METHOD FOR DEPOSITING NANO-OBJECTS ON THE SURFACE OF A POLYMER GEL WITH UNIFORM RIGIDITY

Abstract

The invention relates to a method for depositing nano-objects on the surface of a gel comprising the steps of: a) providing a gel having a polymer matrix and a solvent within the polymer matrix, the polymer matrix forming a three-dimensional network which is capable of swelling in the presence of the solvent, wherein the solubility of the polymer matrix in the solvent at 1 bar and 25 C. is less than 1 g/l, wherein the gel has a rigidity gradient on the micrometer scale of less than 10%, then b) depositing nano-objects on the surface of the gel, the nano-objects having a mean diameter greater than or equal to the mean diameter of the pores of the gel, then c) evaporating the solvent from the gel at least until the content of solvent no longer varies over time, under the proviso that, at the start of evaporation, the content of mineral salts in the solvent is less than 6 g/l, the gel capable of being obtained and the uses thereof.

Claims

1. Method of depositing nano-objects on the surface of a gel comprising the steps of: a) providing a gel comprising a polymer matrix and a solvent within the polymer matrix, the polymer matrix forming a three-dimensional network capable of swelling in the presence of said solvent, where the solubility of the polymer matrix at 1 bar and 25 C. in the solvent is less than 1 g/l, where the standard deviation 6 of the rigidity values Ri is less than 20%, the rigidity values Ri being measured by atomic force microscopy on n points distributed over the entire surface of the gel provided in step a), n being greater to 50, the standard deviation 6 being as defined in formula (I): $\begin{array}{cc}=\sqrt{\frac{1}{n}.Math.{.Math.}_{i=1}^n.Math.{\left(\mathrm{Ri}-\mathrm{mean}\right)}^2}& \left(I\right)\end{array}$ where mean is the arithmetic mean of the rigidity values Ri and is as defined in formula II: $\begin{array}{cc}\mathrm{mean}=\frac{1}{n}.Math.{.Math.}_{i=1}^n.Math.R.Math.i& \left(\mathrm{II}\right)\end{array}$ then b) depositing nano-objects on the surface of the gel, said nano-objects having an average diameter greater than or equal to the average diameter of the pores of the gel, then c) evaporating the solvent from the gel at least until the solvent content no longer varies over time, provided that at the start of evaporation the content of inorganic salts in the solvent is less than 6 g/l.

2. Method according to claim 1, wherein said rigidity values follow 10% a symmetrical distribution, provided that the arithmetic mean mean and the median median of said distribution are such that the deviation e such that defined in formula (III): $\begin{array}{cc}e=2.Math.\frac{\mathrm{mean}-\mathrm{median}}{\mathrm{me}.Math.\mathrm{an}+\mathrm{median}}& \left(\mathrm{III}\right)\end{array}$ is less than 10%.

3. Method according to claim 1, wherein the polymer matrix of the gel comprises a polymer chosen from: polyacrylamides; polyethylene glycols, polypropylene glycols and ethylene glycol or propylene glycol copolymers, these optionally comprising units resulting from the polymerization of (meth)acrylate compounds; polysaccharides, optionally comprising repeating units resulting from the polymerization of (meth)acrylate compounds; (co)polymers resulting from the polymerization of diacrylate and/or (meth)acrylate compounds; polyvinyl alcohols comprising repeating units resulting from the polymerization of (meth)acrylate compounds; dextrans comprising repeating units resulting from the polymerization of (meth)acrylate compounds; polypropylene fumarates and poly(propylene fumarate-co-ethylene glycol); polysiloxanes; and the combinations of these.

4. Method according to claim 1, wherein the solvent present within the polymer matrix of the gel is an aqueous solution.

5. Method according to claim 4, wherein the aqueous solution is water.

6. Method according to claim 1, wherein the solvent present within the polymer matrix of the gel is chosen from among pentane, triethylamine, diisopropylamine and xylene and the polymer matrix comprises poly(dimethylsiloxane).

7. Method according to claim 1, wherein the nano-objects are chosen from among: proteins, peptides and mixtures thereof, polysaccharides, and nanoparticles.

8. Method according to claim 7, wherein the nano-objects are chosen from among polysaccharides, proteins, peptides and mixtures thereof.

9. Method according to claim 8, comprising, a step d) of covalent grafting of proteins and/or peptides and/or polysaccharides on the gel.

10. Gel obtainable by the method according to claim 1, the surface of the gel being at least partially coated with nano-objects, where the standard deviation of the quantities Qj of nano-objects per m.sup.2 of surface is less than 40%, the quantities Qj of nano-objects per m.sup.2 of surface being measured by microscopy on p m.sup.2 of surface distributed over the entire surface of the gel, p being greater than 10, the standard deviation being as defined in formula (IV): $\begin{array}{cc}{}^{}=\sqrt{\frac{1}{p}.Math.{.Math.}_{j=1}^p.Math.{\left(\mathrm{Qj}-{\mathrm{mean}}^{}\right)}^2}& \left(\mathrm{IV}\right)\end{array}$ where mean is the arithmetic mean of the quantities Qj of nano-objects per m.sup.2 of area and is as defined in formula (V): $\begin{array}{cc}{\mathrm{mean}}^{}=\frac{1}{p}.Math.{.Math.}_{j=1}^p.Math.\mathrm{Qj}.& \left(V\right)\end{array}$

11. Gel according to claim 10, wherein said quantities Qj of nano-objects per m.sup.2 of surface follow a symmetrical distribution within 10%, provided that the arithmetic mean mean and the median median of said distribution are such that the deviation e as defined in formula (VI): $\begin{array}{cc}e=2.Math..Math.\frac{{\mathrm{mean}}^{}-{\mathrm{median}}^{}}{{\mathrm{mean}}^{}+{\mathrm{median}}^{}}& \left(\mathrm{VI}\right)\end{array}$ is less than 25%.

12. Gel according to claim 10, which is a photonic or physicochemical sensor, a sensor for the detection of analyte, a protein or peptide chip, or a biomolecule capture chip.

13. Method according to claim 3, wherein the polymer matrix of the gel comprises a polymer chosen from polyacrylamides.

14. Method according to claim 13, wherein the polymer matrix of the gel comprises a polymer obtained from the polymerization of acrylamide and N,N-methylenebisacrylamide.

15. Method according to claim 7, wherein the nano-objects are chosen from among metal, semiconductor and polymer nanoparticles.

16. Method according to claim 8 wherein the nano-objects are chosen from among proteins and/or peptides inducing cell adhesion via integrins.

17. Method according to claim 16, wherein the nano-objects are chosen from among fibronectin, collagen, laminin, vibronectin or RGD type peptides.

18. Gel according to claim 10, wherein the standard deviation of the quantities Qj of nano-objects per m.sup.2 unit of surface is less than 30%.

19. Gel according to claim 11, wherein the deviation e is less than 20%.

20. A cell positioning method for screening active pharmaceutical ingredients, said method comprising bringing pharmaceutical active ingredients into contact with a gel according to claim 10, wherein the nano-objects are chosen from among peptides, proteins and polysaccharides.

Description

EXAMPLE 1

Grafting of Previously Activated Proteins (Fibronectin) on the Surface of Polyacrylamide Hydrogels.

[0143] In this example, a photosensitive crosslinker was grafted to the fibronectin used as protein, to make it reactive with the surface of the hydrogel under exposure to UV A. The grafting of the fibronectin previously activated on the surface of the gel is a photoactivated reaction.

[0144] a) Silanization of Basal Glass Coverslips

[0145] The basal coverslip serves as the basal anchor for the hydrogel.

[0146] The basal glass coverslip, with a diameter of 30 mm, was cleaned in a solution of 0.1 mol/L of sodium hydroxide for 10 min. It was then rinsed extensively with water, then with ethanol, and air dried.

[0147] 500 L of a silane solution comprising 56 L of Bind-Silane (GE Healthcare), 484 L of 10% acetic acid, and 14.46 mL of ultra pure ethanol were placed on the coverslip and rubbed with a knitted polyester cloth until all traces of solution disappear. A glass slide was thus obtained having aldehyde functions at its surface, which allow covalent grafting of the polyacrylamide gel.

[0148] b) Silanization of the Transparent Mask

[0149] The hydrogel is crosslinked by UV, the transparent mask allowing the surface of the hydrogel to be flat. The mask consisted of a microscope coverslip treated with a fluorinated silane to limit its adhesion to the hydrogel.

[0150] An optical microscopy coverslip (26 mm76 mm) was washed in a 1:2 concentrated hydrogen peroxide/sulfuric acid solution for 10 minutes. It was then made hydrophobic by an Optool treatment (Daikin DSX): immersion for 1 minute in Optool diluted to 1/1000 in perfluorohexane. Then the coverslip was left for 1 hour in water vapor at 80 C. Finally, it was immersed with slow stirring for 10 minutes in perfluorohexane.

[0151] c) Preparation of Three Polyacrylamide Hydrogels

[0152] The hydrogel was prepared according to the method described in application WO 2013/079231 from a composition consisting of: [0153] 10% acrylamide (250 L of initially 40% solution) [0154] 0.5% of N, N-methylenebisacrylamide (Bis) (250 L of initially 2% solution) [0155] 0.2% of Irgacure 819 w/v (Ciba, photoinitiator) [0156] 1% propylamine (initiator) [0157] deionized water (490 l)

[0158] Irgacure 819 was weighed in a UV-opaque bottle. Propylamine was added to it. The whole was heated at 50 C. for 2 minutes. After heating, a homogeneous and transparent solution was obtained. Water, acrylamide, and bis acrylamide were added quickly. The whole was homogenized gently with a pipette, to limit the incorporation of oxygen. 30 L were deposited on the 30 mm glass coverslip pretreated according to the above protocol. The coverslip was placed on a sample holder having spacers which maintain a spacing of 40 m between the coverslip and the transparent mask, deposited on the spacers. The whole (mask, solution, coverslip) was illuminated using an Eleco UVP281 fiber lamp (2 W/cm.sup.2) for 7.8 s, 15 s or 20 s, in order to obtain three hydrogels. Each set was then immersed in water to detach the mask from the hydrogel using forceps. Each hydrogel was rinsed 3 times with deionized water and stored in deionized water.

[0159] d) Characterization of the Rigidity of Hydrogels

[0160] The variability in porosity of each hydrogel was estimated by measuring the local rigidity of the hydrogel. The local rigidity was measured by an AFM in aqueous medium (JPK brand). The resistance of the gel to the indentation of the point was recorded. Four 100 m100 m regions spaced several millimeters apart were scanned. The scans were performed with a step of 10 m in order to obtain a series of indentation curves. Each curve was processed according to the manufacturer's protocol with an elastic indentation model.

[0161] The rigidities obtained are dependent on the illumination time to prepare the gel. They are of the order:

[0162] 0.6 kPa with a standard deviation a of the rigidity values Ri of 11.7% for a hydrogel prepared with an illumination time of 7.8 s,

[0163] 11.8 kPa with a standard deviation a of the rigidity values Ri of 11.8% for a hydrogel prepared with an illumination time of 15 s and

[0164] 24.7 kPa with a standard deviation a of the rigidity values Ri of 9.2% for a hydrogel prepared with an illumination time of 20 s.

[0165] e) Deposition of Activated Fibronectin on the Hydrogel Followed by Covalent Grafting

[0166] The fibronectin was previously coupled to the hetero-bifunctional sulfo-NHS-LC-Diazirine (Sulfosuccinimidyl-6-(4,4-azipentanamido) hexanoate crosslinker, ThermoScientific Pierce; trade name: sulfo-LC-SDA), with a molar ratio of 1/480. 5 mg of fibronectin (Roche) was dissolved in 2 mL of ultrapure deionized water, at 37 C. for 30 min. 1.2 mg of sulfo-LC-SDA were weighed in the dark and dissolved in the fibronectin solution for 30 min at room temperature. This operation was repeated a second time, resulting in the molar ratio of 1/480. This protocol made it possible to react the sulfo-NHS function of the sulfo-LC-SDA with the amine groups of the fibronectin while limiting the hydrolysis of the sulfo-LC-SDA. The compound formed is a fibronectin molecule coupled to a photosensitive diazirine function. The compound formed was dialyzed through a 6-8000 membrane in a dark room and at 4 C. against 2 I of PBS +/+ 1 for 48 h with a change of PBS after 24 h. It was then aliquoted in small volumes (25 and 50 l) and stored frozen at 20 C.

[0167] The hydrogel prepared according to the above protocol was dehydrated under a vertical laminar flow hood (Aura) at 26 C. for one hour (step a0)).

[0168] In a room with UV-free lighting, 800 l of conjugated fibronectin solution according to the above protocol was prepared at a concentration of 3.5 g/ml in sterile deionized water, and was deposited using with a pipette on the gel (step b)).

[0169] The whole hydrogel+fibronectin solution was placed on a hot plate at 37 C. under a laminar flow hood (convective flow of 0.5 m/s) until the solution had completely evaporated from the surface of the hydrogel (step c)).

[0170] The gel was immediately illuminated by the ElecoUVP281 UV lamp for 5 min (step d)). It was then gently rinsed 3 times with a solution of PBS +/+ (steps e)). The functionalized gel was stored hydrated in a solution of PBS +/+, at 4 C.

[0171] f) Characterization of the Distribution of Grafted Proteins

[0172] The PBS +/+ solution was aspirated from the gel, and replaced with a saturation solution consisting of a solution of PBS +/+ 1-0.1% Tween20-2% BSA, for 30 min with slow stirring at room temperature.

[0173] The saturation solution was aspirated using a pipette and replaced by a solution of 3 L of primary polyclonal anti fibronectin antibody produced in rabbits (Sigma-Aldrich, F3648) diluted in 1.2 mL of PBS +/+ 1-0.1% Tween20-2% BSA. The antibody was incubated for 1 hour with slow stirring at room temperature. It was then revealed with 1.2 mL of a solution containing 0.6 L of a secondary antibody coupled to Alexa 488 produced in the donkey and directed against the rabbit (Molecular Probes, A21206), supplemented by a 1x PBS +/+ solution-0.1% Tween20 -2% BSA for 1 hour with slow stirring at room temperature and protected from light. The solution was then removed by aspiration and the gel was rinsed 3 times with 1.2mL of PBS +/+ 1-Tween20 0.1%-BSA 2%. The gel was then stored in a solution of PBS +/+ 1at 4 C. and protected from light.

[0174] The characterization of the distribution of the grafted proteins was carried out by confocal fluorescence microscopy (Leica SP microscope). An image stack was acquired for each hydrogel at the wavelength 488 nm with an image spacing of 0.28 m. The various acquisitions were made at constant gain and at constant laser intensity. Each stack of images was then assembled with ImageJ software and sections were extracted. The maximum intensity image was calculated from the image stack, resulting in a two-dimensional projection of the fluorescent surface for 375375 m windows. The antibody markings lead to obtaining pixelated images. To remedy this, the pixelation was limited by a Gaussian filter with a radius of 10 pixels. Then the mean value of the intensity was calculated on this projection (Table 1).

TABLE-US-00001 TABLE 1 Average fluorescence intensity over an area of 375 375 m.sup.2 and variability of the protein surface density for each hydrogel. 0.6 kPa 11.8 kPa 24.7 kPa rigidity gel rigidity gel rigidity gel Average 85.1 13.5 81.1 10.5 80.7 19.8 fluorescence intensity Standard deviation 16% 13% 25% of the quantities Qj of proteins per m.sup.2 of surface area

[0175] The lack of significant variation in intensity between each of the three hydrogels demonstrates that the amount of grafted protein is independent of the rigidity/porosity of the hydrogel.

EXAMPLE 2

Grafting of Proteins (Fibrinogen) on the Previously Activated Surface of a Polyacrylamide Hydrogel.

[0176] In this example, the same crosslinker as that of Example 1 was attached in excess to the surface of the hydrogel by photochemical reaction to obtain an activated surface, wherein the proteins (fibrinogen) reacted with the activated surface of the hydrogel by a coupling reaction with the primary amine functions of the proteins.

[0177] a) Silanization of Basal Glass Coverslips [0178] Same as Example 1a.

[0179] b) Silanization of the Transparent Mask [0180] Same as Example 1b.

[0181] c) Preparation of Three Hydrogels [0182] Same as Example 1c. The illumination times were 7.5, 9, and 10 s.

[0183] d) Characterization of the Rigidity of each Hydrogel [0184] Same as Example 1d. The rigidities obtained were respectively: [0185] 2.9 kPa with a standard deviation of the rigidity values Ri of 10.3%, [0186] 4.6 kPa with a standard deviation of the rigidity values Ri of 4.3% and [0187] 9.5 kPa with a standard deviation of the rigidity values Ri of 9.5%.

[0188] e) Activation of the Surface of the Hydrogels (Step b0))

[0189] In a room with UV-free lighting, each hydrogel prepared according to the above protocol was dehydrated in a vertical laminar flow hood (Aura) at 26 C. for one hour. A solution of the hetero-bifunctional sulfo-NHSLC-Diazirine (Sulfosuccinimidyl 6-(4,4-azipentanamido) hexanoate, ThermoScientific Pierce; trade name: sulfo-LC-SDA) crosslinker was prepared in sterile deionized water at a concentration of 0.44 mg/mL. 800 l of this solution were deposited on the gel using a pipette. This solution was allowed to incubate for 60 min at 26 C. under the laminar flow hood. The residual solution was then gently aspirated with a pipette, and the gel was again allowed to dry for 40 min, still under the laminar flow hood.

[0190] The gel was then illuminated by the ElecoUVP281 UV lamp for 5 min.

[0191] f) Covalent Grafting of Fibrinogen

[0192] A solution of fibrinogen coupled to a fluorescent Alexa Fluor 488 probe (F13191, Invitrogen) was prepared at a concentration of 8.75 g/mL. 800 l of this solution were deposited on the activated surface of the gel using a pipette (step b)).

[0193] The hydrogel+fibrinogen solution assembly was placed on a hot plate at 37 C. under a laminar flow hood (convective flow of 0.5 m/s) until complete evaporation of the solution on the surface of the hydrogel (steps c) and d)).

[0194] The gel was then gently rinsed 3 times with a solution of PBS +/+ (steps e)). The functionalized gel was stored hydrated in a solution of PBS +/+, at 4 C. and protected from light.

[0195] g) Characterization of the Distribution of Grafted Proteins

[0196] The grafted proteins were coupled to a fluorophore. The characterization of the distribution of the grafted proteins was carried out by confocal fluorescence microscopy (Leica SP microscope). An image stack was acquired for each rigidity step at the wavelength 488 nm with an image spacing of 0.28 m. The various acquisitions were made at constant gain and at constant laser intensity. Each stack of images was then assembled with ImageJ software. The maximum intensity image was calculated from the image stack, resulting in a two-dimensional projection of the fluorescent surface. The antibody markings lead to obtaining pixelated images. To remedy this, the pixelation was limited by a Gaussian filter with a radius of 10 pixels. Then the mean value of the intensity was calculated on this projection (Table 2).

TABLE-US-00002 TABLE 2 Average fluorescence intensity over an area of 375 375 m.sup.2 and variability of the protein surface density for each hydrogel. 2.9 kPa 4.6 kPa 9.5 kPa rigidity gel rigidity gel rigidity gel Average 178.4 49.5 167.5 48.7 144.9 40.6 fluorescence intensity Standard deviation 28% 29% 28% of the quantities Qj of proteins per m.sup.2 of surface area

[0197] The lack of significant variation in intensity between each of the three hydrogels demonstrates that the amount of grafted protein is independent of the rigidity/porosity of the hydrogel.