Self-supporting, biodegradable film based on hydrophobized hyaluronic acid, method of preparation and use thereof

Abstract

The invention relates to a self-supporting, biodegradable film comprising a C.sub.10-C.sub.22-acylated derivative of hyaluronic acid according to the general formula (I), where R is H.sup.+ or Na.sup.+, and where R.sup.1 is H or C(O)C.sub.xH.sub.y, where x is an integer within the range from 9 to 21 and y is an integer within the range from 11 to 43 and C.sub.xH.sub.y is a linear or branched, saturated or unsaturated C.sub.9-C.sub.21 chain, wherein in at least one repeating unit one or more of R.sup.1 is C(O)C.sub.xH.sub.y and where n is within the range from 12 to 4000; a method of preparation thereof and use thereof. ##STR00001##

Claims

1. A film based on a hyaluronic acid ester, characterized by that it comprises a C.sub.10-C.sub.22-acylated derivative of hyaluronic acid according to the general formula (I) ##STR00003## where R is H.sup.+ or Na.sup.+, and where R.sup.1 is H or C(O)C.sub.xH.sub.y, where x is an integer within the range from 9 to 21 and y is an integer within the range from 11 to 43 and C.sub.xH.sub.y is a linear or branched, saturated or unsaturated chain C.sub.9-C.sub.21, wherein in at least one repeating unit one or more of R.sup.1 is C(O)C.sub.xH.sub.y and where n is within the range from 12 to 4000; and wherein the film is self-supporting, the thickness of the film is homogeneous and is within a range of 2 m to 100 m, and the Young's modulus of the film is within a range from 1 to 5000 MPa in the dry state, and the surface roughness expressed in the form of a root mean square of at least one of the film surfaces is within the range from 0.5 to 100 nm.

2. The film according to claim 1, characterized by that it comprises palmitoyl hyaluronan or lauroyl hyaluronan.

3. The film according to claim 1 or claim 2, characterized by that the C.sub.10-C.sub.22-acylated derivative of hyaluronic acid has the molecular weight from 110.sup.5 to 110.sup.6 g/mol.

4. The film according to claim 1 or claim 2, characterized by that the C.sub.10-C.sub.22-acylated derivative of hyaluronic acid has the substitution degree within the range from 15 to 160% relative to hyaluronan dimers.

5. The film according to claim 1 or claim 2, characterized by that it has the thickness within the range from 5 to 25 m.

6. The film according to claim 1 or claim 2, characterized by that it further comprises at least one biologically active substance selected from the group including pharmaceutically active substances and cosmetically active substances.

7. The film according to claim 1 or claim 2 for use in medical applications or biotechnological applications.

8. The film according to claim 7 for use in the construction of a medical device.

9. A method of preparation of the film defined in claim 1 or claim 2, characterized by that a solution comprising a C.sub.10-C.sub.22 acylated derivative of hyaluronic acid according to the general formula (I) is prepared in a mixture of water and C.sub.1-C.sub.6 alcohol, which is stirred, and then applied on a substrate and dried in a closed space, and then is removed from the substrate.

10. The method of preparation of the film according to claim 9, characterized by that the ratio of the mixture of C.sub.1-C.sub.6 alcohol and water is within the range from 25-55 vol. % to 45-75 vol. %, wherein the amount of the C.sub.10-C.sub.22 acylated derivative of hyaluronic acid in the solution is within the range from 0.5 to 3 wt. %.

11. The method of preparation of the film according to claim 9, characterized by that the solution is stirred for 20 to 72 hours.

12. The method of preparation of the film according to claim 9, characterized by that the drying takes place at a temperature within the range from 20 C. to 50 C. for 3 to 6 hours.

13. The method of preparation of the film according to claim 9, characterized by that the drying takes place in a temperature gradient, where the lower film surface lying on the substrate is heated to a temperature that is at least 1 C. higher than the temperature to which the opposite upper surface of the film is heated or cooled.

14. The method of preparation of the film according to claim 13, characterized by that the lower surface of the film lying on the substrate is heated to the temperature within the range from 20 C. to 60 C. and the opposite upper surface of the film is heated or cooled to the temperature within the range from 10 C. to 59 C.

15. The method of preparation of the film according to claim 13 or claim 14, characterized by that the film is dried in a temperature gradient for 6 to 12 hours.

16. The method of preparation of the film according to claim 9, characterized by that at least one biologically active substance is admixed to the solution, the substance being selected from the group consisting of pharmaceutically and cosmetically active substances, vitamins, drugs, cytostatics, steroids, phytoextracts, phytocomplexes, and phytoactive substances.

17. The method of preparation of the film according to claim 9, characterized by that the substrate is a polymer selected from the group consisting of polyvinyl alcohol, polypropylene, polyethylene, polyoxymethylene, polystyrene, and hydrophobized glass, wherein the contact wetting angle of the substrate surface by demi water is within the range from 30 to 120.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1: effect of the adhesion of the film (a,b) on the basis of the oleyl derivative of sodium hyaluronate on the surface appearance thereof (c)

(2) FIG. 2: effect of the adhesion of the film on the basis of the palmitoyl derivative of sodium hyaluronate on the surface appearance thereof

(3) FIG. 3: comparison of the films prepared according to Examples 2 (b) and 18 (a) after 3 weeks of degradation in a conditioned medium

(4) FIG. 4: Proof of the presence of HA-based oligosaccharides in the solution of the film according to Example 5 (DS=20%) after incubation in DMEM (Dulbecco's modified Eagle's medium) with an addition of an enzyme, by means of HPLC. The figure shows 3 chromatograms corresponding to (i) separation of HA oligosaccharide standards (standard HA2 (t.sub.R=4.1 min), HA4 (t.sub.R=12 min), HA6 (t.sub.R=18.1 min), HA8 (t.sub.R=22.9 min)), (ii) a blank sampleDMEM with an addition of an enzyme and (iii) solution in which the incubation of the film was carried out.

(5) FIG. 5: morphology of the film prepared according to Example 16 from the substrate side

(6) FIG. 6: viability of the suspension THP-1 cells after 24 and 72 hours of incubation with the film prepared according to Example 1 based on the palmitoyl derivative of sodium hyaluronate

(7) FIG. 7: induction of the cell death after 24 hours of incubation with the film prepared according to Example 1 based on the palmitoyl derivative of sodium hyaluronate

(8) FIG. 8: induction of the cell death after 72 hours of incubation with the film prepared according to Example 1 based on the palmitoyl derivative of sodium hyaluronate

(9) FIG. 9: contact inhibition of the growth of mouse 3T3 Swiss fibroblasts caused by the film prepared according to Example 1.

(10) FIG. 10: cell antiadhesive properties of the film, Athe film prepared according to Example 17, upper surface of the film, Bthe film prepared according to Example 17, lower surface of the film, Cthe film prepared according to Example 2, upper surface of the film, Dthe film prepared according to Example 2, lower surface of the film, CTRLcontrol

EXAMPLES

Example 1. Preparation of the Film Based on Palmitoyl Derivative of Sodium Hyaluronate

(11) 100 mg of palmitoyl derivative of sodium hyaluronate having the substitution degree of 100% and molecular weight 2.810.sup.5 g/mol were dissolved in 20 ml of 55% solution of propan-2-ol and stirred for at least 72 hours. After stirring, the solution was dosed on a hydrophobized glass having the wettability by demi water of 61 (+/2) and dried in a closed space, in a temperature gradient at the temperature of the lower plate 50 C. and the temperature of the upper plate 40 C. for 12 hours. After drying, the film was evaluated, removed from the hydrophobized glass and characterized. The thickness of the thus prepared film was determined to be about 15 m. The dry matter was determined to be around 92%.

Example 2. Preparation of the Film Based on Palmitoyl Derivative of Sodium Hyaluronate

(12) 100 mg of palmitoyl derivative of sodium hyaluronate having the substitution degree of 100% and molecular weight 2.1210.sup.5 g/mol were dissolved in 20 ml of 55% solution of propan-2-ol and stirred for at least 48 hours. After stirring, the solution was dosed on a hydrophobized glass having the wettability by demi water of 61 (+/2) and dried in a closed space, in a temperature gradient at the temperature of the lower plate 50 C. and the temperature of the upper plate 20 C. for 6 hours. After drying, the film was evaluated, removed from the hydrophobized glass and characterized. The thickness of the thus prepared film was determined to be about 15 m.

Example 3. Preparation of the Film Based on Palmitoyl Derivative of Sodium Hyaluronate

(13) 100 mg of palmitoyl derivative of sodium hyaluronate having the substitution degree of 55% and molecular weight 6.010.sup.5 g/mol were dissolved in 20 ml of 50% solution of ethanol and stirred for at least 20 hours. After stirring, the solution was dosed on a polyethylene substrate having the wettability by demi water of 79 (+/4) and dried in a closed space, in a temperature gradient at the temperature of the lower plate 30 C. and the temperature of the upper plate 29 C. for 12 hours. After drying, the film was evaluated, removed from the substrate and characterized. The thickness of the thus prepared film was determined to be about 15 m.

Example 4. Preparation of the Film Based on Palmitoyl Derivative of Sodium Hyaluronate

(14) 50 mg of palmitoyl derivative of sodium hyaluronate having the substitution degree of 31% and molecular weight 9.910.sup.5 g/mol were dissolved in 20 ml of 45% solution of ethanol and stirred for at least 20 hours. After stirring, the solution was dosed on a hydrophobized glass having the wettability by demi water of 61 (+/2) and dried in a closed space by evaporating the solvent at the temperature of 30 C. for 4 hours. After drying, the film was evaluated, removed from the substrate and characterized. The thickness of the thus prepared film was determined to be about 8 m.

Example 5. Preparation of the Film Based on Palmitoyl Derivative of Sodium Hyaluronate

(15) 100 mg of palmitoyl derivative of sodium hyaluronate having the substitution degree of 20% and molecular weight 2.410.sup.5 g/mol were dissolved in 20 ml of 25% solution of ethanol and stirred for at least 20 hours. After stirring, the solution was dosed on a polystyrene substrate having the wettability by demi water of 102 (+/4) and dried in a closed space, in a temperature gradient at the temperature of the lower plate 50 C. and the temperature of the upper plate 20 C. for 12 hours. After drying, the film was evaluated, removed from the substrate and characterized. The thickness of the thus prepared film was determined to be about 15 m.

Example 6. Preparation of the Film Based on Erucoyl Derivative of Sodium Hyaluronate

(16) 100 mg of erucoyl derivative of sodium hyaluronate having the substitution degree of 160% and molecular weight 2.0410.sup.5 g/mol were dissolved in 20 ml of 60% solution of propan-2-ol and stirred for at least 20 hours. After stirring, the solution was dosed on a polypropylene substrate having the wettability by demi water of 105 (+/2) and dried in a closed space by evaporating the solvent at the temperature of 50 C. for 3 hours. After drying, the film was evaluated, removed from the substrate and characterized. The thickness of the thus prepared film was determined to be about 15 m.

Example 7. Preparation of the Film Based on Lauroyl Derivative of Sodium Hyaluronate

(17) 100 mg of lauroyl derivative of sodium hyaluronate having the substitution degree of 64% and molecular weight 3.210.sup.5 g/mol were dissolved in 20 ml of 50% solution of propan-2-ol and stirred for at least 20 hours. After stirring, the solution was dosed on hydrophobized glass having the wettability by demi water of 50 (+/3) and dried in a closed space, in a temperature gradient at the temperature of the lower plate 50 C. and the temperature of the upper plate 20 C. for 6 hours. After drying, the film was evaluated, removed from the substrate and characterized. The thickness of the thus prepared film was determined to be about 15 m.

Example 8. Preparation of the Film Based on Lauroyl Derivative of Sodium Hyaluronate

(18) 100 mg of lauroyl derivative of sodium hyaluronate having the substitution degree of 90% and molecular weight 1.8810.sup.5 g/mol were dissolved in 20 ml of 50% solution of propan-2-ol and stirred for at least 20 hours. After stirring, the solution was dosed on hydrophobized glass having the wettability by demi water of 61 (+/2) and dried in a closed space by evaporating the solvent at the temperature of 20 C. for 6 hours. After drying, the film was evaluated, removed from the substrate and characterized. The thickness of the thus prepared film was determined to be about 15 m.

Example 9. Preparation of the Film Based on Oleyl Derivative of Sodium Hyaluronate

(19) 100 mg of oleyl derivative of sodium hyaluronate having the substitution degree of 20% and molecular weight 2.810.sup.5 g/mol were dissolved in 20 ml of 50% solution of propan-2-ol and stirred for at least 20 hours. After stirring, the solution was dosed on a polyvinylchloride substrate having the wettability by demi water of 95 (+/5) and dried in a closed space, in a temperature gradient at the temperature of the lower plate 50 C. and the temperature of the upper plate 20 C. for 6 hours. After drying, the film was evaluated, removed from the substrate and characterized. The thickness of the thus prepared film was determined to be about 15 m.

Example 10. Preparation of the Film Based on Oleyl Derivative of Sodium Hyaluronate

(20) 300 mg of oleyl derivative of sodium hyaluronate having the substitution degree of 20% and molecular weight 2.810.sup.5 g/mol were dissolved in 20 ml of 30% solution of propan-2-ol and stirred for at least 20 hours. After stirring, the solution was dosed on hydrophobized glass having the wettability by demi water of 57 (+/3) and dried in a closed space, in a temperature gradient at the temperature of the lower plate 50 C. and the temperature of the upper plate 20 C. for 12 hours. After drying, the film was evaluated, removed from the substrate and characterized. The thickness of the thus prepared film was determined to be about 40 m.

Example 11. Preparation of the Film Based on Oleyl Derivative of Sodium Hyaluronate

(21) 300 mg of oleyl derivative of sodium hyaluronate having the substitution degree of 20% and molecular weight 2.810.sup.5 g/mol were dissolved in 20 ml of 30% solution of propan-2-ol and stirred for at least 20 hours. After stirring, the solution was dosed on hydrophobized glass having the wettability by demi water of 107 (+/1) and dried in a closed space, in a temperature gradient at the temperature of the lower plate 50 C. and the temperature of the upper plate 20 C. for 12 hours. After drying, the film was evaluated, removed from the substrate and characterized. The thickness of the thus prepared film was determined to be about 40 m.

Example 12. Preparation of the Film Based on Caprinyl (C10) Derivative of Sodium Hyaluronate

(22) 100 mg of caprinyl (C10) derivative of sodium hyaluronate having the substitution degree of 87% and molecular weight 2.5010.sup.5 g/mol were dissolved in 20 ml of 50% ethanol and stirred for at least 20 hours. After stirring, the solution was dosed on hydrophobized glass having the wettability by demi water of 61 (+/2) and dried in a closed space, in a temperature gradient at the temperature of the lower plate 60 C. and the temperature of the upper plate 40 C. for 10 hours. After drying, the film was evaluated, removed from the substrate and characterized. The thickness of the thus prepared film was determined to be about 15 m, the dry matter was determined to be around 92%. The swelling capacity of the film was determined to be more than 100% (variation of the film area was measured) in equilibrium state.

Example 13. Preparation of the Film Based on Behenoyl Derivative of Sodium Hyaluronate

(23) 100 mg of behenoyl derivative of sodium hyaluronate having the substitution degree of 16% and molecular weight 3.310.sup.5 g/mol were dissolved in 20 ml of 50% solution of propan-2-ol and stirred for at least 20 hours. After stirring, the solution was dosed on a polypropylene substrate having the wettability by demi water of 105 (+/2) and dried in a closed space, in a temperature gradient at the temperature of the lower plate 50 C. and the temperature of the upper plate 20 C. for 6 hours. After drying, the film was evaluated, removed from the substrate and characterized. The thickness of the thus prepared film was determined to be about 15 m.

Example 14. Preparation of the Film Based on Lauroyl Derivative of Sodium Hyaluronate

(24) 100 mg of lauroyl derivative of sodium hyaluronate having the substitution degree of 29% and molecular weight 1.8810.sup.5 g/mol were dissolved in 20 ml of 50% solution of propan-2-ol and stirred for at least 20 hours. After stirring, the solution was dosed on hydrophobized glass having the wettability by demi water of 61 (+/2) and dried in a closed space, in a temperature gradient at the temperature of the lower plate 50 C. and the temperature of the upper plate 20 C. for 7 hours. After drying, the film was evaluated, removed from the substrate and characterized. The thickness of the thus prepared film was determined to be about 15 m.

Example 15. Preparation of the Film Based on Oleyl Derivative of Sodium Hyaluronate

(25) 100 mg of oleyl derivative of sodium hyaluronate having the substitution degree of 15% and molecular weight 2.810.sup.5 g/mol were dissolved in 20 ml of 50% solution of propan-2-ol and stirred for at least 48 hours. After stirring, the solution was dosed on hydrophobized glass having the wettability by demi water of 61 (+/2) and dried in a closed space, in a temperature gradient at the temperature of the lower plate 50 C. and the temperature of the upper plate 20 C. for 12 hours. After drying, the film was evaluated, removed from the substrate and characterized. The thickness of the thus prepared film was determined to be about 15 m.

Example 16. Preparation of the Film Based on Palmitoyl Derivative of Sodium Hyaluronate

(26) 100 mg of palmitoyl derivative of sodium hyaluronate having the substitution degree of 34% and molecular weight 2.6710.sup.5 g/mol were dissolved in 20 ml of 50% solution of ethanol and stirred for at least 48 hours. After stirring, the solution was dosed on hydrophobized glass having the wettability by demi water of 61 (+/2) and dried in a closed space, in a temperature gradient at the temperature of the lower plate 50 C. and the temperature of the upper plate 20 C. for 12 hours. After drying, the film was evaluated, removed from the substrate and characterized. The thickness of the thus prepared film was determined to be about 15 m.

Example 17. Preparation of the Film Based on Palmitoyl Derivative of Sodium Hyaluronate

(27) 100 mg of palmitoyl derivative of sodium hyaluronate having the substitution degree of 60% and molecular weight 2.810.sup.5 g/mol were dissolved in 20 ml of 50% solution of ethanol and stirred for at least 48 hours. After stirring, the solution was dosed on hydrophobized glass having the wettability by demi water of 61 (+/2) and dried in a closed space, in a temperature gradient at the temperature of the lower plate 50 C. and the temperature of the upper plate 20 C. for 12 hours. After drying, the film was evaluated, removed from the substrate and characterized. The thickness of the thus prepared film was determined to be about 15 m.

Example 18. Preparation of the Film Based on Palmitoyl Derivative of Sodium Hyaluronate

(28) 100 mg of palmitoyl derivative of sodium hyaluronate having the substitution degree of 31% and molecular weight 2.710.sup.5 g/mol were dissolved in 20 ml of 50% solution of propan-2-ol and stirred for at least 20 hours. After stirring, the solution was dosed on hydrophobized glass having the wettability by demi water of 61 (+/2) and dried in a closed space, in a temperature gradient at the temperature of the lower plate 50 C. and the temperature of the upper plate 20 C. for 12 hours. After drying, the film was evaluated, removed from the substrate and characterized. The thickness of the thus prepared film was determined to be about 15 m.

Example 19. Preparation of the Film Based on Lauroyl Derivative of Sodium Hyaluronate

(29) 100 mg of lauroyl derivative of sodium hyaluronate having the substitution degree of 58% and molecular weight 1.8810.sup.5 g/mol were dissolved in 20 ml of 50% solution of propan-2-ol and stirred for at least 48 hours. After stirring, the solution was dosed on hydrophobized glass having the wettability by demi water of 61 (+/2) and dried in a closed space, in a temperature gradient at the temperature of the lower plate 20 C. and the temperature of the upper plate 10 C. for 12 hours. After drying, the film was evaluated, removed from the substrate and characterized. The thickness of the thus prepared film was determined to be about 15 m.

Example 20. Comparison of Substrate Wettabilities Obtained by Using Various Hydrophobization Agents

(30) The glass intended for hydrophobization was first cleaned thoroughly so that the resulting demi water wettability would not be above 10. Then the hydrophobization of the glass was conducted. For the hydrophobization of the glass, the following hydrophobization agents were used: chlorotrimethylsilane (CTMS), (3-aminopropyl)trimethoxysilane (APTMS) and octadecyltrichlorosilane (OTS). The resulting values of the glass wettability (with the concentration of the agent being 1%) are listed in Table 1. Moreover, for CTMS, various concentrations of the agent in hexane were tested. The obtained values of the measured glass wettabilities are listed in Table 2.

(31) TABLE-US-00001 TABLE 1 glass wettability expressed by the contact angle upon hydrophobization by an agent having the concentration of 1% Agent (1%) Solvent CA CTMS hexane approx. 70 APTMS hexane approx. 90 acetone approx. 70 dichloromethane approx. 90 OTS ethanol (96%) approx. 66 toluene approx. 105 propan-2-ol approx. 103 CA means contact angle

(32) TABLE-US-00002 TABLE 2 glass wettability expressed by the contact angle upon hydrophobization by various concentrations of CTMS in hexane Concentration of CTMS CA 0.10% approx. 50 0.50% approx. 60 1% approx. 70 3-5% <80 CA means contact angle

Example 21. Determination of the Hydrophobization Agent Residues in the Film Prepared According to Example 1

(33) Trimethylsilanol was analysed as a residuum of trimethylsilyl chloride after its reaction with the OH groups of the hyaluronan derivative. The analysis was carried out on a gas chromatograph equipped with a headspace sampler and a mass spectrometry detector in the form of a simple quadrupole. A sample of the film prepared according to Example 1 was dissolved to the concentration of 6 mg/ml in 50% (vol./vol.) propan-2-ol and upon dissolution, 4.75 ml of the sample and 0.25 ml of n-butanol (1 mg/ml), which acted as an internal standard, were pipetted into a vial. A stock solution of trimethylsilyl chloride (1 mg/ml) was prepared in 50% propan-2-ol as well, which reacted to trimethylsilanol immediately. From this solution, a calibration series ranging from 0.5 to 15.0 g/ml was prepared, with the addition of n-butanol as an internal standard. No analysed film sample proved the presence of trimethylsilanol in a concentration higher than the first calibration point, i.e., the content of trimethylsilanol in the film samples was lower than 0.008 wt. %.

Example 22. Effect of the Adhesion of the Film Based on an Oleyl Derivative of Sodium Hyaluronate on the Surface Appearance Thereof

(34) The films prepared according to Examples 10 and 11 were prepared on two glasses having different wettabilities, namely 57 (+/3) and 107 (+/1). After drying, the surface appearance of the film was evaluated and correlated with the adhesion. In the case of a good adhesion of the film to the substrate, the film surface is flat, without any surface deformations. The film was completely adhered to the substrate having a lower contact angle, on the substrate having a higher contact angle it was partially peeled off and deformed. The results are documented on FIGS. 1a, 1b, 1c. The figures imply that if the film is fully adhered to the surface, it is even and without surface deformations after being peeled off (FIG. 1c right). Conversely, in the case of an imperfect adhesion the film is more or less deformed (FIG. 1c left).

Example 23. Effect of the Adhesion of the Film Based on a Palmitoyl Derivative of Sodium Hyaluronate on the Surface Appearance Thereof

(35) The film based on the palmitoyl derivative of sodium hyaluronate prepared according to Example 2 on a glass having the wettability of 61 (+/2) was evaluated. After drying, the adhesion and the appearance of the film were evaluated. The film was well adhered and its surface was absolutely flat, without any deformations. The result of the adhesion is documented in FIG. 2.

Example 24. Determination of the Residual Propan-2-ol in the Films

(36) The residual concentration of the organic solvent propan-2-ol was determined by means of gas chromatography in the films prepared according to Examples 1, 8 and 12. The principle of the determination of the solvent is the conversion thereof into the gaseous phase at an elevated temperature, the separation thereof on the gas chromatograph and the subsequent detection by the flame ionization detector. The concentration of propan-2-ol in the film was always determined twice (i.e., for two samples), by reading from the calibration curve. The sample weight was always 50 mg. After completing the analysis, the concentration of the residual propan-2-ol was determined in all films to be lower than the lowest calibration curve point and was expressed as <0.02 wt. %. This value safely fulfils the requirements on the amounts of residual solvents of class 3 according to the EU pharmacopoeia.

Example 25. Determination of the WeightHomogeneity within the Area

(37) The film prepared according to Example 1 was cut into 55 squares with an area of 1 cm.sup.2. Prior to the measurement, the individual samples were left at room humidity and temperature for 5 hours. Then the individual squares were weighed on analytical scales. The obtained weights of the individual squares are listed in Table 3. The average, standard deviation and variation coefficient were calculated based on all the values listed in the Table. The calculated values: average 2.35 mg, standard deviation 0.18 mg, variation coefficient 7.51%.

(38) TABLE-US-00003 TABLE 3 determination of the weight homogeneity of the film 2.6 2.3 2.4 2.6 2.4 2.2 2.1 2.6 2.1 2.1 2.5 2.5 2.3 2.7 2.6 2.5 2.6 2.0 2.6 2.1 2.5 2.4 2.5 2.2 2.3 2.4 2.3 2.2 2.3 2.6 2.2 2.2 2.3 2.3 2.4 2.2 2.2 2.5 2.7 2.5 2.3 2.3 2.5 2.3 2.1 2.1 2.1 2.3 2.3 2.3 2.4 2.2 2.2 2.3 2.4

Example 26. Determination of the ThicknessHomogeneity within the Area

(39) A square grid having the area of one square 1 cm.sup.2 and the total number of squares 35 was drawn on the film prepared according to Example 15. On each square, the thickness of the film was measured by means of a mechanical thickness meter Mytutoyo VL-50. The measurement was conducted in a stable environment having the humidity of 50% and the temperature of 25 C. The measured values are listed in Table 4. The average, standard deviation and variation coefficient were calculated based on all values listed in the Table. The calculated values: average 14.6 m, standard deviation 1.17 m, variation coefficient 8.02%.

(40) TABLE-US-00004 TABLE 4 determination of the thickness homogeneity, the listed values are in m 16.2 16.7 16.2 15.2 13.8 13.8 14.7 13.9 13.9 13.9 13.7 13.4 13.2 14.3 14.8 13.9 14.1 13.3 12.8 13.5 14.6 16.9 16.2 16.6 15.7 15.6 15.0 16.2 14.7 14.9 15.0 14.7 13.7 12.5 13.8

Example 27. Comparison of the Swelling Capacities of the Films Prepared from Palmitoyl Derivatives of Sodium Hyaluronate Having Various Substitution Degrees in 0.1M Phosphate Buffer, pH 7.4

(41) Films prepared according to Examples 2, 16 and 17 were cut to precisely defined squares, weighed, measured and inserted into 0.1M phosphate buffer (PBS), pH 7.4. At 37 C., the swelling capacity of the films was monitored; each experiment was done in triplicate. The changes in the weight and dimensions of the film were evaluatedthe results are listed in Table 5. It is evident from this Table that the lower is the substitution degree, the higher is the swelling capacity of the film. In case of using high substitution degrees, only a small change of the film area can be achieved, which may be very important in a number of applications.

(42) TABLE-US-00005 TABLE 5 swelling capacity of the films prepared from palmitoyl derivatives having various substitution degrees area change after weight change after 5 days in 0.1M 5 days in 0.1M PBS, pH 7.4 (%) PBS, pH 7.4 (%) Film prepared according 69 1365 to Example 16 Film prepared according 32 749 to Example 17 Film prepared according 15 496 to Example 2

Example 28. Degradation of the Films Prepared from Palmitoyl Derivative of Sodium Hyaluronate in a Conditioned MediumComparison of Two Substitution Degrees

(43) Films prepared according to Examples 2 and 18 were cut to precisely defined squares, weighed, measured and inserted into a conditioned medium. All preparation proceeded in a laminar box so that no contamination and undesirable reactions of the medium occur. At 37 C., the change of the area of the film and the visual appearance thereof were checked in predetermined intervals, which properties may be associated with the degradation. The conditioned medium was exchanged in regular intervals; the experiment was conducted in triplicate. The film prepared according to Example 2 began to degrade significantly later than the film prepared according to Example 18. The results are shown in FIGS. 3a and 3b, where the appearance of the film is shown after 3 weeks of degradation in the conditioned medium. Table 6 documents the change of the film area after 1 week of degradation for the film according to Example 2, as well as to Example 18, and after three weeks for the film according to Example 2 (the film according to Example 18 was degraded to pieces or even dissolved after three weeks of degradation). Based on the results it is obvious that the degradation rate depends significantly on the substitution degree of sodium hyaluronate by the acyl chain. In the case of a highly substituted film prepared according to Example 2, the degradation proceeded in terms of several months.

(44) TABLE-US-00006 TABLE 6 change of the film area after 1 week in a conditioned medium - comparison of two substitution degrees change of the area of the film after 1 week in the conditioned medium (%) Film prepared according 13 to Example 2 Film prepared according 125 to Example 18

Example 29. Degradation of the Film Based on a Palmitoyl Derivative of Sodium Hyaluronate

(45) Samples of the film prepared according to Example 5 were incubated in a standard medium for cell cultures (Dulbecco's modified Eagle's medium) with the addition of 300 IU of an enzyme per 1 mg of the film. The incubation proceeded at 37 C. and the samples were analysed after 24 hours. The sample analysis was conducted on the HPLC system Alliance (Waters) according to an internal standard operating procedure. After 24 hours, it was still possible to observe non-degraded pieces of the film. In spite of that, oligosaccharides based on hyaluronan were detected in the solution, as shown in FIG. 4.

Example 30. Degradation of the Film Based on a Palmitoyl Derivative of Sodium Hyaluronate

(46) Samples of the film prepared according to Example 2 were incubated in a standard medium for cell cultures (Dulbecco's modified Eagle's medium) with the addition of 300 IU of an enzyme per 1 mg of the film. The incubation proceeded at 37 C. and the samples were analysed after 24 hours. The sample analysis was conducted on the HPLC system Alliance (Waters) according to an internal standard operating procedure. After 24 hours, no oligosaccharides based on hyaluronan were detected in the solution, which is in accordance with Example 28, where a very long degradation time was observed for the film according to Example 2, and which demonstrates the possibility of degradation modulation by means of the substitution degree.

Example 31. Characterization of the Films by Means of Young's Modulus

(47) Young's modulus was determined for films prepared according to Examples 10, 15, 16 and 19 in the dry state. The films were tested for mechanical properties by means of a single stage tensile testing machine INSTRON3343 with a 100N head. The Young's modulus was calculated based on the mean value of at least 9 valid measurements. Table 7 shows that the Young's modulus of dry, non-hydrated films does not depend on the molecular weight, substituent or substitution degree.

(48) TABLE-US-00007 TABLE 7 Young's modulus of the films Young's modulus (MPa) Film prepared according to Example 10 2835 (+/ approx. 10%) Film prepared according to Example 15 2409 (+/ approx. 10%) Film prepared according to Example 16 1800 (+/ approx. 10%) Film prepared according to Example 19 2636 (+/ approx. 10%)

Example 32. Characterization of the Film Surface Morphology and Determination of RMS by Means of AFM

(49) The films prepared according to Examples 8 and 16 were characterized by means of the Atomic force microscopy (AFM) method, wherein especially the appearance and the character of the surface were monitored. Especially the RMS roughness (root mean square roughness) was determined. It was found out that a very smooth surface having the RMS value up to 2 urn may be obtained from the side of the substrate (see FIG. 5 for the film according to Example 16). The side of the film that is exposed to the air during drying is always rougher, wherein RMS is somewhere around 50 or more nm.

Example 33. Comparison of Films Dried in a Temperature Gradient and in a Closed Space

(50) The films prepared according to Examples 8 and 19 were visually compared after drying. The surface of both films was not deformed, the surface crust did not form on any of the films. Both films were qualitatively the same (visual comparison).

Example 34. Viability of THP-1 Suspension Cells after 24- and 72-Hour Incubation with the Film Prepared According to Example 1 Based on a Palmitoyl Derivative of Sodium Hyaluronate

(51) The THP-1 cell line was cultured in a medium with the addition of 10% fetal bovine serum. After achieving a sufficient density and viability (measured by means of an automatic cell calculator CASY TT, Roche), the cells were seeded into a 6-well panel in 2 ml of 10% medium. The tested film was added to the cells in an amount of 1 and 0.5 mg/ml. After 24 and 72 hours of incubation, the cells were washed and their viability and the occurrence of cell death were detected by means of the detection kit ApoFlowEx FITC Kit (Exbio) on a flow cytometer MACSQuant (Miltenyi Biotec). The cells were evaluated as viable in case no propidium iodide fluorescence was detected. FIG. 6 shows a negligible reduction of the viability after 24 hours of incubation, which was not detected anymore after 72 hours. Therefore, the tested film is evaluated as non-cytotoxic in said concentrations.

Example 35. Analysis of the Cell Death of THP-1 Suspension Cells after 24 and 72 Hours of Incubation with the Film Prepared According to Example 1 on the Basis of Palmitoyl Derivative of Sodium Hyaluronate

(52) The THP-1 cell line was cultured in a medium with the addition of 10% fetal bovine serum. After achieving a sufficient density and viability (measured by means of an automatic cell calculator CASY TT, Roche), the cells were seeded into a six-well panel in 2 ml of 10% medium. The tested film was added to the cells in an amount of 1 and 0.5 mg/ml. After 24 and 72 hours of incubation, the cells were washed and their viability and the occurrence of cell death were detected by means of the detection kit ApoFlowEx FITC Kit (Exbio) on the flow cytometer MACSQuant (Miltenyi Biotec). The evaluation of the presence of cell death (apoptosis and necrosis) was conducted according to the recommendation of the kit producer. In brief: the population of the individual cells was divided based on the fluorescence intensity of propidium iodide and Annexin V-FITC into 3 groups: negative in both channels (living cells), positive just in the channel for Annexin V-FITC (apoptotic cells) and positive cells for the channel propidium iodide+/Annexin V-FITC (necrotic cells).

(53) FIGS. 7 and 8 imply that after 24, as well as 72 hours, no greater increase of the number of apoptotic or necrotic cells in the culture occurs and the tested material may therefore be evaluated as not inducing cell death.

Example 36. Contact Inhibition of the Growth of Mouse 3T3 Swiss Fibroblasts

(54) The mouse 3T3 Swiss fibroblast line was cultured in a medium with the addition of 10% fetal bovine serum. In the exponential growth phase, the cells were seeded into a six-well panel in 2 ml of the 10% medium. After achieving confluency, the tested film prepared according to Example 1 having the area of 1 cm.sup.2 was added, which was loaded by a silicon sterile ring so that no significant movement of the film on the monoculture occurs. At the same time, control cells were incubated without any treatment and just with the silicon ring. After 72 hours of incubation, the film samples and the silicon rings were removed, the cells were washed with PBS and fixed by 4% formaldehyde (10 min/room temperature). After washing with deionized water, the cells were coloured with crystal violet (0.1% in water, 30 min/room temperature) and after washing the colour away the cell area was photographed and observed under a light microscope. The cell area under the tested material, the extent of the damage of the cells and the size of the damaged zone were evaluated.

(55) Macrophotographs (FIG. 9) clearly show that the damaged zone of the monolayer is delimited and that only the cells which were directly under the film were damaged, most probably by a slight friction; the details from the light microscope show that the cells tended to re-grow under the film. Neither any damage nor a change in morphology of the cells at the borders of the tested film were observed. Therefore, it can be assumed that the material does not exhibit the contact inhibition of the cell growth.

Example 37. Cell Antiadhesive Properties of the Film

(56) The films prepared according to Examples 2 and 17 (derivatives having two different substitution degrees) were cut in a sterile manner to parts having the dimensions of 1 cm.sup.2. These parts were placed into a six-well culture panel face-up or face-down. Then they were loaded with sterile silicon rings and culture medium (2 ml) for primary human fibroblasts (NHDF) containing 10% fetal bovine serum was pipetted to the thus prepared samples. Meanwhile, a NHDF suspension was prepared and pipetted to the middle of the silicon ring on the film surface in an amount of 100 000 cells/sample. The samples with the cells were incubated for 72 hours and checked in 24-hour intervals under the light microscope. Polystyrene adapted for cell cultures with good adhesion properties was used as a positive control (CTRL). After completion of the incubation, the silicon rings were removed and the films together with the cells were fixed by 4% formaldehyde for 10 minutes and then coloured with 1% crystal violet in water (10 min). After washing the unbound crystal violet away thoroughly (25 min rinse with distilled water), the samples were photographed using an inverted microscope Nikon with 100 magnification. The results are shown in FIG. 10. On the CTRL photo, an almost confluent cell layer may be seen. On the film photos (A-D), a certain structure is observable, probably formed due to the long incubation in the medium and made visible by means of crystal violet. However, no cells are present on the films. It can therefore be stated that the films are completely non-adherent in this system and even the presence of proteins in the culture medium did not promote the adhesion.

Example 38. Preparation of the Film with Octenidine Dihydrochloride

(57) 20 l of a stock solution of octenidine dihydrochloride in ethanol having the weight concentration of 10 mg/ml were mixed with 20 ml of 50% propan-2-ol. After stirring thoroughly, 100 mg of a palmitoyl derivative of sodium hyaluronate having the substitution degree of 57% and molecular weight of 2.6710.sup.5 g/mol were added to the solution. The solution was stirred for 72 hours and after stirring it was dosed on a hydrophobized glass having the wettability value of 65 (+/3) and dried in a closed space, in a temperature gradient at the temperature of the lower plate 50 C. and the temperature of the upper plate 20 C. for 6 hours. After drying, the film was evaluated, removed from the substrate and characterized. The thickness of the film was determined to be 15 m.