Amphiphilic polymers and use thereof in the treatment of surfaces made of hydrophobic materials

Abstract

A straight, branched or cross-linked polymer, including, per 100 mol %: a) a mole fraction from 75% to 99.95% of monomer units from an N,N-dialkyl acrylamide; b) a mole fraction from 0.05% to 1% of monomer units from a monomer of formula (I): CH2=C(Ri)-C(═O)—O—[(CH2-CH(R2)-O]n-R3 (I); c) optionally a mole fraction higher than 0% to 24% either of monomer units from a monomer including a free strong acid function, partially or totally salified, or of monomer units from a monomer of formula (II): CH2=C(R4)-C(═O)—Y—(CH2)m-N(R5)(R6) (II); d) optionally a mole fraction higher than 0% to 1% of a diethylene or polyethylene cross-linking monomer. Also, a method for treating a surface made of a hydrophobic material, using the polymer, and an aqueous, hydro-organic or organic solution including the polymer for modifying interactions between the species contained the solution and the hydrophobic surface.

Claims

1. A process for the treatment of a surface composed, in all or in part, of a hydrophobic material, the process comprising: molding or etching, so as to create, on said surface composed, in all or in part, of a hydrophobic material, channels exhibiting, in one direction, at least one dimension of less than 1 mm; bringing into contact with said surface on which said channels have been molded or etched an aqueous, aqueous/organic or organic solution of a linear, branched or crosslinked polymer comprising, per 100 mol %: a) a molar proportion of greater than or equal to 75% and less than or equal to 99.95% of monomer units resulting from an N,N-dialkylacrylamide, the alkyl radicals each comprising from 1 to 4 carbon atoms, b) a molar proportion of greater than or equal to 0.05% and less than or equal to 1% of monomer units resulting from a monomer of formula (I):
CH.sub.2═C(R.sub.1)—C(═O)—O—[(CH.sub.2—CH(R.sub.2)—O].sub.n—R.sub.3  (I) wherein n represents a number between 1 and 50, R.sub.1 represents a hydrogen atom or a methyl radical, R.sub.2 represents a hydrogen atom, a methyl radical or an ethyl radical and R.sub.3 represents a saturated or unsaturated and linear or branched aliphatic hydrocarbon radical comprising from 8 to 30 carbon atoms, and c) optionally a molar proportion of greater than 0% and less than or equal to 24% either of monomer units resulting from a monomer comprising a free, partially salified or completely salified strong acid functional group or of monomer units resulting from a monomer of formula (II):
CH.sub.2═C(R.sub.4)—C(═O)—Y—(CH.sub.2).sub.m—N(R.sub.5)(R.sub.6)  (II) wherein m represents a number between 1 and 4, Y represents O or NH, R.sub.4 represents a hydrogen atom or a methyl radical and R.sub.5 and R.sub.6, which are identical or different, represent a methyl radical or an ethyl radical, and d) optionally a molar proportion of greater than 0% and less than or equal to 1% of a diethylenic or polyethylenic crosslinking monomer, and allowing the solution of polymer and said surface to incubate; rinsing said surface, after allowing the solution of polymer and said surface to incubate, in order to remove said polymer not adsorbed on the surface from said surface; and optionally, drying said rinsed surface resulting from stage b).

2. The process as defined in claim 1, wherein said surface is a surface of a chip, of a microfluidic system, of a microsystem or of a lab-on-a-chip.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrate a design of microchannels forming a cross v with branches i, ii, iii, and iv,

(2) FIG. 2a illustrates MCF-7 cells cultured on COC slides treated with a HEC+PLL solution. The membranes of the cells, visible by EpCAM labeling, are not well defined or are not observable.

(3) FIG. 2b illustrates MCF-7 cells subjected to the cationic DMAM-C22 treatment (without PLL). A suitable cell density and doublets or multiplets of cells. which are characteristics of divided cells, are observed.

DETAILED DESCRIPTION OF THE INVENTION

(4) The following examples illustrate the invention without, however, limiting it.

Example 1: Preparation of a DMAM/BEM-25(EO) Copolymer (Polymer 1)

(5) The polymer 1 is prepared as follows:

(6) The monomers are mixed in water in the amounts shown in the following table:

(7) TABLE-US-00001 Material Stoichiometry Charge DMAM 95% by weight (~99.65 mol %)   95 g BEM-25(EO)  5% by weight (~0.35 mol %)   5 g Water  900 g V50 0.1 mol %/monomers 0.26 g

(8) After deoxygenating for 45 minutes, the reaction medium is brought to 50° C. and the V50 is introduced into the reactor. The reaction medium slightly whitens and becomes viscous. An increase in the temperature of approximately 3.6° C. is recorded. The reaction is halted after 2 hours. After filtration, washing and drying, polymer 1 is obtained. Polymer 1 is characterized as follows:

(9) Visual appearance: cloudy viscous liquid;

(10) Viscosity of a 3% by weight dispersion of polymer 1 in water at 25° C. (Brookfield™ RVT, spindle 3, speed: 5 revolutions per minute): 440 mPa.Math.s;

(11) Viscosity of a 3% by weight dispersion of polymer 1 in water at 25° C. (CAP2000™, cone 1, speed: 50 revolutions per minute): 196 mPa.Math.s;

(12) Residual content of N,N-dimethylacrylamide: less than 0.05% by weight;

(13) Weight of polymer obtained: 100 g.

Examples 2 and 3: Preparation of Polymers 2 and 3

(14) Other tests were carried out under the same operating conditions as in example 1, the contents by weight of the various compounds employed being modified, as shown in the following table, in order to result in polymers 2 and 3.

(15) TABLE-US-00002 Material Polymer 2 Polymer 3 DMAM/BEM-25(EO) ratio by weight 97.5/2.5 90/10 Molar content of BEM-25(EO) 0.175% 0.7% DMAM 97.5 g 90 g BEM-25(EO)  2.5 g 10 g Water  900 g 900 g  V50 0.26 g 0.26 g  

(16) The operating conditions are identical to those of the preceding example 1. Polymers 2 and 3 are characterized as follows:

(17) Polymer 2

(18) Visual appearance: cloudy viscous liquid;

(19) Viscosity of a 3% by weight dispersion of polymer 2 in water at 25° C. (Brookield™ RVT, spindle 3, speed: 5 revolutions per minute): 2240 mPa.Math.s;

(20) Residual content of N,N-dimethylacrylamide: less than 0.05% by weight;

(21) Weight of polymer obtained: 100 g.

(22) Polymer 3

(23) Visual appearance: white, very viscous liquid;

(24) Viscosity of a 3% by weight dispersion of polymer 3 in water at 25° C. (Brookfield™ RVT, spindle 3, speed: 5 revolutions per minute): 3800 mPa.Math.s;

(25) Residual content of N,N-dimethylacrylamide: less than 0.05% by weight;

(26) Weight of polymer obtained: 100 g.

Example 4: Preparation of a DMAM/ATBSNa/BEM-25(EO) Anionic Polymer (Polymer 4)

(27) Polymer 4 is prepared as follows:

(28) The monomers are mixed in water in the amounts shown in the following table:

(29) TABLE-US-00003 Material Polymer 4 DMAM/ATBS, Na salt/BEM-25(EO) ratio by weight 75/20/5   DMAM/ATBS, Na salt/BEM-25(EO) molar ratio 90/10/0.35 DMAM 75.25 g ATBS, Na salt, 55% 35.4 g BEM-25(EO) 5 g Water q.s. for 1000 g V50 (0.1 mol %/monomers) 0.23 g

(30) After deoxygenating for 45 minutes, the reaction medium is brought to 50° C. and the V50 is introduced into the reactor. The reaction medium rapidly whitens after the introduction of the initiator and becomes viscous. Stirring is reduced. After reacting for 2 hours, heating is halted and the product is left for 2 hours before emptying. The DMAM content is <0.05% (552-385B method), which means that the polymerization reaction has indeed taken place.

Example 5: Preparation of a DMAM/DMAPANBEM-25(EO) Cationic Polymer (Polymer 5)

(31) TABLE-US-00004 Material Polymer 5 DMAM/DMAPAA/BEM-25(EO) ratio by weight 80/15/5   DMAM/DMAPAA/BEM-25(EO) molar ratio 90/10/0.35 DMAM 80.2 g DMAPAA 14.3 g BEM-25(EO) 5 g 1N HCl q.s. for pH = 7 Water q.s. for 1000 g V50 (0.1 mol %/monomers) 0.24 g

(32) The procedure used is that described above. The reaction medium is maintained at pH=7; it rapidly whitens and becomes viscous. After reacting for 1 h 30, heating is halted and the reaction medium is left to cool overnight. The DMAM content is <0.05%, which means that the polymerization reaction has indeed taken place.

Example 6: Treatment of a Surface Made of COG

(33) A series of Topas™ 8007 plates (5 cm×5 cm×5 mm) sold by Topas Advanced Polymers is cleaned with acetone and isopropanol and then dried with compressed air.

(34) 200 Microliters of a treatment solution A.sub.1 according to the invention, comprising 0.01% by weight of polymer 1 in a phosphate buffer at pH=7.4, sold by Life Technologies under the name Phosphate-Buffered Saline (PBS, pH=7.4), are subsequently spread homogeneously over one of the faces of a plate and then the surface thus treated is left to incubate at ambient temperature for 1 hour.

(35) In another experiment, 200 microliters of a treatment solution B.sub.1 according to the invention, comprising 0.1% by weight of polymer 1 in ethanol, are spread homogeneously over one of the faces of a second plate and then the surface thus treated is left to incubate at ambient temperature for 1 hour.

(36) In both cases, the plate is subsequently rinsed with osmosed water, in order to remove the nonadsorbed polymer, and then dried with compressed air.

(37) The plates thus prepared are ready for their subsequent use.

(38) Two new plates are also prepared according to the same protocol using treatment solutions A.sub.2 and B.sub.2, which are identical to the solutions A.sub.1 and B.sub.1 except that the polymer used is polymer 2.

Example 7: Measurement of Contact Angle

(39) The contact angle of water on the plates treated with the solutions A.sub.1, B.sub.1, A.sub.2 and B.sub.2 was measured using an optical goniometer.

(40) A drop of water of 3 microliters is deposited using a syringe and the image of the drop during its formation is analyzed in grazing incidence by a high resolution camera and then processed using software.

(41) A COC surface originally from the same source but which has not been subjected to the treatment of the polymers of the invention is used as reference.

(42) In all cases, a mean over 10 measurements is taken in order to determine the error bar. The results, recorded in the table below, reveal that all of the polymers and embodiments make it possible to significantly reduce the contact angle, that is to say to render markedly more wetting the initially hydrophobic surfaces.

(43) TABLE-US-00005 Treatment solution Contact angle (degrees) Untreated surface 89.4 +/− 2.7 Surface treated with the solution A.sub.1 72.7 +/− 1.2 Surface treated with the solution A.sub.2 72.1 +/− 1.1 Surface treated with the solution B.sub.1 74.8 +/− 1.3 Surface treated with the solution B.sub.2 77.7 +/− 1.8

Example 8: Treatment of Microchannels

(44) Preparation of the Microchannels

(45) An aluminum mold comprising a cross is prepared by micromachining using a Minitec Machinery Corporation device and a cutting tool with a diameter of 100 μm.

(46) The design of the microchannels is represented in FIG. 1. The lengths of each of the branches of cross v are as follows:

(47) branch i: 4 mm; branch ii: 4 mm; branch iii: 4 mm; branch iv: 50 mm.

(48) A COC plate as presented in example 7 is brought into contact with the mold (master) within a heated hydraulic press (SPECAC™). The embossing is carried out under a pressure of 50 kPa at 130° C. for 10 minutes. It is followed by cooling to 40° C. under the same pressure and then the press is opened and the plate carrying the microchannels is withdrawn. The reservoirs are drilled using the drilling machine and the plate is washed in an ultrasonic isopropanol bath and then dried.

(49) The microchannels are subsequently closed using a COC film. The plate carrying the microchannels and the film are placed for 4 minutes above a bath of cyclohexane in a petri dish provided with a lid, in order to be exposed to the cyclohexane vapor, and are then pressed against one another at 50 kPa at 60° C. for 20 minutes. The width of the channels is 100 μm and their depth is 50 μm. The reservoirs at the end of each branch of the cross have a diameter of 3 mm and a depth of 5 mm. Tygon™ tubes are connected on the one hand to the reservoirs of the chip and on the other hand to the reservoirs of an MFCS Fluigent™ pressure control system, in order to cause the fluids in the chip to move from the end of the longest branch of the cross.

(50) The treatment of the microchannels with the aqueous solutions (A.sub.1 or A.sub.2) is carried out as follows: the microchannels are first filled with 500 μl of ethanol filtered under pressure [(ΔP=3.5×10.sup.4 Pa (350 mbar*)]; the ethanol is subsequently removed by rinsing them with 500 μl of 1×PBS buffer at 2×10.sup.4 Pa (200 mbar); 500 μl of the treatment solution are then injected at a pressure of 10.sup.4 Pa (100 bar) and left to incubate for 1 hour at the temperature of the room without flow; finally, the channels are rinsed with 500 μl of PBS at a thrusting pressure of 10.sup.4 Pa (100 mbar).

(51) The treatment of the microchannels with the alcoholic solutions (B.sub.1 or B.sub.2) is carried out as follows: the alcoholic solution comprising the polymer is injected directly under the same conditions as those mentioned above; the channel is dried under vacuum and then rinsed with an aqueous PBS solution.

Example 9: Measurement of Electroosmosis in the Microchannels Prepared in Example 8

(52) The electroosmosis properties are measured by the technique of the measurement of the current, as described in Yasui T. et al “Characterization of low viscosity polymer solutions for microchip electrophoresis of non-denatured proteins on plastic chips”, Biomicrofluidics, Vol. 5, Issue 4, page 044114. the microchannels, the sample reservoir i, the buffer reservoir ii and the sample outlet reservoir iii are filled with a first phosphate buffer 20 mM, pH=7.5; the buffer outlet reservoir iv is filled with the buffer diluted 5-fold, i.e. 4 mM, pH=7.5; a voltage source HVS448 1500V Labsmith, Livermore, is used to apply a field of 270 V/cm and to cause a buffer front to migrate, by electroosmosis, from the buffer outlet reservoir, and the current is measured during the operation using the software supplied by Labsmith; the time necessary to reach a plateau is used to measure the linear rate of electroosmosis. For this experiment, three independent measurements were carried out.

(53) The results, recorded in the table below, show that, in all cases, the treatments according to the invention made it possible to reduce the electroosmosis, which is characteristic of a lasting presence of a layer of hydrophilic polymer at the surface of the microchannel.

(54) TABLE-US-00006 Rate of electroosmosis Treatment solution (×10.sup.−4 cm.sup.2/V/s) Untreated surface 2.76 +/− 0.14 Surface treated with the solution A.sub.1 0.85 +/− 0.14 Surface treated with the solution A.sub.2 1.43 +/− 0.19 Surface treated with the solution B.sub.1 1.23 +/− 0.04 Surface treated with the solution B.sub.2 0.75 +/− 0.15

Example 10: Effects on the Adsorption of Proteins of the Treatment of a Microchannel Made of COC with the Solution B2

(55) Simple linear channels were prepared according to the same protocol as that described in example 8, except for the shape of the mold, which exhibits a simple straight channel with a length of 3 cm and a width of 500 μm.

(56) After treatment of the surface of the microchannel with the solution B.sub.2 according to the protocol described in example 8, the microchannel is filled by circulation of 500 μl 1×PBS. A solution of BSA (fluorescent bovin serum albumin, from Life Technologies™) in suspension at 0.1% by weight in 1×PBS is then introduced into the channel at a pressure of 10.sup.3 Pa (10 mbar) and is incubated for 10 minutes at ambient temperature. The channels are subsequently rinsed for 10 minutes with protein-free 1×PBS under a pressure of 10 mbar. The fluorescence of the microchannel before and after incubation is recorded using a Nikon Eclipse™ microscope equipped with a Coolpix Roper Scientific™ camera, an HGFIL™ 130 W lamp and a set of FITC™ filters, with a fixed exposure time of 200 ms. 3 Different measurements were carried out at different points for each condition and the signal was corrected for the background noise of the camera and for the self-fluorescence of the COC (recorded outside the microchannel). In order to evaluate the reversible nature of the treatment and to confirm that the surface has not been detrimentally affected, 3 cycles of fixing the protein, rinsing with PBS, drying with alcohol and then again treating were carried out, without giving rise to a significant variation in the results. The latter, recorded in the table below, reveal that the treatment according to the invention greatly reduces the adsorption of this protein, which is known to adhere strongly to surfaces, in particular hydrophobic surfaces.

(57) TABLE-US-00007 Treatment Fluorescence (arbitrary units) Number of beads adsorbed None 14.3 +/− 1.6  3326 +/− 930 Solution B.sub.2 2.9 +/− 0.3 139 +/− 49

Example 11: Adsorption of Microparticles within a Microchannel

(58) The same protocol as for example 10 was used, except for the solution of fluorescent protein, which was replaced with 70 μl of a solution of microbeads at a concentration of 0.13 bead/μl (“Dynabeads Epithelial Enrich”) in PBS enriched with 0.1% of Tween 20™ in order to ensure the colloidal stability of the beads.

(59) This solution was first introduced at 100 mbar and then the pressure was reduced in order to achieve a flow rate of 1 μl/min in order to promote the sedimentation of the beads. Finally, the nonadsorbed beads were removed by rinsing with a solution of PBS enriched with 0.1% of Tween 20™ at a pressure of 300 mbar.

(60) Images of an untreated microchannel and of a microchannel pretreated with the solution B.sub.2 were taken using the same microscope as described in example 10, provided with a 10× objective, and were processed automatically in order to count the beads. The results, given in the third column of table 10.1, show that the treatment spectacularly reduces the adsorption of microparticles.

Example 12: Determination of the CMC of the Polymers 1 and 2

(61) The CMC is determined by the Wilhelmy plate method (K10 device, Kruss), with a series of dilutions of the polymers in a 1×PBS buffer, varying from 10.sup.−8% to 1%. For the two polymers, the CMC determined by this method is between 0.5% and 2×10.sup.−3% by weight of polymer.

Example 13: Use of Polymers According to the Invention of the Charged Polymer Type for Facilitating the Adhesion of Cells

(62) This study was carried out on open surfaces made of COC, consisting of Topas 8007 COG sheets, with a thickness of 145 μm. They were cut out according to the dimensions 2 mm×4 mm and were adhesively bonded to StarFrost glass slides with an adhesive which can be cured by UV irradiation (NOA81, Norland Optical Adhesive), in order to facilitate the handling thereof under a microscope. The insolation time used is 10 min with a UV lamp (Fisher Scientific).

(63) Choice of the Treatments:

(64) Use is made, as reference of the state of the art, of a treatment in the form of a double layer of hydroxyethyl cellulose (HEC, average molecular weight ˜90 000, Sigma Aldrich)-poly-L-lysine (PLL, molecular weight: 150 000-300 000, concentration: 0.01%, sterile-filtered, Sigma Aldrich). The HEC treatment is biocompatible and it is adsorbed on the COG to render it hydrophilic (contact angle of the treated COC=56°). The PLL is adsorbed on the COC treated with HEC, conferring on it a positive charge, which improves the adhesion of the cells to the glass.
In this example, two charged polymers according to the invention were used:
another batch of DMAM-022 M polymer was grafted with 10% of anionic charges (sodium sulfonate) and a third batch of polymer was grafted with 10% of cationic charges (tertiary amine hydrochloride type). Thus, the anionic polymer can be used to couple the PLL to the surface of the COC and the cationic polymer could be used alone.
Preparation of the Slides:
The following solutions were prepared in order to treat the surface of the COO: Solution 1: 2% by weight HEC in 1×PBS Solution 2: neutral DMAM-C22 M (polymer 3 described in example 3) at 0.1% by weight in Milli-Q water Solution 3: anionic DMAM-C22 (polymer 4 described in example 4) at 0.1% by weight in Milli-Q water Solution 4: cationic DMAM-C22 (polymer 5 described in example 5), 0.1% by weight in Milli-Q water.

(65) The polymer solutions were incubated over the COC overnight, then rinsed once with 50 μl of 1×PBS and then in a bath of 1×PBS for 5 minutes.

(66) The COC slides treated with solutions 1, 2 and 3 were subsequently treated with a solution of PLL at 37° C. in an incubator with a humid atmosphere with 5% CO.sub.2 for 2 hours and then rinsed with 1×PBS.

(67) The COC slides treated with solution 4 were used directly without PLL.

(68) For each slide, a silicone microchamber was adhesively bonded in order to confine the cells in a volume of 50 μl and to limit the surface area for adhesion of the cells in order to facilitate the imaging.

(69) 10 μl of a solution of epithelial cells (MCF-7 line, breast cancer) with a concentration of 2000 cells/μl were injected into each chamber.

(70) After culturing for 24 hours in an incubator with a humid atmosphere with 5% of CO.sub.2, the slides were washed twice with 1×PBS in order to remove the cells which have not adhered to the substrate. The cells which have adhered to the COC were subsequently fixed for 30 min in a 3.7% paraformaldehyde solution. After fixing, the slides were washed twice in 1×PBS.

(71) EpCAM-FITC Labelings+DAPI/Vectashield:

(72) In order to compare the effectiveness of the treatments, the cells attached to the slides after having labeled them with fluorescence were observed by fluorescence microscopy. The cell membranes were labeled with anti-EpCAM antibodies (20 μl EpCAM Ab+500 μl PBS+1% BSA) at ambient temperature for 30 min. A first washing was subsequently carried out with 50 μl of a 1×PBS solution comprising 1% of BSA and then in a bath of 1×PBS at ambient temperature for 5 minutes. Finally, a drop of DAPI/Vectashield was applied to each spot of cells and everything was covered with a cover slip having dimensions of 50×24 mm. The samples were retained at 4° C. until the acquisition of the fluorescence images (DAPI+FITC).

(73) Results:

(74) The HEC+PLL solution improves the adhesion of the cells to the surface of the COC. On the other hand, this treatment does not appear suitable for the culturing of MCF-7 cells. In our experiments, we observed a low density of cells adhered to the COC. Many cells appear to have burst or to have undergone an apoptotic process, the membranes visible by EpCAM labeling are not well defined or are not observable, as is apparent in FIG. 2a. For COC+DMAM-C22 M (0.35%)+PLL, a high background noise in the FITC channel, a suitable density of cells but many lysed or apoptotic cells are observed. This treatment appears to be a good candidate for attaching cells to the COC but not for their culturing. For COC+anionic DMAM-C22+PLL, the background noise is low, which shows that this treatment does not promote the nonspecific adsorption of antibodies. However, the density of cells attached to the surface of the COC is low, which implies that the cells have trouble adhering to the substrate. Semiapoptotic cells but no lysed cells are also observed. The cationic DMAM-C22 treatment (without PLL) appears suitable for promoting the adhesion and the division of the MCF-7 cells. A suitable cell density and doublets or multiplets of cells, which are characteristics of divided cells, are observed in FIG. 2b.

(75) It is thus found that the charged polymers according to the invention can constitute an improvement with respect to the state of the art, either in the anionic form combined with a cationic polymer, for improving the cell adhesion, or used alone, for promoting cell culturing.