BIOCOMPATIBLE DEVICE WITH AN ADSORBED LAYER OF CATIONIC COMB COPOLYMER

Abstract

The present invention relates to a biocompatible device which comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide or a polyethylene glycol (meth)acrylamide, at least one monomer M selected from alkyl (meth)acrylate, aryloxyalkyl (meth)acrylate, alkyl (meth)acrylamide or aryl (meth)acrylamide, and at least one cationic monomer C selected from cationic ethylenically unsaturated N-containing monomers. It further relates to a process for making a biocompatible device which comprises on its surface an adsorbed layer of the polymer P comprising the following steps: providing a biocompatible device, and applying to the surface of the biocompatible device a solution S of the polymer Pin a solvent L. It further relates to a solution S comprising the polymer P in the solvent L, where the solvent L comprises an alcohol; and to a process for cultivating cells, comprising the following steps: providing the biocompatible device and cultivating the cells in the supernatant medium above the surface of the biocompatible device.

Claims

1. A biocompatible device which comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide or a polyethylene glycol (meth)acrylamide, at least one monomer M selected from alkyl (meth)acrylate, aryloxyalkyl (meth)acrylate, alkyl (meth)acrylamide or aryl (meth)acrylamide, and at least one cationic monomer C selected from cationic ethylenically unsaturated N-containing monomers.

2. The biocompatible device according to claim 1, where the biocompatible device is a biosensor or a device for cultivating cells.

3. The biocompatible device according to claim 2, where the device for cultivating cells is selected from cell culture flasks, cell culture dishes, cell culture plates, cell culture bags, reactors, tubes, probes, vials, bottles, pipettes, pipette tips, syringes, microscopic slides, microfluidic devices, glass capillaries, biochips, SiO2 wavers or SiO2 arrays.

4. The biocompatible device according to claim 1, where the macromonomer is the ester E of (meth)acrylic acid and polyethylene oxide.

5. The biocompatible device according to claim 1, where the monomer M is C.sub.1-C.sub.6 alkyl (meth)acrylate.

6. The biocompatible device according to claim 1, where the cationic monomer C is a monomer of formula (I) ##STR00002## where R.sub.1 is H or C1-C4-alkyl; R.sub.2 is H or methyl; R.sub.3 is C1-C4-alkylene; R.sub.4, R.sub.5 and R.sub.6 are each independently H or C1-C30-alkyl; X is —O— or —NH—; and Y is Cl; Br; I; hydrogensulfate or methosulfate.

7. The biocompatible device according to claim 1, where the polymer P comprises 5-90 mol % of the macromonomer; 15-75 mol % of the monomer M; and 5-60 mol % of the cationic monomer C, and where the molar amounts of the monomers sums up to 100%.

8. The biocompatible device according to claim 1, where the surface is free of other layers beside the adsorbed layer of the polymer P.

9. The biocompatible device according to claim 1, where the surface is at least partly made of glass.

10. The biocompatible device according to claim 1, where there are no covalent chemical bonds between the polymer P and the surface.

11. A process for making a biocompatible device which comprises on its surface an adsorbed layer of a polymer P which is a copolymer of at least one macromonomer selected from an ester E of (meth)acrylic acid and polyethylene oxide or an polyethylene glycol (meth)acrylamide, at least one monomer M selected from alkyl (meth)acrylate, aryloxyalkyl (meth)acrylate, alkyl (meth)acrylamide or aryl (meth)acrylamide, and at least one cationic monomer C selected from cationic ethylenically unsaturated N-containing monomers, comprising the following steps: A) providing a biocompatible device, and B) applying to the surface of the biocompatible device a solution S of the polymer P in a solvent L.

12. The process according to claim 11, further comprising the following step: C1) removing the supernatant solution S; or C2) removing the solvent L.

13. The process according to claim 11, where the solvent L comprises an alcohol.

14. A solution S comprising the polymer P as defined in claim 1 in a solvent L, where the solvent L comprises an alcohol.

15. The solution S according to claim 14 comprising 0.01 to 5 wt % of the polymer P and 5 to 90 wt % of the alcohol.

16. A process for cultivating cells, comprising the following steps: a) providing the biocompatible device which is a device for cultivating cells as defined in claim 1, and b) cultivating the cells in the supernatant medium above the surface of the biocompatible device.

Description

EXAMPLES

[0195] Molecular weights were determined by Size Exclusion Chromatography using a mixed bed scouting column for water soluble linear polymers at 35° C. The eluent used was 0.01 M phosphate buffer at pH=7.4 containing 0.01 M sodium azide. The polymer used as 1.5 mg/mL concentrated solution in the eluent. Before injection all samples were filtered through a 0.2 μm filter. The calibration was carried out with narrow poly(ethylene glycol) samples having molecular weights between 106 and 1,378,000 g/mol.

Example 1: Polymer Preparation

[0196] 32.3 parts by weight of methoxy polyethylene glycol methacrylate (50% aqueous solution, molecular weight (av.) 2100 g/mol) dissolved in 12.7 parts by weight of isopropanol and 2 parts by weight of 2-Trimethylammoniumethyl methacrylate chloride (TMAEMC, 80% aqueous solution) and 2.2 parts by weight of n-butyl methacrylate dissolved in 5.8 parts by weight of isopropanol were added to 400 parts by weight of isopropanol. The reaction mixture was stirred at 125 rpm under nitrogen and heated to 80° C. Simultaneously, 291.2 parts by weight of the methoxy polyethylene glycol methacrylate dissolved in 113.8 parts by weight of isopropanol were added within 3 h, 18.2 parts by weight of TMAEMC (80% aqueous solution) and 19.9 parts by weight of n-butyl methacrylate dissolved in 51.9 parts by weight of isopropanol were added within 2 h and 2.7 parts by weight of tert-butyl peroxypivalate (75% solution) in 47.3 parts by weight of isopropanol were added within 4 h. Subsequently, 2.7 parts by weight of tert-butyl peroxypivalate (75% solution) in 17.3 parts by weight of isopropanol were added within 1 h. Afterwards, the reaction was kept for 2 hours at 80° C. Subsequently, the reaction was cooled down to the ambient temperature and subjected to purification by water steam distillation. The molar ratio of methoxy polyethylene glycol methacrylate:TMAEMC:n-butyl methacrylate was of 1:1:2. The resulting polymer had a Mn 6500 g/mol and about Mw 9000 g/mol.

Example 2: Polymer Preparation

[0197] The polymer preparation of Example 1 was repeated with the same monomers, but a different molar ratio of methoxy polyethylene glycol methacrylate:TMAEMC:n-butyl methacrylate of 2:1:1.

Example 3: Coating of Model Surfaces and Anti-Adhesive Evaluation

[0198] Method: The amounts of polymer P or milk proteins absorbed on the surface were determined by Quartz-Crystal Microbalance (QCM), which measures the resonance frequency of a freely oscillating quartz sensor after excitation in contact with the liquid medium. QCM measurements were performed using standard flow-through setups with a flow rate of 50 μL/min at 23° C. A measured shift in resonance frequency scales inversely with mass changes at the sensor surface. The adsorbed amounts were calculated from the shift of the 7th overtone of the resonance frequency according to the method of Sauerbrey. The mass sensitivity or mass changes is estimated to ˜10 ng/cm.sup.2 in all experiments. Measurements were performed on a quartz sensor quartz coated with silicon dioxide, soda lime glass, or with borosilicate glass.

[0199] Adsorption of Polymer P and of Milk Proteins:

[0200] The experiments comprised the following steps and used an aqueous HEPES (4-(2-Hydroxyethyl)-1-piperazine ethanesulfonic acid) buffer (10 mmol/L) with pH 7 (“buffer”): [0201] 1) buffer until a stable baseline was achieved; [0202] 2) 2 h 0.1 wt % polymer P solution in buffer; [0203] 3) 2 h buffer; [0204] 4) 0.5 h 0.1 wt % milk powder in buffer; [0205] 5) 0.5 h buffer.

[0206] Milk fouling was monitored during exposure of the samples to 0.1 wt % solutions of milk powder for 0.5 h. The final mass change was recorded after another 0.5 h of rinsing with buffer (steps 4) and 5) above). The results (based on double determination) are given in Table 1-3.

TABLE-US-00001 TABLE 1 Silicon dioxide surface Amount of Milk Protein Milk Protein polymer P Adhesion Adhesion Polmyer P [ng/cm.sup.2] [ng/cm.sup.2] [wt %] No polymer (comparative) — 364 100 Ex. 1 401 53 14.6 Ex. 2 315 41 11.3

TABLE-US-00002 TABLE 2 on soda lime glass surface Amount of Milk Protein Milk Protein polymer P Adhesion Adhesion Polmyer P [ng/cm.sup.2] [ng/cm.sup.2] [wt %] No polymer (comparative) — 457.5 100 Ex. 1 436 72 15.7 Ex. 2 442 54 11.8

TABLE-US-00003 TABLE 3 on borosilicate glass surface Amount of Milk Protein Milk Protein polymer P Adhesion Adhesion Polmyer P [ng/cm.sup.2] [ng/cm.sup.2] [wt %] No polymer (comparative) — 697 100 Ex. 1 543 33 4.7 Ex. 2 532 −34 −4.9

Example 4: Evaluation of Cell Adherence in Cell Culture

[0207] The adsorbed layer of polymer P on the glass plates have been made as follows. As basis plate a SiO.sub.2 coated plate was used (96 PLATE+GL CT 7 MM RD FLAT BASE, Greiner: CRMA60180-P304).

[0208] Solid polymer P powder was dissolved in 20% ethanol (v/v) and 80% Milli-Q-water at a final concentration of 1% and passed through a filter for sterilization. Sterile solution was pipetted into wells (200 μl per well) and incubated at room temperature for 3 min. After incubation the polymer solution was removed using a standard pipette and dried for 30 min, followed by cell seeding (HUVEC, 6000 cell/200 μl/well) as described previously.

[0209] The adherence was characterized using an automated light microscopy (IncuCyte System, Sartorius) with a 4× objective, phase contrast and photo documentation taken whole well picture per well. Quantitative analysis of cell adherence was performed by means of automatic microscopy and image analysis using the corresponding software of the automated microscope (IncuCyte S3 2018B, confluence mask). The percentage of adherent cells was determined after 2 days of culture via surface analysis within the well. Cells that do not adhere to the surface of the plate, accumulate into cell aggregates. In contrast, adherent cells spread on the surface of the cell culture plate can be quantified via a confluence analysis, which determines the surface covered by cells. The results were summarized in Table 4.

[0210] The results demonstrate that the Polymer P reduces the deposition of cells on glass surfaces.

TABLE-US-00004 TABLE 4 Polymer P % Area covered by adherent cells no polymer (comparative) 78 ± 1.7 Ex 1 10 ± 0.9