MATRICES COMPRISING A MODIFIED POLYSACCHARIDE

Abstract

The present invention discloses a matrix comprising a modified polysaccharide consisting of repeating disaccharide units whereby in at least 11% of the disaccharide units one primary alcohol group is oxidized into a carboxylic acid group.

Claims

1-19. (canceled)

20. A method, comprising: injecting, into a subject, a plastic surgery implant comprising a composition comprising a modified agarose, wherein at least 11% of the modified agarose exhibits a β-sheet structure.

21. The method of claim 20, wherein the modified agarose comprises a carboxylated agarose.

22. The method of claim 20, wherein the modified agarose has a shear modulus G′ in the range of from 10 Pa to 10.sup.7 Pa.

23. The method of claim 20, wherein the implant further comprises an unmodified polysaccharide.

24. The method of claim 23, wherein the unmodified polysaccharide comprises agarose.

25. The method of claim 23, wherein the unmodified polysaccharide comprises one or more of a member of the carrageenan family, hyaluronic acid, heparin sulfate, dermatan sulfate, chondroitin sulfate, alginate, chitosan and pullulan.

26. The method of claim 20, wherein the modified agarose further comprises living cells.

27. The method of claim 20, wherein the modified agarose is obtained by oxidizing at least one primary alcohol group in at least 11% of an unmodified agarose.

28. The method of claim 20, wherein 20% to 99% of the modified agarose exhibits a β-sheet structure.

29. The method of claim 20, wherein 50% to 95% of the modified agarose exhibits a β-sheet structure.

30. The method of claim 20, wherein the modified agarose further comprises a pharmaceutically active agent.

31. A method, comprising: performing plastic surgery on a subject by injecting, into the subject, a modified agarose, wherein at least 11% of the modified agarose exhibits a β-sheet structure.

32. The method of claim 31, wherein the modified agarose comprises a carboxylated agarose.

33. The method of claim 31, wherein the modified agarose has a shear modulus G′ in the range of from 10 Pa to 10.sup.7 Pa.

34. The method of claim 31, wherein the implant further comprises an unmodified polysaccharide.

35. The method of claim 34, wherein the unmodified polysaccharide comprises agarose.

36. The method of claim 34, wherein the unmodified polysaccharide comprises one or more of a member of the carrageenan family, hyaluronic acid, heparin sulfate, dermatan sulfate, chondroitin sulfate, alginate, chitosan and pullulan.

37. The method of claim 31, wherein the modified agarose is obtained by oxidizing at least one primary alcohol group in at least 11% of an unmodified agarose.

38. The method of claim 31, wherein 20% to 99% of the modified agarose exhibits a β-sheet structure.

39. The method of claim 31, wherein 50% to 95% of the modified agarose exhibits a β-sheet structure.

40. A composition, comprising: a liquid formulation comprising hyluraonic acid and modified agarose, wherein at least 11% of the modified agarose exhibits a β-sheet structure.

Description

DESCRIPTION OF THE DRAWINGS

[0135] FIG. 1 shows a mechanism for polysaccharide modification.

[0136] FIGS. 2A-2C shows examples of polysaccharides.

[0137] FIG. 3 shows a scheme illustrating the strategies for preparation of modified polysaccharides. In the box on the left hand side modifications made by the supplier are shown. In the box on the right hand side two blendings made by the final user are represented.

[0138] FIG. 4A shows a reaction mechanism of D-Galactose primary alcohol oxidation into a carboxyl acid.

[0139] FIG. 4B shows FTIR spectra of the modified agarose (blacksolid line) and the native agarose (dotted line).

[0140] FIG. 4C shows .sup.13C-MAS-NMR spectra of the modified agarose (black) nad the native agarose (grey).

[0141] FIG. 4D is a plot of the percentage of modification as determined by FTIR against the amount of NaOH solution added to the reaction mixture as a mean of 12 reactions. Error bars represent the standard deviation.

[0142] FIG. 5A shows a temperature sweep of native agarose (black) and 60% modified agarose (squares and rhomboids), squares and rhomboids represent G′ and triangles and squares represent G″.

[0143] FIG. 5B shows a frequency sweep of native agarose (dots and triangles) and 60% modified agarose (squares and rhomboids), dots and rhomboids represent G′ and triangles and squares represent G″.

[0144] FIG. 5C represents a plot showing the relationship between the temperature of gelation of modified agarose and the percentage of its modification. The error bars represent the standard deviation.

[0145] FIG. 5D is a diagram showing the shear modulus G′ of agarose samples having different percentage of modification. The error bars represent the standard deviation.

[0146] FIG. 5E Photographs of gels containing 2% w/v respectively: 1: 93% modified agarose; 2: 60% modified agarose; 3: 28% modified agarose; 4: native agarose.

[0147] FIG. 6A shows a CD spectrum of native agarose (black) and 93% modified agarose (grey).

[0148] FIG. 6B shows a CD spectrum of 93% modified agarose at 5° C. (dashed) and 93% modified Agarose at 90° C. (solid).

[0149] FIG. 6C shows CD of agarose gels having a different degree of modification of at 203 nm. Error bars represent the standard deviation.

[0150] FIG. 6D shows CD spectra of native agarose (dashed), 93% modified agarose (dashed; one point), native k-carrageenan (dashed; two points), 93% modified k-carrageenan (dotted).

[0151] FIG. 6E shows a plot of zeta potential (mV) of modified agaroses vs. their degrees of modification. Error bars represent the standard deviation.

[0152] FIG. 6F shows plots of polydispersity (black) and size (grey) measured by light scattering of diluted solutions of different modified agaroses vs. their degrees of modification. Error bars represent the standard deviation.

[0153] FIG. 7A shows a RMSD from the first frame for native agarose (black) and 100% modified agarose (grey).

[0154] FIG. 7B represents the sum of cumulated H-bonds between the two strand of polysaccharide during the MD simulation for native agarose (dashed) and 100% modified agarose (solid).

[0155] FIG. 7C shows a Ramachandran plot of native agarose AG link in chain 1 and GA link in chain 2.

[0156] FIG. 7D shows a Ramachandran plot of totally modified agarose AG link chain 1 and GA link in chain 2.

[0157] FIG. 7E shows the MD simulation for native Agarose (left) and from the middle of the simulation (right).

[0158] FIG. 7F shows the MD simulation for totally modified Agarose (left) and from the middle of the simulation (right).

[0159] FIGS. 8A-8E. FIG. 8A: native polysaccharide, FIG. 8B: 28% of modification, Row 1 is the ESEM of 2% w/v freeze dried agarose gel; Row 2 is the AFM of the height of agarose gels, Row 3 is the 3D reconstruction of AFM pictures. FIG. 8C: 60% of modification, FIG. 8D: 93% of modification Row 1 is the ESEM of 2% w/v freeze dried agarose gel; Row 2 is the AFM picture of the height of agarose gels, Row 3 is the 3D reconstruction of AFM pictures, FIG. 8E: Row 1 is the ESEM of 2% w/v freeze dried k-carrageenan gels at low magnification; Row 2 is the ESEM of 2% w/v freeze dried k-carrageenan at high magnification. FIG. 8F is a diagram showing the surface roughness of the AFM sample of agarose gels plotted against the percentage of modification.

EXAMPLES

[0160] The following non-limiting examples will illustrate representative embodiments of the invention in detail.

[0161] Methods Description

[0162] a) Infrared Spectroscopy

[0163] FTIR spectra have been recorded on a Brucker Vector 22 FT-IR spectrometer in KBr pellets at 20° C. The pellets were prepared with 2 mg of the substance in 200 mg of KBr, then grinded and pressed under a pressure of 10 tons press for 10 min.

[0164] b) Nuclear Magnetic Resonance

[0165] Magic Angle Spinning NMR spectra were recorded at room temperature (20° C.) in the solid state using a Brucker Avance DRX 500 spectrometer. For this purpose freeze dried samples were put in a ceramic holder and spined at 7500 U/s.

[0166] c) Rheology

[0167] Rheology experiments were performed with a MCR rheometer Anton Paar Physica MCR 301 equipped with a Peltier temperature cell. Sample were prepare as 2% w/v in deionized water, heated at 90° C. and stirred for 10 min until a clear solution was obtained. The liquid was then poured on the rheometer plate, pre-heated at 80° C., using a pipette. The solution was allowed to stabilize for 10 min before the recording was started. A plate tool from Anton Paar: PPR25 was used for all experiments. Sol-gel transition and frequency sweep were made using the same sample in a single cycle: 10 min equilibrium at 80° C., cooling down to 5° C. in 30 min and record of G′ and G″ every 1.5° C. at 1 rad/s with a deformation of 10%, stabilized at 5° C. for 30 min, heated up at 37° C. and stabilized for 30 min then the frequency sweep was recorded at 37° C. by increasing the rotation frequency from 0.01 rad/s up to 10 rad/s over 30 min with a deformation of 10%. Sol-gel transition temperature was calculated as the temperature where tan θ=0.

[0168] d) Circular Dichroism

[0169] Circular dichroism spectra were obtained using a Jasco J-810 spectropolarimeter equipped with a Peltier temperature cell Jasco PFD-425S. Solution of 0.15% w/v of agarose was made in Milli-Q water at 90° C. for 15 min then solution have been cooled down to 5° C. in the CD chamber for 30 min prior measurement. Each spectrum was recorded three times and the obtained spectra were summed together. Each spectrum for a given modification is a mean of three batches obtained by three different syntheses.

[0170] e) Dynamic Light Scattering

[0171] Dynamic light scattering (DLS) measurements were carried out on a Beckman Coulter Delsa™ Nano C particle analyzer with a polystyrene cuvette of 1 cm. Agarose was dissolved in Milli-Q water at 90° C. for 15 min to obtain a 0.15% w/v solutions that was cooled down at room temperature for a day. The light scattering was done at 15° C. and samples were equilibrated for 30 min prior to measurement. For each value three measurements were done and an average was calculated. Each point is a mean of three batches obtained by three different syntheses.

[0172] f) Zeta Potential

[0173] Zeta potential was measured on a Beckman Coulter Delsa™ Nano C particle analyzer. The same solutions were used as for the light scattering experiment. Measurements were made in a flow cell that was aligned with the laser prior every measurement. Each measurement was made three times and an average was calculated. Each spectrum for a given modification is a mean of three batches obtained by three different syntheses.

[0174] g) ESEM

[0175] ESEM images were obtained with a ref agarose gels. 2% w/v solutions were prepared and 2 ml of these solutions were freeze-dried for 24 hours under 0.1 mbar vacuum in a 5 ml glass vial. The samples were vertically cut and the inside of the sample was imaged at different magnification. Images shown here are representative images of different areas of a given sample at different magnification, which were reproduced with three different gels prepared from different batches.

[0176] h) AFM

[0177] AFM images were obtained with a scanning probe microscope Veeco Dimension 2100. The samples were prepared on a 3 mm microscopic glass holder that was previously passivated. The glass slide was washed with 0.1 M NaOH solution and dried in the oven. The dry slides were then passivated with a few drops of dichloromethylsilane. Two slides were sandwiched together to have a uniform passivation. After 10 min the slides were washed with water and the excess of dichloromethylsilane was washed away with soap after what the slides were dried. Slides side was prepared in a hydrophobic way. Agarose samples were prepared as 2% w/v gels and 25 μl of the obtained solution was poured on an unmodified glass slide. A dichloromethylsilane passivated slide was then adjusted on top of the solution. Slides of 0.5 mm were put as spacers between the hydrophobic and the normal glass slide, the whole montage was then allowed to gel for 30 min at 4° C. The upper slide (hydrophobic) was after that removed and a thin layer of agarose gel was obtained. This gel was then allowed to stabilize at room temperature for 30 min before measurement in order to avoid any shrinkage or dilatation of the gel during the measurement.

[0178] i) Molecular Dynamic Simulations

[0179] MD simulations were done using the Desmond package of the Maestro, Version 8.5 from Schrödinger. Initial conformation was been obtained from the X-ray structure of the agarose that was downloaded from the protein database (PDB) library. Modified agarose was drawn from the PDB file directly inside the Maestro software. Implicit water model was build using the Desmond tool, resulting in a 10 Å square box build by following the TIP3 solution model. The simulations were run in the model NPV at 300° K at atmospheric pressure for 15 ns. Analysis of the results was done using the VMD software and the tools available in the standard package.

[0180] j) Elementary Analysis

[0181] Samples have been analyzed on an element analyzer Elementar Vario EL

Example 1

Modification of Agarose

[0182] Agarose type I was obtained from Calbiochem. (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl (TEMPO), NaOCl, NaBH.sub.4, NaBr, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2-(N-morpholino)ethanesulfonic acid (MES) buffer were obtained from Sigma Aldrich and used as received. Solution of 0.5 M NaOH as well as solution of 5 M HCl were freshly prepared every three-month. Ethanol technical grade was used without any further purification. Deionized water was used for non-sterile synthesis.

[0183] Agarose was modified under sterile conditions: all the chemicals were dissolved in autoclaved water and filtered with a 0.2 μm filter. All the glassware was autoclaved and the reaction was conducted under a laminar flow. Agarose (1 g) was autoclaved in MilliQ water. Autoclaved agarose was poured into a 3 necked round bottom flask, which was used as a reactor. A mechanical stirrer was adapted to one of the neck. A pH-meter was adapted another neck of the round bottom flask. The reaction mixture was then cooled down to 0-5° C. and vigorously stirred. TEMPO (0.160 mmol, 20.6 mg) was added, NaBr (0.9 mmol, 0.1 g) and NaOCl (2.5 ml, 15% solution) was as well poured inside the reactor. The resulting solution was adjusted to pH=10.8 with HCl and NaOH solution. The pH was maintained at 10.8 by adding NaOH solution. At the end of the reaction NaBH.sub.4 (0.1 g) was added and pH=8 was reached. The solution was stirred for 1 hour and NaCl (0.2 mol, 12 g) and ethanol (500 ml) was added. The agarose was precipitated and extracted in a funnel. The two layers were then filtered on a frit glass. The agarose was then dialyzed in Spectra Pore 4 membranes, MWCO=12-14000 for 2 days and the water was changed two times. Prior dialysis, the membranes were left overnight in a 70% ethanol solution, 2 hours before use they were rinsed in autoclaved water. Finally, the product was put on a freeze-drier Christ LD 2-8 LD plus at 0.1 mbar for the main drying and at 0.001 mbar during the desorption phase. Samples were put in round bottle flask and freezed in liquid nitrogen bath on a Rotary evaporator modified for this purpose. Thin layer of frozen solution was obtained on the flask wall reducing the lyophilization time.

Example 2

Modification of K-Carrageenan

[0184] K-carrageenan was obtained from Sigma Aldrich and used as such. K-carrageenan was objected to a TEMPO-mediated oxidation, according to the synthetic protocol of Example 1.

[0185] The resulting modified k-carrageenan was dialyzed and freeze-dried, as specified in Example 1 above.

Example 3

Blending of Modified Agarose with Unmodified Agarose

[0186] Blends of modified agarose and native agarose in different proportions were prepared. The resulting blends were studied using CD spectrometry. The obtained results indicate that the blending of two polysaccharides lead to the same change in tertiary structure as the modified polysaccharide. This suggests that these two polysaccharides are miscible and can be used for engineering new matrices.

[0187] The rheology studies show a specific behavior of the gel. Indeed the blended gels have a higher shear modulus than the unmodified gel of agarose. These results suggest that the organization of the modified chains with the unmodified chains follows a new mechanism, that is different from modified agarose.

[0188] The ESEM image illustrates the structure of the gels and reveals a different organization of the fibers than for the unmodified agarose. As well for the surface roughness, the roughness of the blended gels is not dependent of the proportion suggesting a new mechanism of organization.

Example 4

Covalent Biding of Peptide to Modified Agarose

[0189] Peptide GGGGRGDSP (SEQ ID NO: 3) was obtained from Peptide International.

[0190] Functionalization of agarose with the G.sub.4RGDSP peptide (SEQ ID NO: 3) was done by EDC peptide coupling. Agarose (30 mg, 0.25 μmol) was dissolved in autoclaved water; all the chemical and buffer were sterilized on a 0.2 μm filter, MES buffer was added and the solution reached a pH=4. The peptide (500 μg) was added thereto, followed by EDC (200 mg) and the resulting solution was stirred for two hours at 40° C. to avoid any gel formation. The solution was then dialyzed in Spectra Pore 4 MWCO=12-14 kDa, whereby water was changed three times. Subsequently, the sample was freeze-dried. Prior dialysis the membranes were left overnight in a 70% ethanol solution, 2 hours before use they were rinsed in autoclaved water.

Example 5

Use of Modified Extracellular Matrix for Biological Tests

[0191] Cell Culture:

[0192] Modified gels were prepared at a 2% w/v concentration in DMEM media and heated at 60° C. for 30 minutes in order to avoid any degradation of the RGD peptide. The temperature was adjusted to 37° C. and the culture media was completed with usual nutrients and cytokins. Human chondrocytes were obtained from ATCC and used between passage 3 and 5. Solution of agarose was mixed with the cells and then seeded in a 48 wells plate. The plate was then stored at 4° C. for up to 30 min in order to allow the sol-gel transition to occur. The plates were then cultivated in a incubator at 37° C., 4% CO.sub.2 for two weeks.

[0193] Cell Shape Factor:

[0194] All images were taken on an Axio Observer A1, from Carl Zeiss, equipped with a differential interference contrast (DIC) filter. The images were taken after 1, 5 and 14 days at different magnification in different area of the sample. The cell perimeter and cell area were then measured and the cell shape factor (CSF) was calculated. The results are a mean of three different batch of peptide modified agarose which was reproduced two times, which represent a total of 6 wells. Per well more than 100 cells were measured in order to have a meaningful CSF.

[0195] Real Time PCR:

[0196] At 7 days, 14 days and 21 days of cell culture in the gel, the media was removed and the gels were frozen at −80° C. overnight. The solids obtained were then crushed and digested with a CT AB buffer. Then extract using the Qiagen kits.