Patent Application
20060046590
1. Field of Invention
The present invention relates to methods for crosslinking hyaluronan solutions and nanofibrous membranes without the use of additional chemical cross-linking agent.
2. Discussion of the Background
Hyaluronan (HA) is an associated polymer, having the following structure:
HA has an acidic group as well as a glucosamine segment. As shown above, HA is a linear polysaccharide consisting of alternating 1,4-linked units of 1,3-linked glucoronic acid and N-acetylglucosamine. The presence of the weak acid group makes the polymer a polyelectrolyte, i.e., its charge density depends on the degree of dissociation, that can be influenced by factors including, but not limited to:
The degree of association can be disturbed by physical and/or chemical means. For example:
The occurrence of HA is widespread in animals and bacteria, particularly in synovial fluid, vitreous humor, rooster comb, and the umbilical cord. HA is a polyanionic acid that has unique physicochemical properties and distinctive biological functions. Its unusually high viscosity has been utilized for surgical treatments in ophthalmology as a viscoelastic biomaterial protecting ocular cells from damage during the surgery.1-7 The lubricious polysaccharide is also used in orthopedics for the treatment of osteoarthritis of the knee through injection of the aqueous solution.8-11 In addition, HA is applied in cosmetics to prevent the skin from drying because of its high retention ability12 and in drug delivery systems because of its biodegradability.13-16
Although HA can be produced on a large scale by biotechnology,17 applications of HA are still limited because the hydrophilic HA can be readily dissolved in water and undergoes relatively rapid biodegradation in vivo. Many attempts have been made to introduce cross-linking sites into HA molecules to produce insoluble or gel-like HA materials. The cross-linking reagents used in previous studies included diopeoxy,18 glutaraldehyde,19 carbodiimide20,21 and disulfide.22
Unfortunately, conventional chemical modifications and subsequent cross-linking of HA have an inevitable problem of extra risks, such as toxicity and bio-incompatibility intrinsic to most chemical modifications. However, one method of producing HA gels by freezing and thawing acidic aqueous solution of HA has been proposed in a US patent (Patent No.: U.S. Pat. No. 6,387,413B1)23. The obtained HA gel kept its shape for at least one day in a neutral aqueous solution at 25° C.
Previously, the present inventors successfully prepared electrospun HA nanofiber membranes by a blowing-assisted electro-spinning method.24 However, such electro-spun HA nano-fibrous membranes, due to the very large surface-to-volume ratios, dissolve instantly in water. Accordingly, a pathway is needed to fabricate hydrophilic, but essentially water-resistant, electro-spun HA membranes without chemical modifications.
Accordingly, one object of the present invention is to provide a method for production of hydrophilic, but substantially water-insoluble, HA.
A further object of the present invention is to provide a method for production of substantially water-insoluble hydrophilic HA, with a high effective viscosity but without significant chemical modification to the HA structure itself.
A further object of the present invention is to provide a non-chemically cross-linked HA that is substantially water-insoluble.
These and other objects of the invention have been satisfied by the discovery of a process for crosslinking hyaluronan, comprising:
A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:
FIGS. 1 (a)-(d) show SEM images of electro-spun HA membranes treated with HCL vapor from 37% (w/v) HCl (at a height of 5 cm) for: (a) 0 minute; (b) 5 minutes; (c) 7 minutes and (d) 10 minutes.
FIGS. 3(a)-(d) show SEM images of electro-spun HA membranes treated with HCl vapor for 3 minutes (at a height of 5 cm from HCl solution) from HCl aqueous solutions at different HCl concentrations of: (a) 29, (b) 24, (c) 20 and (d) 26% (w/v)
FIGS. 4(a) and 4(b) show SEM images of electro-spun HA membranes: (a) treated by 24% (w/v) HCl aqueous solution for 3 min and then kept at −20° C. for 25 days; and (b) treated by 20% (w/v) HCl aqueous solution for 3 min and then kept at −20° C. for 40 days.
FIGS. 5(a)-(d) show SEM images of electro-spun HA membranes treated with HCl vapor from 26% (w/v) aqueous solution (with the membrane at a height of about 8 cm from the liquid surface) for 10 min (a, b and c) and 25 min (d), then kept at −20° C. for: (a) 0 day; (b) 10 days; (c) 35 days; and (d) 35 days.
FIGS. 7(a) and 7(b) show SEM images of HA membrane soaked in: (a) 3:1 Ethanol/HCl mixture for 60 hrs at 4° C.; (b) 5:1 Ethanol/HCl mixture for one hour, then kept at −20° C. for 60 hrs.
FIGS. 8(a) and 8(b) show SEM images of HA membrane: (a) soaked in 3:1 Ethanol/HCl mixture for one hour, then heated in the oven at 40° C. for 20 minutes; (b) soaked in 5:1 Ethanol/HCl mixture for one hour, then heated in a microwave oven for 1 minute.
FIGS. 9(a) and 9(b) show SEM images of HA membrane treated by: (a) ‘11-mL H2O+17-mL 3:1 Ethanol/HCl (37% (w/v))’ at 4° C. for 16 hrs; (b) ‘2-mL H2O+15-mL 4:1 Ethanol/HCl (37% (w/v))’ at 4° C. for 20 hrs.
FIGS. 10(a)-(d) show SEM images of HA membrane treated by ‘2-mL H2O+15-mL 4:1 Ethanol/HCl at 4C. for 20 hrs: (a) sample dried by filter paper; (b) soaked in water for 5 min; (c) soaked in water for 10 min; and (d) soaked in water for one day.
The present invention relates to a method for production of HA products that are substantially reduced in their water-solubility comprising contacting HA with an acidic solvent/water mixture (preferably alcohol/water mixture) having sufficient solvent present to prevent dissolution of the HA sample and for a time and at a temperature sufficient to effect crosslinking of the HA.
Within the context of the present invention, the term “chemically crosslinked” indicates that the polymer chains of HA have been connected by insertion of a chemical moiety, by reaction with a chemical crosslinking agent, such as carbo-diimide or glutaraldehyde. The term “crosslinked” as used in relation to the present invention, indicates that the polymer chains of HA have been connected without insertion of a chemical moiety, such as by an acid-based reaction between groups on two chains in proximity to one another.
The present invention process permits the preparation of HA nano-fibrous membranes that have greatly increased longevity in neutral water or in aqueous solutions without substantial degradation or dissolution.
In the present method, the initial HA sample is contacted with an acidic solution of a solvent/water mixture. The solvent used in the solvent/water mixture must be completely miscible with water and also must be compatible with use in biomedical applications (i.e. must be non-toxic in biomedical applications). Suitable preferred solvents include, but are not limited to, lower alkyl alcohols, dimethyl sulfoxide (DMSO), and dimethylformamide (DMF). A more preferred solvent/water mixture is a lower alkyl alcohol/water mixture, wherein the lower alkyl alcohol is present in an amount sufficient to prevent substantial dissolution (preferably no greater than about 10% by weight dissolution, more preferably no greater than about 5% by weight) of the initial HA sample in the mixture. As a lower alkyl alcohol useful in the present process, any of the alcohols having from 1 to 4 carbon atoms may be used, individually or as mixtures of two or more, including methanol, ethanol, n-propanol, i-propanol, n-butanol, s-butanol, t-butanol and their mixtures. More preferred alcohols of these are ethanol and methanol, with ethanol being most preferred
The solvent/water mixture is made acidic with any protic acid, including organic and inorganic protic acids and mixtures thereof. The protic acid (and preferably its conjugate base) should be non-toxic with respect to use in biomedical applications. Suitable inorganic protic acids include, but are not limited to, hydrobromic acid (HBr), hydrochloric acid (HCl), sulfuric acid, nitric acid and their mixtures. Suitable organic protic acids include, but are not limited to, acetic acid, propionic acid, methane sulfonic acid, phosphoric acid and their mixtures. More preferred are the hydrohalic acids, with hydrochloric acid being most preferred.
In preparing the more preferred acidic alcohol/water mixture, the amount of alcohol is preferably 65 vol % or more, most preferably from 68-74 vol %, depending on the particular alcohol chosen. With the most preferred alcohol ethanol, the amount of ethanol in the ethanol/water mixture is preferably from 65-80 vol %, more preferably from 68-72 vol %, most preferably about 71 vol %.
The amount of acid used is again dependent on the particular acid chosen. For the most preferred hydrochloric acid, the acid is preferably used as a 37% (w/v) aqueous solution. Of this 37% (w/v) aqueous solution, it is preferred to use a ratio of alcohol:acid of from 3:1 to 5:1, most preferably from 4:1 to 5:1. This provides, for the most preferred ethanol embodiment, an amount of HCl from about 12% (w/v) to about 7.5% (w/v) of HCl in the final acidic alcohol/water mixture.
The process comprises bringing the uncrosslinked initial HA sample into contact with the acidic alcohol/water mixture under temperature conditions and for a time sufficient to effect crosslinking of the HA. The temperature for the reaction is preferably from greater than −20° C. to ambient temperature (approximately 25° C.), more preferably from −10 to 10° C., most preferably from 2 to 5° C. The time for contacting the HA with the acidic alcohol/water mixture is preferably from 60 min to 50 hours, more preferably from 10-25 hours, most preferably about 15-20 hours. Of course, the time for contacting the HA with the acidic alcohol/water mixture is dependent on the temperature, with lower temperatures requiring longer reaction times, and higher temperatures requiring shorter reaction times. Most preferably the reaction is performed by immersing the HA sample in the acidic alcohol/water solution at 0-10° C. for a period of about 15-25 hours.
The contacting step of the present process may be performed by immersion of the HA sample in the acidic alcohol/water mixture, or by continuous spraying of a stream or aerosol of the acidic alcohol/water mixture onto one or more sides of the HA sample. The HA sample itself may be in any form, including but not limited to, membranes, films or fibers. Membranes, films and fibers may be formed using any conventional process for forming HA membranes or films, such as casting or extrusion. Additionally, membranes and fibers may be prepared using the electro-blowing process described by the present inventors in U.S. application Ser. No. 10/674,464, filed Oct. 1, 2003, the contents of which are hereby incorporated by reference. The contacting step is more preferably performed by simple immersion of the sample into the acidic alcohol/water mixture for the desired time and at the desired temperature.
The crosslinked HA resulting from the present process preferably is a water resistant non-woven hyaluronan fibrous membrane having controlled void volume and mean pore size comprising:
The crosslinked HA resulting from the present process has significantly increased stability in neutral water. Un-crosslinked HA dissolves instantly in neutral water, whereas the crosslinked HA produced by the present process is stable for one week in neutral water.
Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.
Material
HA powders with an average molecular weight of 3.5×106 g/mole were provided by Denki Kagaku Kogyo Kabushiki Kaisha (Tokyo, Japan).
Preparation of Electro-Spun HA Membrane
HA cast films were prepared by casting the 1.5% (w/v) acidic aqueous solution of HA onto a clean glass plate, followed by drying at 25° C. for 3 days. The thickness of the HA cast film was about 100 μm.
Freezing of Acidic HA Solution
HA powder was dissolved in Mill-Q water to prepare a 1˜2% (w/v) HA aqueous solution and then the pH of the solution was adjusted to 1.5 with 0.1N hydrochloride acid. This acidic solution was frozen at −20° C. over different time periods, ranging from 0 to 3, 6, 8, 10, 40 hours, and then thawed at 25° C. A 40-hour freezing period was used as a reference that yields a spongy HA gel.
Treatment of HA Film in Acidic Environments
Electro-spun HA membrane or cast film was pasted onto the inner surface of a petri dish, and then placed on the top of a beaker (HA membrane or cast film side to the interior of the beaker) containing 10-ml HCl aqueous solution at different HCl concentrations. After several minutes of HCl vapor treatment, the film was taken out and kept in covered bottles in the freezer at −20° C. for a few weeks.
The LEO-1550 scanning electron microscope was used for SEM analysis.
Viscosity Measurements
Shear viscosity measurements of HA solutions were performed on a Rheometrix mechanical spectrometer (RMS, Model 605) at room temperature with a parallel plate (50 mm diameter) fixture. The intrinsic viscosity of diluted HA solution after freezing was measured in an Ubbelohde capillary viscometer at 25° C.
FT-IR Spectroscopic Characterization
FT-IR spectra measurements were performed with a Nicolet 760 spectrometer.
Results and Discussion
1. Cross-Linking of Electro-Spun HA Membrane by HCl-Vapor Treatment
a) Optimization of HCl Vapor Treatment
The Method to produce water-insoluble HA gels by freezing and thawing acidic aqueous solutions (pH ˜1.5) of HA has been reported (Patent No.: U.S. Pat. No. 6,387,413B1). As the electro-spun HA membrane could be dissolved almost instantly in water, we tried to introduce the hydrochloric acid (HCl) into the membrane first by exposing the electro-spun HA membrane in HCl vapor.
b) Influences of HCl Vapor Treatment and Freezing Condition on the Cross-Linking of Electro-Spun HA Membranes
After the HCl-vapor treatment, the electro-spun HA membranes were kept in covered bottles in the freezer at −20° C. for weeks.
The above results indicate that the cross-linking of electro-spun HA membranes can be performed by HCl vapor treatment. However, the desired mechanical strength has not yet been achieved.
2. Cross-Linking of Electro-Spun HA Membranes by Ethanol/HCl/H2O Mixture Treatment
The cast film of HA has been chemically cross-linked with glutaraldehyde19 or water-soluble carbodiimide20 in ethanol or acetone-water mixtures. The organic solvent content in the mixture was at least 65 vol % in order to prevent the dissolution of HA. In the following experiments, we tried to “cross-link” the HA membrane in acidic ethanol-water mixtures, i.e., the presence of sufficient amounts of ethanol to prevent the dissolution of HA in the ethanol/water mixture, the presence of sufficient amounts of HCl to destabilize HA, and the cooling of the mixture to slow down the reaction.
a) Ethanol/H2O (pH 1.5)
By using the ethanol/H2O (pH 1.5) solution treatment, the volume percent of ethanol varied from 65 to 80 vol % and the soaking temperatures were −20° C., 4° C. and 20° C., with time treatment varied from one day to one month. The fibrous structure was relatively intact for thus treated membranes (
b) Ethanol/HCl (37% (w/v)) Mixture
In this case, the volume ratios of ethanol and 37% (w/v) HCl were 3:1, 4:1, and 5:1. The electro-spun HA membranes were immersed into the above ethanol-37% (w/v) HCl mixtures and kept at different temperatures for different time periods.
In another set of experiments, HA membranes were soaked in the ethanol/HCl mixture, and the membranes were taken out and heated in an oven or a microwave oven with minutes. The SEM results are shown in
All samples treated by this very strong acidic ethanol/HCl mixture showed some degree of crosslinking. These treated membranes could float on the surface of water for minutes before immersing into the liquid. The cross-linking of HA membranes was still relatively weak even when treated by using strong acidic ethanol/HCl (37% (w/v)). It should be noted that the slightly swollen HA nano-fibers in the non-woven membrane could probably not yet be fully exposed in the strong acid solution. Thus, the cross-linking reaction has not been fully initiated in the interior of those fibers.
c) Ehanol/37% (w/v) HCl/H2O
In this set of experiments, extra water was introduced into the ethanol/37% (w/v) HCl mixture. Different ratios (3:1, 4:1, 5:1) in the ethanol/37% (w/v) HCl mixed solution were mixed with water. The three component mixture was checked continuously by dropping one small piece of electro-spun HA membrane until the HA membrane changed from complete dissolution to no significant shrinkage. The contents of ethanol, H2O and HCl were then calculated as shown in Table 1. The electro-spun HA membranes were immersed into the above prepared three component mixture at different temperatures (−20° C., 4° C., 20° C.) for different time periods.
It was found that such prepared membranes were well cross-linked in the mixture of ‘3:1 ethanol/HCl+H2O’ and ‘4:1 ethanol/HCl+H2O’ at 4° C., as shown in
For the content of ethanol in the mixture, it should be about 71 vol %. The HA membrane would dissolve or shrink significantly if the ethanol content were a little lower than 71 vol %. The water content should be about 24 vol %. If the water content was lower than 23 vol %, there would not have been enough water to ensure the membrane to be sufficiently swollen and the cross-linking of HA membranes would be less resistant to water dissolution.
3. IR Spectroscopy
FTIR spectroscopy was conducted on the electrospun membranes before and after cross-linking. The results are shown in
4. Viscosity Changes of Acidic HA Aqueous Solution After Freezing
As reported previously, when acidic HA solution was allowed to freeze at −20° C. and followed by the thawing process, insoluble HA gel could be obtained. In the present work, we tried to find out the optimal freezing time for acidic HA solution under which the viscosity of HA solution could be increased significantly but in the absence of gel formation. The acidic HA aqueous solution was frozen at −20° C. for different time period. After freezing, the frozen samples were thawed at room temperature. The final solutions after thawing were transparent similar to that of the starting solution, if the freezing time was no more than 6 hours. However, if freezing time was longer than 8 hours, the final solution after thawing looked like putty, indicating the onset of gel formation. Spongy gels were obtained when the freezing time was longer than 40 hours.
The shear viscosity of thawed HA solution after freezing for different time periods increased with increasing of freezing time from 0 to 6 hrs. The measured apparent shear viscosity then decreased for the samples frozen for more than 8 hours, as shown in
With the cross-linking reaction occurring in acidic HA solution after a short time period of freezing (less than 6 hours), we tried to estimate the molecular weight changes after the freezing process were estimated by viscosity measurements. When the thawed solution was neutralized with 0.2 M NaOH solution and then diluted with 0.1 M NaCl solution, the final solution was clear if the freezing time was lower than 6 hours. A substantial amount of gel-like particles would appear if the freezing time was longer than 8 hours. The intrinsic viscosity of diluted HA solution with 0.1M NaCl before and after 6 hours of freezing showed very similar values, as shown in
Obviously, additional modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.
