage of protein in the supernatant and attached to the NR after irradiation c) Effect of the melting temperature of the DNA on the amount of BSA released from the NR. Quantifications were done by measuring the fluorescence of DL-BSA (n=3). d) Laser induced sequential release of DL.sub.488-BSA and DL.sub.550-BSA from AuNR. A suspension of DL.sub.488;550-BSA-dsDNA-AuNR-BSA was first irradiated for 2 min at 1.25 W/cm.sup.2 and then centrifuged in order to collect the supernatant. The AuNR were resuspended and irradiated again at 2 W/cm.sup.2 for 3.5 min and then centrifuged. The fluorescence of both supernatants was measured in a Biotek HT plate reader.

[0082] FIG. 3: A) Confocal images of SC-1 cells incubated with DL.sub.488-BSA-dsDNA-AuNR.sub.TRITC (50 g/mL; DNA Tm 68.9 C.) without and with laser irradiation (1.25 W/cm.sup.2 and 2 W/cm.sup.2 for 2 min). b and c) Intensity and coefficient of variation of the signal of BSA-DL.sub.488. d) colocalization between AuNR.sub.TRITC and DL488-BSA.

[0083] FIG. 4: Intracellular delivery of two proteins. Fibroblasts were incubated with AuNR-TRITC conjugated with DL650-BSA-dsDNA51.7 and DL488-BSA-dsDNA68.9 for 4 h. Cells were then washed with cell culture media and irradiated for 2 min at 1.25 W cm.sup.2. A subset of samples was fixed with 4% PFA after irradiation and the other group was incubated for additional 5 min before being irradiated for 2 min at 2 W/cm.sup.2 and fixed afterwards. The amount of protein released was calculated as % BSAR=100(MCbeforeMCafter)/MCbefore, where MCbefore is the Manders' colocalization coefficient before irradiation and MCafter is the Manders' colocalization coefficient after irradiation. Results are AverageSEM, n=3 (3 samples, 5 microscope fields per sample). Unpaired t-test was used to compare each condition (p value0.0001).

[0084] FIG. 5: Activity of beta-galactosidase in SC-1 after 4 h incubation with Gal-dsDNA.sub.51.7AuNR (50 g/mL) After irradiation, the activity of beta-galactosidase was determined with a beta galactosidase Detection Kit (Abcam). Briefly, the cells were fixed incubated overnight with X-Gal substrate and then observed in confocal microscope using 633 nm laser excitation. A) Fluorescence of Xgal staining assessed by confocal microscopy. B) corrected total cell fluorescence of SC-1 cells incubated with Xgal substrate after incubation with Gal-dsDNA.sub.51.7AuNR and irradiation with different laser powers. C) coefficient of variation of the xGal staining.

[0085] FIG. 6: Distribution of beta-galactosidase assessed by immunocytochemistry. Confocal images of SC-1 cells incubated with Gal-dsDNA.sub.51.7AuNR (50 g/mL) with and without laser irradiation. After laser treatment the cells were fixed and immunostained using a rabbit anti-beta galactosidase primary antibody and anti-rabbit alexa-fluor 488 as secondary antibody.

[0086] FIG. 7: Cytotoxicity of BSA-dsDNA.sub.51.7-AuNR. Mouse fibroblasts were seeded on a 96 well plate (410.sup.3 cells/well), left to adhere for 24 h and then incubated with different concentrations of BSA-dsDNA.sub.51.7-AuNR for 4 h. After incubation, the cells were washed with medium to remove non-internalized AuNR. Some of the conditions were irradiated for 2 min with 1.25 or 2 W/cm.sup.2. Then, the plate was left in the incubator for 24 h. The ATP production was measured by a Celltiter-Glo Luminescent Cell Viability Assay (Promega).

[0087] FIG. 8: Colocalization between calcein and lysotracker red in SC-1 cells incubated with BSA-dsDNA.sub.51.7-AuNR. Cells were incubated with 50 g/mL of BSA-dsDNA.sub.51.7-AuNR and 25 mM calcein for 4 h. After replacing the medium, cells were incubated with lysotracker red (100 nM) for 15 min and then irradiated for 2 min with 1.25 or 2 W/cm.sup.2 and observed in confocal microscope.

[0088] FIG. 9: Enzymatic activity of beta-galactosidase followed by absorbance at 420 nm. After laser irradiation with 0.57 W/cm.sup.2, NR suspension was centrifuged at 9000 g, the supernatant was collected and the pellet was resuspended in 10 mM phosphate buffer. Then, 50 L of supernatant or suspension were added to 100 L of ONPG (13 mg/mL) prepared in 0.1 M phosphate buffer The absorbance at 420 nm was measured for 30 min at 37 C. in a 96 well plate using a Synergy HT microplate reader. The slope of the curves was used to calculate the amount of active protein.

[0089] FIG. 10: Activity of beta-galactosidase in SC-1 cells after 4 h incubation with Gal-dsDNA.sub.51.7AuNR (50 g/mL). a) effect of the time between laser activation and the activity assay. B) influence of the time between the end of incubation and laser activation.

[0090] FIG. 11: Activity of beta-galactosidase in SC-1 mouse fibroblasts after 4 h incubation with Gal-dsDNA.sub.51.7AuNR (50 g/mL) in the presence of 100 M chloroquine. After irradiation, the activity of beta-galactosidase was determined with a beta galactosidase Detection Kit (Abcam).

[0091] FIG. 12: Enzymatic activity of beta-galactosidase followed by absorbance at 420 nm. 50 L of beta galactosidase solutions (0.4 g/m L) prepared in 0.1 M phosphate buffer (pH 6.0; 6.8; 7.0) were added to 100 L of ONPG (13 mg/mL) also prepared in 0.1 M phosphate buffer (pH 6.0; 6.8; 7.0). The absorbance at 420 nm was measured for 30 min at 37 C. in a 96 well plate using a Synergy HT microplate reader.

[0092] FIG. 13: Preparation of miR-dsDNA-AuNR conjugates. (a) Preparation of ssDNA-miR conjugates. miR-155 or miR302a were initially reacted with a heterofunctional linker (Sulfo-GM BS) by its terminal succinimide ester. The miR conjugate was then reacted with a ssDNA having a terminal thiol group. After reaction, the miR conjugate (miR155-ssDNA or miR302a-ssDNA) was purified by HPLC. (b) Preparation of AuNR-ssDNA. AuNRs were reacted with HS-ssDNA complementary to the strands of miR155-ssDNA or miR302a-ssDNA conjugates. The miR-ssDNA conjugates were then bound to the ssDNA-AuNR by hybridization. The surface of AuNR was then filled with thiol-PEG. Upon NIR irradiation, there is an increase in the temperature at the AuNR leading to the DNA de-hybridization and the release of miRs with different kinetics. The release kinetic depends on the heat generated (which depends on the power of NIR laser used) and the melting temperature of the oligonucleotides.

[0093] FIG. 14: Characterization of miR-ssDNA conjugates. Chromatograms of the HPLC purification of a) miR-155-ssDNA and b) miR-302a-ssDNA. c) and d) Characterization of HPLC fractions by electrophoresis. Lines a, b and c are relative to the reaction mixture, control miR and control ssDNA respectively. Lines 1-5 are relative to the HPLC fractions represented in each chromatogram.

[0094] FIG. 15: a) Fluorescence microscopy images of hEK-293T cells transfected with miR-ssDNA conjugates. Images correspond to 24 h transfection with 2.5 nM miR-ssDNA conjugates (60 h after transfection). Scale bar corresponds to 50 m. b and c) HEK.293T cells were transfected with lipofectamine RNAimax and miR-155-ssDNA or miR-302a-ssDNA. Cells were exposed to the conjugates for 4 h or 24 h and their fluorescence was monitored afterwards in a high-content fluorescence microscope. EGFP and mCherry fluorescence were normalized to the control (cells transfected with scramble miR).

[0095] FIG. 16EGFP and mCherry knockdown in HEK-293T after laser induced release of miR-ssDNA conjugates. a) mCherry fluorescence of cells transfected with supernatants from irradiated suspension of miR-155-dsDNA51.7-AuNR. b) EGFP fluorescence of cells transfected with supernatants from irradiated suspension of miR-302a-dsDNA.sub.68.9-AuNR. Briefly, suspensions of miR-155-dsDNA51.7-AuNR and miR-302a-dsDNA.sub.68.9-AuNR (20 g/mL in 10 mM phosphate buffer pH 7.4 with 30 mM NaCl) were irradiated with different laser powers and times of irradiation and immediately centrifuged. Then the supernatants were complexed for 20 min with lipofectamine RNAimax (35 L of supernatant complexed with 35 L of RNAimax diluted 1:50 in DMEM) and 20 L of this mixture was added to each well containing 100 L of DMEM with 10% FBS. After incubation, cells were kept in DMEM (10% FBS, 0.3 g/mL of Hoechst) and their fluorescence was monitored in a high-content fluorescence microscope (IN Cell 2200, GE Healthcare)

[0096] FIG. 17a) Confocal images of cells stained with lysotracker green after 4 h incubation with miR-155-dsDNA-AuNR-TRITC (50 g/mL) and cecropin-melittin. Scale bar is 30 urn. b) Intensity of the signal of AuNR-TRITC. c) Colocalization between AuNR-TRITC and lysotracker green expressed as Manders' overlap coefficient assessed by Image) analysis. In b and c, the results are expressed as AverageSD (n=3). *** denotes statistical significance (p<0.001) assessed by one-way ANOVA followed by Tukey's post-hoc test.

[0097] FIG. 18Cytotoxicity of DNA51.7-AuNR. HEK-293T cells were incubated with DNA51.7-AuNR (50 g/mL) without or with cecropin-melittin (5 and 10 M) for 4 h. Then cells were washed and new medium was added (DMEM with 10% FBS). Subsequently, cells were irradiated for 2 min at 1.25 W/cm.sup.2 and left in the incubator. After 2 h, cells were irradiated for 2 min at 2 W/cm.sup.2 and then incubated for additional 24 h at 37 C. Cell metabolism was evaluated by an ATP assay.

[0098] FIG. 19EGFP and mCherry knockdown after laser induced release of miR-ssDNA conjugates in HEK-293T. a) Normalization of mCherry fluorescence relative to EGFP fluorescence in cells incubated for 4 h with miR-155-dsDNA.sub.51.7-AuNR with and without laser irradiation. b) Normalization of EGFP fluorescence relative to mCherry fluorescence in cells incubated for 4 h with miR-302a-dsDNA.sub.68.9-AuNR with and without laser irradiation. Cell fluorescence was monitored in a high-content fluorescence microscope.

[0099] FIG. 20a) and b) Fluorescence microscopy images of cells incubated with miR-155-dsDNA-AuNR and miR-302a-dsDNA-AuNR for 4 h in the presence of cecropin melittin (10 M). After incubation cells were exposed to one laser stimulus (2 min at 1.25 W/cm.sup.2) or two laser stimuli (2 min at 1.25 W/cm.sup.2 and 2 min at 2 W/cm.sup.2) with an interval of 2 h between each stimulus. c) and d) quantification of cell fluorescence 48 h after laser irradiation.

[0100] The present disclosure relates to the modulation of cell activity the sequential release of several proteins from a given nanocarrier using a wavelength, in particular this disclosure relates with a system that is able to release sequentially several proteins from the same nanocarrier using only one wavelength and just varying the laser power or exposure time. Moreover, the formulation as well as the stimuli used for the controlled release are not cytotoxic. Finally, proteins immobilized on the NR surface are protected from degradation and light stimulation does not decrease their activity.

[0101] Au NR synthesis. Au NRs were prepared using the seed mediated method. For the preparation of the seed solution, chloroauric acid (HAuCl.sub.4) (0.1 M, 12.5 L) was added to a hexadecyltrimethylammonium bromide (CTAB) solution (0.1 M, 5 mL) and stirred vigorously for 5 min, after which an ice-cold sodium borohydride (NaBH4) (10 mM, 0.3 mL) was added. After stirring for 2 min the solution was kept at 25 C. For the preparation of growth solution of silver nitrate (AgNO.sub.3) (5 mM; 3.2 mL) was added to CTAB solution (0.1 M; 200 mL) and mixed gently, after which HAuCl.sub.4 (50 mM, 2 mL) was added. After mixing, ascorbic acid (0.1 M; 1.5 mL) was added. The solution changed from dark yellow to colourless. Finally, 1.5 mL of the seed solution (aged for 8 min at 25 C.) was added to the growth solution. The solution was kept at 28 C. for 2 h. The NRs were washed by centrifugation at 8.500 rpm and resuspended in water.

[0102] The CTAB on the NR surface was replaced using a method already reported with some modifications, in particular hexanethiol (1.5 mL) was added to the NR-CTAB suspension (1 mL; 2.5 nM). Then, acetone (3 mL) was added and the mixture was swirled for a few seconds. The aqueous phase became clear indicating ligand exchange and the organic phase containing the NRs was extracted. Then, a mixture of toluene (2 mL) and methanol (5 mL) was added to the organic phase. The solution was centrifuged at 5.000 g, 10 min, and the pellet was resuspended in 0.5 mL of toluene by brief sonication. The organic to aqueous phase was performed as follows. NR-hexanethiol (1 mL) in toluene was added to 9 mL of mercaptohexanoic acid (MHA; 5 mM; 9 mL) in toluene at 95 C. The reaction proceeded under reflux with magnetic stirring for 15 min. The precipitation of NRs indicated successful coating by MHA. After cooling to room temperature, the aggregates were washed twice with toluene by decantation. Finally, the NRs were washed with isopropanol to deprotonate the carboxylic groups and then the aggregates were redispersed in 1TBE. The ligand exchange was confirmed by zeta potential measurements.

[0103] Functionalization of NR-MHA with single strand DNA (ssDNA). Thiolated ssDNA (SEQ. ID. 1:5-HS-C6-TTTTTTTTTTTTTTTATAACTTCGTATA-3 or SEQ. ID. 2:5-HS-C6-TTTTTTTTTTTTGTCCGGGTCCAGGGC-3, purchased from Sigma-Aldrich) were deprotected with 100-fold excess of tris(2-carboxyethyl)phosphine (TCEP) over ssDNA. The NR suspension (0.5 mL; 0.5 nM) was incubated with the deprotected ssDNA in a molar ratio of 1:400 in 10 mM phosphate buffer containing 0.3% (w/v) of sodium dodecyl sulfate (SDS) In order to compensate for the repulsion between DNA and gold NR, a charge screening was performed after 3 h of incubation by the addition of 22.5 l of 0.45 M NaCl every 60 min. This was repeated 4 times and the incubation proceeded overnight. The NR suspension was centrifuged at 9000 g, the supernatant was collected and the pellet was resuspended in 10 mM phosphate buffer with 30 mM NaCl.

[0104] Labelling of BSA with a fluorescent dye. A solution of BSA (2 mg/mL, 4.8 nmol, in PBS) was mixed with DyLight 488 (50 g, 49.4 nmol), DyLight 550 (50 g, 48.07 nmol) or DyLight 650 (50 g, 46.9 nmol) and kept under orbital shaking for 2 h. After reaction, the solution was dialysed against PBS in a dialysis cassette (MWCO 10 kDa) for 48 h at 4 C. The final protein concentration and degree of labelling were determined by measuring the absorbance in Nanodrop at 280 nm and at the DyLight absorbance maximum.

[0105] Preparation of protein conjugated with ssDNA. Protein-ssDNA conjugates were prepared using N-[-maleimidobutyryloxy]sulfosuccinimide ester (sulfo-GMBS, Thermo Scientific) as linker. Briefly, a solution of protein (BSA-DyLight at 7.5 M or -galactosidase at 3.5 M in PBS pH 7.4) was reacted with sulfo-GMBS in a 20-fold molar ratio for 30 min at room temperature. The excess of linker was removed by ultrafiltration with Nanosep 30 kDa (Pall Corporation) and the purified protein (7.5 M) was reacted with thiolated DNA (22.5 M) in a final volume of 150 L of PBS for 2 h at room temperature. DNA strands were complementary to the strands immobilized on the NR surface (SEQ. ID. 3:5-HS-C6-TATACGAAGTTATAAAAAAAAAA-3; SEQ. ID. 4:5-HS-C6-TGCCCTGGACCCGGAC-3). The conjugate was purified by size exclusion HPLC using a Shimadzu-LC-20AD system with a Superdex 200 5/150 GL column (GE Healthcare). PBS was used as eluent at a flow rate of 0.3 mL/min. Labelling of NR-ssDNA with TRITC. Thiol-PEG-amine 1 kDa (Creative PEGworks, 20 nmol) was reacted with tetramethylrhodamine (TRITC, 20 nmol) in 1 mL of 10 mM carbonate buffer at pH 9.0 for 2 h at room temperature. Then 500 L of NR-ssDNA (0.5 nM) were incubated overnight with thiol-PEG-TRITC in a molar ratio of 1:1000. The excess of fluorophore was removed by centrifugation at 9000 g.

[0106] Immobilization of protein-ssDNA conjugates in Au NRs. For the hybridization of complementary oligonucleotide strands conjugated with a protein, a suspension of NR-ssDNA (0.5 nM) was incubated with DNA-protein conjugates (150 nM) for 1 h at 37 C. and then the temperature was slowly decreased to 25 C. The excess of DNA-protein conjugate was removed by centrifugation. The amount of DyLight-BSA immobilized on the NRs was determined by measuring the fluorescence in the supernatant. The amount of -galactosidase immobilized was determined by measuring the enzymatic activity in the supernatant. Briefly, 50 L of supernatant or NR suspension were added to 100 L of o-nitrophenyl -d-galactopyranoside (ONPG, 13 mg/mL in 0.1M phosphate buffer pH 7.0) and incubated at 37 C. for 30 min in a Synergy HT microplate reader. The absorbance at 420 nm was measured every 3 min.

[0107] Light induced release of proteins from Au NRs. A suspension of DL-BSA-dsDNA-AuNR was placed in a 96 well plate and irradiated with a fiber coupled Roithner laser (continuous wave at 785 nm) with different laser powers (0.8, 1.25, 2 W/cm.sup.2). After irradiation, the suspension was immediately centrifuged at 9000 g. The fluorescence of the supernatant was measured in order to determine the amount of protein released.

[0108] To test the multiple release system, AuNR conjugated with DL.sub.488-BSA and DL-.sub.550-BSA were first irradiated for 2 min at 1.25 W/cm.sup.2. The supernatant was collected and after resuspending the pellet, the suspension was irradiated for further 3.5 min at 2 W/cm.sup.2.

[0109] A release kinetics was also done using AuNR conjugated with beta-galactosidase. For that, after irradiation the supernatant was collected and its enzymatic activity was measured using ONPG as substrate.

[0110] Cytotoxicity of BSA-dsDNA.sub.51.7-AuNR. SC-1 mouse fibroblasts were grown in DMEM supplemented with 10% fetal bovine serum (FBS), at 37 C. in a fully humidified air containing 5% CO.sub.2. To assess the cytotoxicity of gold NRs, fibroblasts were seeded on a 96 well plate (410.sup.3 cells/well), left to adhere for 24 h and then incubated with BSA-dsDNA.sub.51.7-AuNR for 4 h. After incubation, the cells were washed with medium to remove non-internalized NR. In some conditions, after incubation with BSA-dsDNA.sub.51.7-AuNR, the cells were washed and irradiated with a fiber coupled Roithner laser (785 nm). Each well was placed below the end of the fibre and irradiated with a power density of 1.25 and 2 W/cm.sup.2 for 2 min. Then cells were left in the incubator for 24 h and the ATP production was measured by a Celltiter-Glo Luminescent Cell Viability Assay (Promega).

[0111] Uptake kinetics of BSA-dsDNA.sub.51.7-AuNR. SC-1 mouse fibroblasts were plated in a 24 well plate at a density of 510.sup.4 cells/well and left to adhere overnight. The cells were incubated with BSA-dsDNA.sub.51.7-AuNR (50 g/mL) for 1, 2, 4, 6 and 24 h. After incubation, in order to remove non-internalized nanorods, the cells were washed three times with PBS, dissociated with trypsin and counted. Finally, the samples were freeze-dried and the amount of gold was determined by inductive coupled plasma mass spectrometry (ICP-MS).

[0112] Light-induced release of proteins in cells. SC-1 mouse fibroblasts were grown in DMEM supplemented with 10% fetal bovine serum (FBS), at 37 C. in 5% CO.sub.2. Cells were incubated with 50 g/mL of NR conjugated with one or two fluorescent proteins (DL.sub.650-BSA Tm 51.7 C.; DL.sub.488BSA Tm 68.9 C.). After 4 h incubation, the medium was replaced and cells were irradiated with fiber coupled Roithner laser (785 nm) with different laser powers (1.25; 2 W/cm.sup.2) for 2 min. For the dual release experiments, the time between the application of the first and second stimulus was 5 min.

[0113] For the transfection studies with Gal-dsDNA.sub.51.7-AuNR-, cells were grown in a 15 well IBIDI slide at an initial density of 3000 cells/well for 24 h. After 4 h incubation with NR-DNAGal at 50 g/m L, cells were and irradiated with different laser power densities (0.57; 1.25; 2 W/cm.sup.2) for 2 min. After irradiation, the activity of beta-galactosidase was determined with a beta galactosidase Detection Kit (Abcam) following the manufacturer's protocol. The presence of beta-galactosidase was also detected by immunocytochemistry. Cells were seeded in gelatin coated coverslips and left to adhere for 24 h. After incubation with NR-DNAGal, cells were irradiated, fixed with paraformaldehyde 4% (v/v) for 15 min at room temperature and washed three times with PBS. After blocking (PBS solution with 1% BSA), cells were incubated with a rabbit beta-galactosidase antibody (Invitrogen) for 60 min, washed three times with blocking buffer and incubated with alexa-fluor488 conjugated goat anti-rabbit IgG (dilution 1:1000) for 60 min. The excess of antibody was removed by washing with PBS before staining with DAPI (1 g/mL) for 5 min. Coverslips were analyzed in a confocal microscope (LSM 710, Carl Zeiss). The corrected total cell fluorescence was quantified with ImajeJ and corrected for background fluorescence. Manders overlap coefficient was calculated using Image J and JACoP plugin.

TABLE-US-00002 TABLE 1 Oligonucleotide strands used for conjugation with AuNRs and for modification of microRNAs. The melting temperature refers to a theoretical melting temperature relative to the portion of the strand that is able to hybridize with the complementary strand Tm/ C.* Sequences AuNR conjugation 51.7 SEQ. ID 1 68.9 SEQ. ID 2 miR ssDNA conjugates or 51.7 SEQ. ID 3 protein-ssDNA conjugates 68.9 SEQ. ID 4 *melting temperature of complementary strands after hybridization (SEQ. ID3 hybridizes with SEQ. ID1 and SEQ. ID4 hybridizes with SEQ. ID2)

[0114] Preparation of Micro-RNAs Conjugated with ssDNA.

[0115] microRNAs conjugated with ssDNA (miR-ssDNA) were prepared using N-[-maleimidobutyryloxy]sulfosuccinimide ester (sulfo-GM BS, Thermo Scientific) as linker. miR-155 or miR-302a (60 l at 100 M in PBS pH 8.0) were reacted with sulfo-GM BS in a 100-fold molar ratio for 30 min at room temperature. The excess of linker was removed by ultrafiltration with Nanosep 30 kDa (Pall Corporation). The buffer was exchanged by PBS pH 7.0 and the purified miR (60 l; 100 M in PBS pH 7.0) was reacted with thiolated DNA (60 l; 200 M in PBS pH 7.0) in a final volume of 200 l of PBS for 2 h at room temperature. Before conjugation DNA strands were reduced with 100-fold excess of TCEP for 1 h at 37 C. DNA strands were complementary to the strands immobilized on the NR surface (Table 1) (SEQ. ID. 3:5-HS-C6-TATACGAAGTTATAAAAAAAAAA; SEQ. ID. 4:5-HS-C6-TGCCCTGGACCCGGAC). miR-155 was conjugated with ssDNA SEQ. ID. 3(Tm 51.7 C.) and miR-302a was conjugated with ssDNA SEQ. ID. 4 (Tm 68.9 C.).

[0116] Purification of miR-ssDNA conjugates by reverse-phase ion-pair liquid chromatography. The products of miR and ssDNA conjugation were separated in a Shimadzu-LC-20AD system using a 4.6250 mm XBridge C18 column packed with 3.5 m particles, average pore diameter 130 (Waters). The mobile phases were as follows: 0.1 M TEAA pH 7.0 (A) and acetonitrile (B). The gradient started in 14% of B to 19% of B in 23 min. The flow rate was 0.55 mL/min. The acetonitrile present in the fraction containing the miR-ssDNA conjugate was removed in a rotary evaporator. The final volume was aliquoted and stored at 20 C.

[0117] Characterization of miR Conjugated with ssDNA by Non-Denaturing PAGE.

[0118] The reaction mixture obtained after reacting miR-155 or miR-302 with ssDNA and the fractions obtained after HPLC purification of the reaction mixture were analysed by gel electrophoresis. Reaction mixture (15 L) and reaction mixture fractions obtained after HPLC purification (15 L) were mixed with glycerol (5 l; glycerol in 50% v/v of H.sub.2O), loaded in a polyacrylamide gel (12%, w/v) and run for 45 min in 0.5TBE at 140 V. The gel was stained with SyBr Gold (1:5000 in 1TBE) for 10 min and imaged in a UV transilluminator (Molecular Imager Gel DOC, Biorad).

[0119] Immobilization of miR-ssDNA Conjugates in AuNRs.

[0120] For the hybridization of complementary oligonucleotide strands conjugated with miR, a suspension of AuNR-ssDNA (0.5 nM) was incubated with DNA-miR conjugates (200 or 400 nM) for 1 h at 37 C. and then the temperature was slowly decreased to 25 C. The excess of DNA-protein conjugate was removed by centrifugation. The amount of miR-ssDNA immobilized on the AuNRs was determined indirectly by measuring the concentration in the supernatant. For that, the supernatant was collected and incubated with SyBr Gold (diluted 1:10000). The fluorescence was measured in a Synergy HT microplate reader (excitation 495 nm, emission 537 nm) and the concentration was extrapolated from a calibration curve.

[0121] Backfill with Thiol-PEG.

[0122] After conjugation with miR-ssDNA conjugates, the surface of AuNRs was backfilled with thiolated PEG (2 kDa). Briefly, a suspension of AuNR-DNA-miR (500 L, 0.5 nM) was incubated with thiol-PEG at 25 M corresponding to a ratio of 1:50000 between AuNR and thiol-PEG. The reaction proceeded for 5 h at room temperature under orbital agitation. Then, the suspension was centrifuged (9000 g, 30 min) and resuspended in 10 mM phosphate pH 7.4 with 30 mM NaCl. The suspension was stored at 4 C.

[0123] Cell Culture.

[0124] HEK-293T transfected with a reporter vector were used for activity assays with microRNAs. The reporter vector encodes EGFP conjugated to the targets of miR-302a and miR-302d, and mCherry conjugated to the targets of miR-142-3p, miR-155 and miR-223. Cells were cultured in T-75 culture flasks at 37 C. in a humidified atmosphere of 5% CO.sub.2 in DMEM cell culture media containing 10% fetal bovine serum (FBS) and 0.5% penicillin-streptomycin. Cells were grown to 80-90% confluency before splitting and re-seeding 24 h before each experiment.

[0125] Cytotoxicity of miR-dsDNA-AuNR and Cecropin-Melittin.

[0126] To assess the cytotoxicity of AuNRs, HEK-293T cells were seeded on a 96 well plate (1210.sup.3 cells/well), left to adhere for 24 h and then incubated with dsDNA.sub.51.7-AuNR (50 g/mL) without or with cecropin-melttin (5 M and 10 M) for 4 h in serum free medium. After incubation, cells were washed with PBS to remove non-internalized AuNRs. In some conditions, after incubation with AuNRs, cells were washed and irradiated with a 780 nm laser at 1.25 W/cm.sup.2 for 2 min. After 2 h in the incubator at 37 C., a subset of samples received a second laser stimulus for 2 min at 2 W/cm.sup.2. Then, cells were left in the incubator for 24 h and the ATP production was measured by a Celltiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions.

[0127] Transfection of Micro-RNAs and DNA-miR Conjugates with Lipofectamine RNAimax.

[0128] The ability of miR-155, miR-302a and miR-ssDNA conjugates to induce the knockdown of mCherry and EGFP respectively, was evaluated via transfection with lipofectamine RNAimax. HEK-293-T cells were seeded in a collagen coated 96 well plate (6500 cells/well) in DMEM (10% FBS, without antibiotics) 24 h before transfection. miR-155, miR-302a, ssDNA-miR-155 and ssDNA-miR-302a (35 L, concentrations ranging from 0.05 to 5 nM) were complexed for 20 min with lipofectamine RNAimax diluted in DMEM (0.7 L of RNAimax in 35 L of DMEM). Then, each of the complexes was added to cells (20 L/well) and incubated for 4 h or 24 h. Finally, cells were washed, new culture medium was added and cell fluorescence was monitored in a high-content fluorescence microscope (IN Cell 2200, GE Healthcare) each 12 h during 3 days.

[0129] Activity of DNA-miR Conjugates Released from AuNRs.

[0130] The activity of miR-155 and miR-302a released from AuNR surface after irradiation was evaluated in HEK-293T cells seeded in a 96 well plate (6500 cells/well). In order to study the laser induced release and activity of miR-DNA conjugates, we used AuNRs hybridized with: I) miR-155 conjugated with ssDNA with a melting temperature of 51.7 C. and II) miR-302a conjugated with ssDNA with a melting temperature of 68.9 C. Each suspension of miR-dsDNA-AuNR was irradiated and immediately centrifuged. Then the supernatant was complexed for 20 min with lipofectamine RNAimax (35 L of supernatant complexed with 35 L of RNAimax diluted 1:50 in DMEM) and 20 L of this mixture was added to each well containing 100 L of DMEM with 10% FBS. After incubation, cells were kept in DMEM (10% FBS, 0.3 g/mL of Hoechst) and their fluorescence was monitored in a high-content fluorescence microscope (IN Cell 2200, GE Healthcare) each 12 h during 3 days.

[0131] Intracellular Localization of miR-dsDNA-AuNR-TRITC.

[0132] Cells were seeded in an IBIDI 15 well slide (80% confluency), left to adhere for 24 h and then incubated with miR-dsDNA-AuNR-TRITC (50 g/mL) for 4 h without or with cecropin-melittin (5 or 10 M) in DMEM (0.5% penstrep, without FBS). After incubation, cells were washed with medium to remove non-internalized AuNRs. Then, the cells were incubated with LysoTracker Green (100 nM) for 30 min to stain the endosomes and with Hoechst 33342 (0.3 g/m L) to stain the nuclei. Cells were then observed under confocal microscope. Images were analyzed in Image) and the colocalization was determined by calculating the Manders' colocalization coefficient between AuNR-TRITC and Lysotracker green.

[0133] Light-Induced Release of DNA-miR Conjugates in Cells.

[0134] HEK cells were seeded in a 96 well plate (6500 cells/well), left to adhere for 24 h and then incubated with 50 g/mL of AuNR conjugated with miR-155-ssDNA or miR-302a-ssDNA. First, AuNRs modified with miR-155 or miR-302a were tested separately. A suspension of miR-dsDNA-AuNR was prepared in serum free DMEM. Before adding to cells, the suspension was mixed with cecropin-melittin peptide (final concentration of 5 or 10 M). After 4 h incubation, the medium was replaced and cells were irradiated with a fiber coupled laser (780 nm) at 0.8, 1.25 or 2 W/cm.sup.2 for 2 min. Then, cells were incubated in DMEM (10% FBS, 0.5% penstrep and 0.3 g/mL of Hoechst 34580) and cell fluorescence was monitored in a high-content fluorescence microscope (IN Cell 2200, GE Healthcare).

[0135] All references recited in this document are incorporated herein in their entirety by reference, as if each and every reference had been incorporated by reference individually.

[0136] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0137] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[0138] Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims.

[0139] The present disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above described embodiments are combinable. The following claims further set out particular embodiments of the disclosure.