United States Patent (19)
`Balazs et al.
`
`54)
`
`CROSS-LINKED GELS OF HIYALURONIC
`ACID AND PRODUCTS CONTAINING SUCH
`GELS
`
`75
`
`Inventors: Endre A. Balazs, Riverdale, N.Y.;
`Adolf Leshchiner, Fairview, N.J.
`
`(73)
`
`Assignee: Biomatrix, Inc., Ridgefield, N.J.
`
`(21)
`
`Appl. No.: 678,895
`
`22
`
`Filed:
`
`Dec. 6, 1984
`
`(51)
`(52)
`(58)
`
`Int. Cl. ................................................ C08F 8/00
`U.S. Cl. ....................................... 524/29; 536/4.1;
`524/27; 514/781
`Field of Search ...................... 524/27, 29; 536/4.1
`
`Patent Number:
`11
`(45) Date of Patent:
`
`4,582,865
`Apr. 15, 1986
`
`56
`
`
`
`References Cited
`U.S. PATENT DOCUMENTS
`3,357,784 12/1967 Kasper .................................... 8/129
`4,141,973 2/1979 Balazs ......
`... 536/55.1
`4,303,676 12/1981 Balazs ......
`... 424/359
`4,487,865 12/1984 Balazs et al. ....
`... 524/29
`4,500,676 2/1985 Balazs et al. .......................... 424/81
`Primary Examiner-Ronald W. Griffin
`Attorney, Agent, or Firm-Sheldon Palmer
`57)
`ABSTRACT
`Disclosed are cross-linked gels of hyaluronic acid, alone
`or mixed with other hydrophilic polymers and contain
`ing various substances or covalently bonded low molec
`ular weight substances and processes for preparing
`them. These products are useful in numerous applica
`tions including cosmetic formulations and as drug deliv
`ery systems.
`
`28 Claims, 2 Drawing Figures
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`IPR2019-01505 et al.
`
`

`

`U.S. Patent Apr. 15, 1986
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`U.S.'Patent Apr. 15,1986
`
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`
`4,582,865
`
`4000
`
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`
`3000
`
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`

`

`1.
`
`CROSS-LINKED GELS OF HYALURONIC ACID
`AND PRODUCTS CONTAINING SUCH GELS
`
`5
`
`10
`
`15
`
`25
`
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`The present invention relates to gels and mixed gels
`of hyaluronic acid (HA), formulations containing them
`and methods for preparing them.
`2. The Prior Art
`Hyaluronic acid is a well known, naturally occurring
`polysaccharide containing alternating N-acetyl-D-
`glucosamine and D-glucuronic acid monosaccharide
`units linked with 31-4 bonds and the disaccharide
`units linked with 31->3 glycoside bonds. Hyaluronic
`acid usually occurs as the sodium salt. The molecular
`weight of HA is generally within the range of 50,000 up
`to 8X 106 and even higher.
`The prior art describes the cross-linking of HA with
`20
`the use of 1,2,3,4-diepoxybutane in alkaline medium at
`50° C. (T. C. Laurent, K. Helsing, and B. Gelotte, Acta
`Chem. Scand. 18 1984), No 1, 274-5). The product
`obtained by that method is a gel which substantially
`swells in water.
`It is also known that divinylsulfone (DVS) is used for
`cross-linking polysaccharides, especially cellulose (U.S.
`Pat. No. 3,357,784).
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 is a graphical representation of the experimen
`tal data set forth in Example 3 below; and
`FIG. 2 is a graphical representation of the experimen
`tal data set forth in Example 4 below.
`SUMMARY OF THE INVENTION
`In one aspect thereof, the present invention provides
`highly swollengels of cross-linked hyaluronic acid.
`In another aspect, the invention provides mixed
`cross-linked gels of hyaluronic acid and other hydro
`40
`phillic polymers.
`In yet another aspect, the invention provides cross
`linked gels of hyaluronic acid and other polymers filled
`with various substances.
`In still another aspect, the invention provides cross
`45
`linked gels of hyaluronic acid containing low molecular
`weight substances covalently attached to the macro
`molecules.
`In still yet another aspect, the invention provides
`various formulations containing cross-linked hyaluronic
`50
`acid gels.
`Finally, the invention provides the methods of pre
`paring the products of the invention.
`The present invention is based on the observation that
`divinyl sulfone (DVS) reacts readily with HA in aque
`55
`ous alkaline solutions at room temperature, i.e., about
`20 C., thereby providing cross-linked HAgels. As used
`herein, the term HA means hyaluronic acid and its salts
`such as the sodium, potassium, magnesium, calcium, etc.
`salts. These gels swell in water and water containing
`60
`media. The swelling ratio depends upon the degree of
`cross-linking of the gel. We have found that the degree
`of cross-linking can be controlled by changing several
`factors including the molecular weight of the HA, its
`concentration in the reaction mixture, the alkali concen
`65
`tration and the polymer/DVS ratio. The reaction is
`very fast and in most cases a strong gel can be obtained
`in several minutes. The swelling ratio of these gels can
`
`4,582,865
`2
`be from 20 up to 8000, and more, depending upon the
`reaction parameters.
`It has also been found that the swelling ratio of cross
`linked HAgels is substantially greater than the swelling
`ratio of cross-linked gels of other polysaccharides ob
`tained under the same reaction conditions. This can
`probably be explained by the unique nature of HA (as
`compared to other polysaccharides) and its water solu
`tions. We have found that in water, a large molecule of
`HA forms a very flexible, long random coil which takes
`up an extremely large volume in the solution. For exam
`ple, the specific volume of a hydrated HA molecule in
`a physiological salt solution is about 2-6X 10 ml/g.
`That means that in a quite low concentration water
`solution of HA, a steric exclusion phenomenon occurs
`which will substantially affect not only the physico
`chemical properties of the solution, but the reaction of
`the HA with low molecular weight substances as well.
`In other words, the nature of the HA solutions affects
`the degree of cross-linking and the behavior of the
`cross-linked gel, in a manner quite unlike anything that
`occurs with other polysaccharides.
`We have also found that this unique property of HA
`to give highly swollen cross-linked gels can be used to
`effect modification of the properties of cross-linked gels
`made of mixtures of HA with other hydrophillic poly
`mers. These polymers include other polysaccharides,
`synthetic and natural, such as hydroxyethyl cellulose,
`carboxymethyl cellulose, xanthan gum, chondroitin
`sulfate, heparin, proteins of various types, such as colla
`gen, elastin, albumin, a globulin, etc., sulfated proteins
`such as keratin sulfate and sulfated aminoglycosaminog
`lycans, synthetic water-soluble polymers, such as poly
`vinyl alcohol and its co-polymers, co-polymers of poly
`(hydroxethyl)methacrylate and the like. In other words,
`any polymer soluble in water or water alkaline solutions
`and containing groups capable of reacting with DVS,
`namely, hydroxyl, amino or sulfyhydryl groups, can be
`used to obtain highly swollen cross-linked mixed gels of
`HA.
`We have further found that useful products can easily
`be obtained by carrying out the cross-linking reaction of
`HA in the presence of low-molecular weight substances
`containing reactive groups of the mentioned types.
`Another type of material according to the present
`invention is a cross-linked hydrophillic gel filled with
`various water insoluble substances including hydrocar
`bons, such as petrolatum; an oil or fat such as beeswax,
`conconut oil or lanolin, pigments, such as kaolin, ferric
`oxide; insoluble dyes, polymers, such as polyethylene,
`polyetrafluro ethylene, etc. In this type of product fine
`particles of a filler are immobilized in a gel network or
`in what we call a "polymer cage'. This latter product
`can be very useful for several purposes which will be
`discussed in more detail below.
`DESCRIPTION OF THE PREFERRED
`EMBODINEMT
`The processes by which the hereinabove described
`products are obtained will now be discussed in detail.
`In order to obtain a cross-linked HAgel, a sample of
`sodium hyaluronate or hyaluronic acid from any source
`is dissolved in dilute alkaline solution. The molecular
`weight of HA can be from 50,000 up to 8X 106 and even
`higher. The molecular weight affects the reation-the
`higher the molecular weight the greater the possibility
`to obtain a cross-linked gel.
`
`30
`
`35
`
`

`

`10
`
`15
`
`4,582,865
`4.
`3
`tion mixture before the addition of DVS. These inert
`The alkali concentration in the reaction mixture can
`substances are, preferably, water-insoluble liquids or
`be from 0.005M to 0.5M and higher. The lower limit is
`solid substances. Examples of such substances are petro
`dictated by the necessity to have the pH of the medium
`latum and kaolin. To obtain a filled cross-linked gel, a
`not lower than 9 and the upper limit by the hydrolysis
`chosen substance (based on a consideration of the de
`of HA in an alkaline solution. Usually, a decrease in
`sired properties of the gel) is emulsified or dispersed in
`alkali concentration results in gels with a greater swell
`an alkaline solution of HA or other polymer or mixture
`ing ratio, probably because a small amount of DVS
`of HA with other polymer or polymers and DVS is
`takes part in the cross-linking reaction.
`added to the mixture. The amount of DVS and the
`The concentration of HA in the starting solution can
`other parameters of the reaction are selected depending
`vary from 1% by weight up to 8% by weight and
`upon the desired properties of the gel. The relative
`higher. When the concentration is below the lower
`amount of filler in the gel can vary over a broad range
`limit, a cross-linked gel cannot be obtained even at a
`and is from 1 to 95 wt.% calculated on the total amount
`low HA/DVS ratio. When the concentration is too
`of polymers and filler, preferably from 5 to 90 wt.%.
`high, the solution becomes so viscous that it is difficult
`Cross-linked gels containing low molecular weight
`to handle it. The HA concentration substantially affects
`substances such as drugs, dyes and others covalently
`the swelling behavior of the gels (FIG. 1). It was found
`attached to the macromolecular network are obtained,
`that the shape of the curve for the swelling ratio-the
`preferably by incorporating the named substances into
`HA concentration dependence is essentially the same
`an HA or HA and other polymers solution before the
`for various HA/DVS ratios but the lower this ratio (i.e.,
`addition of DVS. An example of such a substance is
`more DVS in the mixture), the less the swelling ratio of
`20
`carminic acid, an FDA approved substance for use in
`the cross-linked gel for the same concentration of HA in
`food and drug preparations.
`the starting mixture.
`It is probably the presence of a glucosidic moiety of
`We have found that HA/DVS in the reaction mixture
`the carminic molecule which takes part in the cross
`is another parameter which can be conveniently used to
`linking reaction with DVS. It should be understood that
`control the swelling ratio of the cross-linked HA gel.
`a great number of substances can be used to obtain a
`An increase in the ratio results in highly swollen soft
`modified cross-linked gel of this type. The only essential
`gels (the swelling ratio is about 4000 and higher)
`feature of these substances is that they contain chemical
`whereas hard and less swollengels are obtained when
`groups with active hydrogen atoms reactive to DVS.
`this ratio is decreased. In general, the HA/DVS weight
`The amount of such low molecular weight substances
`ratio can be from 15:1 to 1:5 and lower.
`which can be used in the reaction depends upon the
`The cross-linking reaction is usually carried out at
`desired level of that substance in the gel. This amount
`room temperature, i.e., about 20 C., but it can be per
`can be in the range of from 1 to 99 wt.% as calculated
`formed at a lower or higher temperature, if desired.
`on polymer content in the gel, preferably, 5 to 90 wt.%,
`However, it should be kept in mind that HA can de
`The cross-linked HA and mixed gels obtained ac
`grade relatively rapidly in alkaline solutions at elevated
`35
`cording to the present invention can be used for many
`temperatures and, if such degradation occurs, the de
`purposes. We have found that these highly swollengels
`crease in MW can affect the properties of the obtained
`are very useful in cosmetic formulations and can be
`gels.
`considered as water-retaining and water-delivering in
`The cross-linking reaction is relatively fast and strong
`gredients in these formulations.
`gels are formed usually in several minutes when the HA
`40
`As HA is known to be a biologically tolerable poly
`concentration is high enough and the HA/DVS ratio is
`mer in the sense that it does not cause any immune or
`low. But even at low HA concentration in the reaction
`other kind of response when introduced into a human
`mixture, the gel formation starts usually 5-10 minutes
`body, the cross-linked HA gels can be used for various
`after addition of DVS. We have found that in most
`medical applications. The cross-linked gels modified
`cases one hour is enough for completion of the cross
`45
`with other polymers or low molecular weight sub
`linking reaction.
`stances can be used as drug delivery devices. For exam
`Another method of controlling the swelling ratio of
`ple, we have found that heparin introduced in a cross
`cross-linked HA gels involves adding neutral salt to the
`linked HA gel retains its antithrombogenic activity.
`reaction mixture. We have found that the swelling ratio
`We have also found that cross-linked gels of HA can
`of the gels obtained in the presence of water soluble
`slow down the release of a low molecular weight sub
`neutral salts, such as the chlorides, sulfates, phosphates
`stance dispersed therein but not covalently attached to
`and acetates of alkali metals, decreases with the increase
`the gel macromolecular matrix.
`of salt concentration. A salt can be used in concentra
`The domain of the cross-linked hyaluronic acid
`tion up to 20 wt.% and higher, depending upon the
`(alone or co-polymerized with other polyanionic or
`nature of the salt and its effect on the solubility of HA
`55
`neutral polymers) forms a molecular cage. In this cage,
`in the reaction mixture.
`hydrophilic or hydrophobic molecules of various phar
`To obtained cross-linked gels of other hydrophillic
`macological or biological activity can be dispersed.
`polymers the same reaction conditions as for HA can be
`Thus, the cage constitutes a depot for these substances
`used. The swelling ratio of these gels can be conve
`niently controlled by incorporating HA into the gel
`of various molecular size. The substances contained in
`60
`the domain of the molecular cage will be delivered into
`structure. When the mixed gels are obtained, the com
`the environment by diffusion. The delivery process is
`position of the polymer mixture can vary over a broad
`controlled by such factors as the exclusion volume ef
`range depending on the swelling ratio of the cross
`fect and the pore size of the molecular cage and by the
`linked gel desired. The preferred content of HA in the
`molecular interaction between the polymeric network
`mixture is from 5 to 95 wt.%.
`and the substance contained therein. Thus, the molecu
`Cross-linked gels of HA or other polymers or mixed
`lar cage forms a depot for the controlled delivery of
`cross-linked gels filled with inert substances are ob
`drugs or other substances to the skin or other tissues.
`tained by incorporating these substances into the reac
`
`50
`
`25
`
`30
`
`65
`
`

`

`15
`
`6
`EXAMPLE 2
`This example illustrates the effect of alkali concentra
`tion on the cross-linking of HA.
`A sample of HA with a MW of about 3X106 was
`dissolved in a calculated amount of 0.2M NaOH solu
`tion to give 4% viscous solution to which DVS was
`added in an amount providing an HA/DVS ratio of
`about 5:1. The cross-linking and treatment of the gel
`was carried out as described in the preceding example.
`The swelling ratio of the gel in water was 990.
`The example was repeated but the alkali concentra
`tion was 0.01M. A gel was obtained with a swelling
`ratio in water of 3640. Thus, a decrease in the alkali
`concentration in the reaction mixture results in a gel
`with substantially greater swelling in water.
`EXAMPLE 3
`This example illustrates the effect of varying the HA
`concentration in the starting mixture on the swelling
`behavior of the resulting gel.
`Eight solutions of sodium hyaluronate in 0.2M so
`dium hydroxide solution were prepared with the HA
`concentration being 2.0, 2.5, 3.0, 3.5, 4.0, 5.5, 8.0 and
`10.0% by weight respectively. To each solution a calcu
`lated amount of DVS was added to have a weight ratio
`of HA/DVS about 1 (molar ratio about 0.33). The
`cross-linked gels were obtained as described in the
`above examples and treated accordingly. The swelling
`ratio was determined for each sample and plotted
`against starting HA concentration. The results are
`shown in FIG. 1.
`
`25
`
`4,582,865
`5
`There is one additional property of the cross-linked
`HA gels which makes them potentially very useful as
`drug delivery devices. The swelling ratio of these gels
`in water depends substantially upon the salt concentra
`tion in the medium and decreases several times with an
`increase in salt concentration. This means that a gel
`swollen in water will contract substantially when intro
`duced into the body (because of the normal salt content
`of the body fluids and tissues), thus delivering its con
`tents, i.e., an incorporated drug, into the body tissue.
`10
`The cross linked gels filled with various substances
`can also be used in cosmetic formulations. For example,
`a gel with petrolatum incorporated therein gives all the
`benefits of using petrolatum in cosmetic formulations
`without the unpleasant greasy feeling which is normally
`observed with petrolatum containing formulations.
`DETALED DESCRIPTION OF THE
`INVENTION
`20
`The present invention is described in more detail in
`the following examples, wherein all parts given are by
`weight unless otherwise indicated. These examples are
`given merely by way of illustration and are not intended
`to limit the invention as set forth in the claims.
`EXAMPLE
`This example illustrates the effect of varying HA
`molecular weight on the cross-linking reaction.
`0.3410 g. of sodium hyaluronate obtained from
`30
`rooster combs (intrinsic viscosity in 0.15M solution of
`NaCl m) 3850, MW about 2.5x 106) was mixed with
`8.1840 g. of 0.2M NaOH solution to give a 4% by
`weight solution after stirring for 30 minutes. Then,
`0.0721 g of DVS was stirred into the solution. The
`35
`weight ratio HA/DVS was about 4.7. A strong gel
`formed in about 15 minutes. The gel was left for one
`hour and then put into one liter of distilled water. The
`gel was left to swell in water overnight. Then it was
`broken into small particles by vigorous stirring in water.
`40
`The gel particles were filtered off and washed several
`times with water. Colorless, water clear particles were
`obtained. To determine the swelling ratio of the gel, a
`sample weighing about 1 g, was centrifuged in a glass
`filter at 3,000 rpm for two hours. Then the particles left
`45
`on the filter were hydrolyzed with 2 ml of 1N H2SO4
`solution for three hours at 95-98 C. The clear solution
`obtained was neutralized upon cooling with 2 ml of 1N
`NaOH solution and the glucuronic acid content was
`determined by the carbazole method (An Automated
`50
`Method For The Determination Of Hexuronic Acids,
`Analytical Biochemistry, 2, 517-558 (1965). The HA
`content in the starting gel was calculated and the swell
`ingratio was expressed as 100/HA)%, where HA%
`is a percent of HA in the swollengel.
`55
`The swelling ratio in water of the gel obtained was
`820.
`This example was repeated with the exception that
`the solution of HA in alkali was kept at room tempera
`ture for 24 hours. This led to a HA hydrolysis. The
`60
`intrinsic viscosity m) of the polymer was 1064 which
`corresponded to a MW of about 0.5X106. A cross
`linked gel could not be obtained from this polymer at
`the HA/DVS ratio used above.
`The example with the degraded HA was repeated but
`65
`the HA/DVS ratio used was about 2. A cross-linked gel
`was obtained which had a swelling ratio in water of
`2910.
`
`EXAMPLE 4
`This example illustrates the effect of varying the
`HA/DVS ratio on the swelling behavior of the result
`ing gel.
`Six solutions of sodium hyaluronate in 0.2M sodium
`hydroxide solution were prepared with a concentration
`of 4.0% by weight. To each solution a calculated
`amount of DVS was added to have the following
`HA/DVS ratios: 0.2, 0.3, 0.5, 1.0, 1.5 and 2.0 mole/-
`mole. The cross-linked gels were obtained and treated
`as described in the preceding examples. The swelling
`ratio was determined for each sample and plotted
`against HA/DVS ratio in the reaction mixture. The
`results are shown in FIG. 2.
`EXAMPLE 5
`This example illustrates the effect of sodium chloride
`in the reaction mixture on the swelling ratio of the
`cross-linked gel.
`Two samples of the cross-linked HA gel were pre
`pared with the use of the above described procedure.
`Sodium hyaluronate concentration in 0.2M sodium hy
`droxide was 4% by weight. The HA/DVS ratio was
`about 5:1, the reaction time one hour. To the second
`reaction mixture sodium chloride was added in an
`amount to have a 1.0 molar salt concentration. The
`swelling ratio of the first gel was 2380, whereas the gel
`obtained in the presence of salt had a swelling ratio in
`water of 650.
`
`EXAMPLE 6
`This example illustrates the cross-linking of hydroxy
`ethyl cellulose with the use of DVS.
`0.4312 g of air-dry hydroxyethyl cellulose (Cellosize
`QP-100000 (R), Union Carbide) was dissolved with stir
`
`

`

`HA Content in the Starting
`Mixture, Wt. 26
`70
`50
`20
`0
`
`Swelling Ratio
`in Water
`8196
`6757
`117
`623
`
`Example 10
`Example 11
`Example 12
`Example 13
`
`15
`
`25
`
`4,582,865
`8
`7
`cific ratios of the two polymers In all cases the total
`ring in 10.3.g. of 0.2N sodium hydroxide to give 4% by
`polymer concentration was 4% by weight and the poly
`weight. 0.0855 g. of DVS was stirred into this solution
`mer/DVS ratio was about 5:1. The gels were obtained
`(polymer/DVS ratio was about 5:1 by weight) and the
`and processed as described above. The polymer content
`mixture was left for one hour at room temperature. A
`in the gels was determined as described in Example 1,
`cross-linked gel was obtained which was processed as
`with the exception that the hexosamine concentration
`described in Example 1. To determine the polymer
`(instead of glucoronic acid) was determined by a known
`concentration in the gel and, hence, the swelling ratio, a
`weighed sample of the gel was put into acetone, kept
`method (A Rapid Procedure for the Estimation of
`Amino Sugars on a Micro Scale, Analytical Biochemis
`overnight, washed several times with acetone and dried
`try 15, 167-171 (1966) in the hydrolyzate. The polymer
`in a vacuum oven at 50° C. to a constant weight. The
`10
`swelling ratio of the gel obtained was 43 which is sub
`content was calculated from the HA concentration and
`the ratio of the two polymers.
`stantially less than for cross-linked HA gel obtained
`under the same reaction conditions.
`EXAMPLE 7
`This example illustrates the cross-linking of xanthan
`gum with the use of DVS.
`0.4935 g. of air-dry xanthan gum (Kelzan (R), Kelco)
`was dissolved in 11.3 g of 0.2M sodium hydroxide
`solution to give a 4% by weight solution. To this solu
`20
`tion 0.0718g. of DVS was added (the polymer/DVS
`ratio was about 7:1 by weight). The mixture was kept
`for an hour at room temperature. The cross-linked gel
`finally obtained was put into a large volume of water,
`left to swell overnight and broken into small pieces
`which were extensively washed with water.
`The swelling ratio of the gel determined by the
`weight method described in the preceding example was
`526, which is substantially less than for cross-linked HA
`gel obtained under the same reaction conditions.
`30
`EXAMPLE 8
`This example illustrates the cross-linking of a cationic
`water-soluble cellulose polymer with the use DVS.
`0.5483 g. of a cationic cellulose polymer obtained by
`35
`chemical modification of hydroxyethyl cellulose (Poly
`mer Ucare JR (R), Union Carbide) was dissolved in
`13.71 g. of 0.2M sodium hydroxide solution to give a
`4% by weight solution to which 0.0849 g. of DVS was
`added (the polymer/DVS ratio was about 6.5:1). The
`reaction mixture was left to stand for an hour at room
`temperature and the gel obtained was processed and
`analyzed as described in the preceding example. The
`swelling ratio of the gel in water was 386, which is
`substantially less than that for a cross-linked HA gel
`obtained under the same reaction conditions.
`EXAMPLE 9
`This example illustrates the cross-linking of carboxy
`methyl cellulose with the use of DVS.
`50
`0.4703 g. of carboxymethyl cellulose sodium salt (9H
`4F, Hercules) was dissolved in 11.76 g. of 0.2M NaOH
`to give a 4% by weight solution to which 0.0651 g of
`DVS was added (the polymer/DVS ratio was about
`7:1). The reaction mixture was kept for an hour at room
`55
`temperature and the gel obtained was processed and
`analysed as described in the preceding example. The
`swelling ratio in water was 893, which is more than that
`obtained for other cellulosic polymers but less than for
`cross-linked HA gel.
`EXAMPLES 10-13
`These examples illustrate mixed cross-linked gels
`made of HA and carboxymethyl cellulose and the effect
`of the HA content on the swelling ratio of the gels.
`65
`In each example, sodium hyaluronate and carboxy
`methyl cellulose 9H4F were dissolved in 0.2M sodium
`hydroxide solution in such amounts as to provide spe
`
`As can be seen from these data, an increase in the HA
`content in the starting mixture results in an increase in
`the swelling ratio of the resulting gels.
`EXAMPLE 14
`This example illustrates mixed cross-linked gels ob
`tained from HA and collagen. 0.2531 g of dry sodium
`hyaluronate was dissolved in 2.5 ml of 0.1M sodium
`hydroxide solution. 0.063 g of collagen obtained from
`human umbilical cord was dissolved in 2.3 ml of 0.1M
`acetic acid and the two solutions were combined. The
`total polymer concentration was 6 wt. % and the
`weight ratio HA/collagen was about 4:1. 0.05 g. of dry
`KCl was dissolved in the mixed solution and DVS was
`stirred into the reaction mixture in an amount providing
`a polymer/DVS ratio of about 5:1. The reaction mix
`ture was kept at room temperature for an hour and the
`gel obtained was treated as described above. The poly
`mer content in the swollengel was calculated from the
`HA content which was found by the glucuronic acid
`assay. A strong and resilient gel was obtained which
`had a swelling ratio in water of 321.
`EXAMPLE 15
`This example illustrates a mixed cross-linked HA-col
`lagen gel with a higher content of collagen and a lower
`swelling ratio than the gel described in Example 14.
`0.2544 g. of sodium hyaluronate was dissolved in 3.5
`ml of 0.2M sodium hydroxide solution. 0.1192 g of
`collagen obtained from human umbilical crod was dis
`solved in 1.5 ml of 0.2M acetic acid solution and the
`solutions were combined. The total polymer concentra
`tion was 7.5 wt.% and the weight ratio HA/collagen
`was about 2:1. 0.05 g. of sodium chloride was dissolved
`in the mixed solution to which 0.1189 g. of DVS was
`added, thus providing a polymer/DVS ratio of about
`3:1 by weight. The gel was obtained and processed as
`described in the preceding example. A strong gel was
`obtained with a swelling ratio of 35.
`EXAMPLE 16
`This example illustrates a mixed cross-linked gel of
`HA and heparin.
`0.2968 g. of dry sodium hyaluronate was dissolved in
`6.92 g of 0.2M sodium hydroxide solution to give a 4
`wt.% solution to which 0.0503g. of heparin was added.
`The heparin content calculated on the basis of the total
`
`45
`
`60
`
`

`

`5
`
`10
`
`15
`
`4,582,865
`9
`10
`amount of polymers was 14.5 wt.%. 0.0590 g. of DVS
`tained was processed as described in the preceding
`was stirred into the mixture. The reaction was carried
`examples.
`out for an hour at room temperature. The obtained gel
`Red colored transparent gel particles were obtained
`was processed as described in the preceding examples.
`and the color did not disappear after extensive washing
`The swelling ratio of the gel was 625.
`with water. The swelling ratio in water determined by
`the weight method was 115.
`EXAMPLE 1.7
`This example illustrates a cross-linked hydroxyethyl
`EXAMPLE 21
`cellulose gel filled with petrolatum.
`This example illustrates the effect of salt concentra
`0.5292 g. of dry hydroxyethyl cellulose was dissolved
`tion in water on the swelling behavior of a cross-linked
`in 10.58 g. of 1M sodium hydroxide solution and 1.058
`HA gel.
`g. of white petrolatum was stirred into the solution. The
`petrolatum/polymer ratio was about 2. A solution of
`A cross-linked HA gel was obtained as described in
`0.1771 g of DVS in 1.0 g. of 1M sodium hydroxide
`the preceding examples with an HA concentration in
`solution was added to the emulsion with vigorous stir
`0.2M NaOH of 4 wt.%; HA/DVS ratio 5:1, at room
`ring. The reaction mixture was left for an hour at room
`temperature for one hour. The gel particles were put
`temperature and the gel obtained was treated as de
`into water and aqueous sodium chloride solution of
`scribed in the above examples. To find the petrolatum
`different concentrations and the swelling ratios were
`content in the gel, a gel sample was digested with 2 ml
`determined. The following results were obtained:
`of 1N H2SO4 at 95 C. for three hours. Then 2 ml of 1N
`20
`NaOH was added to the mixture followed by 4 ml of
`xylene to extract the petrolatum. The extract was dried
`off in vacuum and the residue was weighed. The calcu
`lated petrolatum content in the gel was 6 wt.%.
`EXAMPLE 18
`This example illustrates a mixed HA-carboxymethyl
`cellulose gel filled with petrolatum.
`0.1830 g. of dry sodium hyaluronate and the same
`amount of carboxymethyl cellulose were dissolved in
`30
`9.1 g of 0.2N sodium hydroxide solution to give a 4 wt.
`% solution of polymer. 0.3660 g. of petrolatum was
`stirred into the solution and 0.0730 of DVS was added
`to the resulting emulsion with vigorous stirring. The
`polymer/DVS ratio was about 5:1. The reaction mix
`35
`ture was left for an hour at room temperature. The
`obtained gel was processed as described in the preced
`ing example. The swelling ratio of the gel determined
`through hexosamine content was 738 and the petrola
`tum content determined as in the preceding example
`was 0.1 wt.%.
`
`NaCl Concentration, M
`Water
`0.05
`0.15
`0.50
`1.00
`
`25
`
`Swelling Ratio
`990
`43
`384
`219
`176
`
`EXAMPLE 22
`This example illustrates the biological activity of a
`mixed HA-heparin cross-linked gel.
`Fine particles of the mixed HA-heparin cross-linked
`gel prepared according to Example 16 were mixed with
`normal human plasma in amounts providing concentra
`tions of cross-linked HA of 0.01, 0.02 and 0.04% and the
`clotting time of the samples increased respectively by
`1.4, 2.8 and 5.0 times. Identical concentrations of non
`heparin containing, cross-linked gel particles had no
`effect on clotting time.
`These data indicate that heparin does not lose the
`ability to inhibit thrombin-catalyzed fibrin formation
`when it is incorporated into a cross-linked gel structure.
`EXAMPLE 23
`This example illustrates a product containing cross
`linked HA gel particles useful for cosmetic formula
`tions.
`A cross-linked HA gel was prepared as described in
`Example 1 under the following reaction conditions: HA
`concentration 3.0 wt.%, sodium hydroxide concentra
`tion 0.2M, HA/DVS ratio about 3:1, room temperature,
`time one hour. The gel was allowed to swell in a large
`volume of water overnight, then was broken into small
`particles by pushing through a syringe with a needle of
`18 gauge and then through a syringe with a needle of
`25 gauge. The particles were thoroughly washed with
`water. Optically clear, colorless particles were ob
`tained. the swelling ratio of the gel was 1980. The HA
`concentration of the fil