United States Patent r19J
`Balazs et al.
`
`[54] CROSS-LINKED GELS OF HYALURONIC
`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.: 755,976
`
`[22] Filed:
`
`Jul. 18, 1985
`
`Related U.S. Application Data
`[62] Division of Ser. No. 678,895, Dec. 6, 1984, abandoned.
`
`[51]
`Int. CI.4 .............................................. C08B 37/08
`[52] U.S. CI . ........................................ 524/27; 524/29;
`536/4.1
`[58] Field of Search ............................ 536/4.1; 524/27
`
`[I I] Patent Number:
`[45] Date of Patent:
`
`4,605,691
`Aug. 12, 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 ofhyaluronic acid, alone
`or mixed with other hydrophilic polymers and contain(cid:173)
`ing various substances or covalently bonded low molec(cid:173)
`ular weight substances and processes for preparing
`them. These products are useful in numerous applica(cid:173)
`tions including cosmetic formulations and as drug deliv(cid:173)
`ery systems.
`
`6 Claims, 2 Drawing Figures
`
`2/)()()
`
`/DI)()
`
`~(cid:173)~
`~
`~
`'i L_ ____ ,.._:::::==::===:::=~---
`
`.5
`EXAMPL.£ .3. SWEl.LJNG li'-ATI() (),t:' Gi'a,S"S-UNita> JIA GEI.S Ar
`/)J,t:'~Nr CONC',EN'Tie-4770/VS OF KA IN ,sT,,tA'TIN(; SOt.UTIONS
`(/,IA/tws WEIG/IT .t'Al70 ABoVT 1.-1, R,:uur TE"MP.QATV,('£, / M:>V.R).
`
`Exhibit 1062
`Prollenium v. Allergan
`
`

`

`U.S. Patent Aug. 12, 1986
`
`Sheet 1 of2
`
`4,605,691
`
`FIG.I
`
`4000
`
`JO(JO
`
`2000
`
`/0{)0
`
`•
`
`•
`
`s
`10 /IA CtJNCENT.:eAT/tJ/11, WT.~
`- £XAAfPL£ 3. SWELLING RATIO OF C/i'tJSS-LINl<ED /.IA GELS AT
`01,CrERENT CONCeNTR.4770NS OF f/A IN STARTING SOLLJTIONS
`(/.IA/DVS WE/6/ITR,4T/tJ //BOUT /:0 ROOM T£MPERATVRE, / I/OUR).
`
`

`

`U.S. Patent· Aug.12, 1986
`
`Sheet 2 of2
`
`4,605,691
`
`F!G.2
`
`4000
`
`3000
`
`2000
`
`/000
`
`•
`
`•
`
`/.0
`
`/.IA/011S tllO/JIR R4TIO
`£X4A,f PL£ 4. SWEL.LING RATIO OF CROSS-LINKED 1./A GELS AT .D/,t:'FERENT
`/.IA/OVS MOL.AR RATIOS
`{ t/A CONCEN'TJ?ATION IN STARTING
`SOLl/TIONS 4 WT. % 1 ROOM TEMPERATURe1
`I-IOVR).
`f
`
`ZO
`
`

`

`1
`
`4,605,691
`
`CROSS-LINKED GELS OF HYALURONIC ACID
`AND PRODUCTS CONTAINING SUCH GELS
`
`This application is a division of application Ser. No. 5
`678,895, filed Dec. 6, 1984, now abandoned.
`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- 15
`glucosamine and D-glucuronic acid monosaccharide
`units linked with ~1-4 bonds and the disaccharide
`units linked with ~1-+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 20
`to 8 X 106 and even higher.
`The prior art describes the cross-linking of HA with
`the use of 1,2,3,4-diepoxybutane in alkaline medium at
`50° C. (T. C. Laurent, K. Hellsing, and B. Gelotte, Acta
`Chem. Scand. 18 [1984), No 1, 274-5). The product 25
`obtained by that method is a gel which substantially
`swells in water.
`It is also known that divinyl sulfone (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(cid:173)
`tal data set forth in Example 3 below; and
`FIG. 2 is a graphical representation of the experimen- 35
`tal data set forth in Example 4 below.
`SUMMARY OF THE INVENTION
`In one aspect thereof, the present invention provides
`highly swollen gels of cross-linked hyaluronic acid.
`In another aspect, the invention provides mixed
`cross-linked gels of hyaluronic acid and other hydro(cid:173)
`phillic polymers.
`In yet another aspect, the invention provides cross(cid:173)
`linked gels of hyaluronic acid and other polymers filled 45
`with various substances.
`In still another aspect, the invention provides cross(cid:173)
`linked gels ofhyaluronic acid containing low molecular
`weight substances covalently attached to the macro(cid:173)
`molecules.
`In still yet another aspect, the invention provides
`various formulations containing cross-linked hyaluronic
`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(cid:173)
`ous alkaline solutions at room temperature, i.e., about
`20° C., thereby providing cross-linked HA gels. As used
`herein, the term HA means hyaluronic acid and its salts 60
`such as the sodium, potassium, magnesium, calcium, etc.
`salts. These gels swell in water and water containing
`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 65
`factors including the molecular weight of the HA, its
`concentration in the reaction mixture, the alkali concen(cid:173)
`tration and the polymer/DYS ratio. The reaction is
`
`40
`
`55
`
`2
`very fast and in most cases a strong gel can be obtained
`in several minutes. The swelling ratio of these gels can
`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 HA gels is substantially greater than the swelling
`ratio of cross-linked gels of other polysaccharides ob(cid:173)
`tained under the same reaction conditions. This can
`probably be explained by the unique nature of HA (as
`10 compared to other polysaccharides) and its water solu(cid:173)
`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 1Q3 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-
`30 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(cid:173)
`gen, elastin, albumin, a globulin, etc., sulfated proteins
`such as keratin sulfate and sulfated aminoglycosaminog(cid:173)
`lycans, synthetic water-soluble polymers, such as poly(cid:173)
`vinyl alcohol and its co-polymers, co-polymers of poly(cid:173)
`(hydroxyethyl)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(cid:173)
`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 hydrophilic gel filled with
`50 various water insoluble substances including hydrocar(cid:173)
`bons, such as petroleum; 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
`EMBODIMENT
`The processes by which the hereinabove described
`products are obtained will now be discussed in detail.
`In order to obtain a cross-linked HA gel, 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 8 X 106 and even
`higher. The molecular weight affects the reation-the
`
`

`

`4,605,691
`
`10
`
`25
`
`3
`higher the molecular weight the greater the possibility
`to obtain a cross-linked gel.
`The alkali concentration in the reaction mixture can
`be from 0.005M to 0.5M and higher. The lower limit is
`dictated by the necessity to have the pH of the medium 5
`not lower than 9 and the upper limit by the hydrolysis
`of HA in an alkaline solution. Usually, a decrease in
`alkali concentration results in gels with a greater swell(cid:173)
`ing ratio, probably because a small amount of DVS
`takes part in the cross-linking reaction.
`The concentration of HA in the starting solution can
`vary from 1% by weight up to 8% by weight and
`higher. When the concentration is below the lower
`limit, a cross-linked gel cannot be obtained even at a
`low HA/DVS ratio. When the concentration is too 15
`high, the solution becomes so viscous that it is difficult
`to handle it. The HA concentration substantially affects
`the swelling behavior of the gels (FIG. 1). It was found
`that the shape of the curve for the swelling ratio-the
`HA concentration dependence is essentially the same 20
`for various HA/DVS ratios but the lower this ratio (i.e.,
`more DVS in the mixture), the less the swelling ratio of
`the cross-linked gel for the same concentration of HA in
`the starting mixture.
`We have found that HA/DVS in the reaction mixture
`is another parameter which can be conveniently used to
`control the swelling ratio of the cross-linked HA gel.
`An increase in the ratio results in highly swollen soft
`gels (the swelling ratio is about 4000 and higher) 30
`whereas hard and less swollen gels are obtained when
`this ratio is decreased. In general, the HA/DVS weight
`ratio can be from 15:1 to 1:5 and lower.
`The cross-linking reaction is usually carried out at
`room temperature, i.e., about 20° C., but it can be per- 35
`formed at a lower or higher temperature, if desired.
`However, it should be kept in mind that HA can de(cid:173)
`grade relatively rapidly in alkaline solutions at elevated
`temperatures and, if such degradation occurs, the de(cid:173)
`crease in MW can affect the properties of the obtained 40
`gels.
`The cross-linking reaction is relatively fast and strong
`gels are formed usually in several minutes when the HA
`concentration is high enough and the HA/DVS ratio is
`low. But even at low HA concentration in the reaction 45
`mixture, the gel formation starts usually 5-10 minutes
`after addition of DVS. We have found that in most
`cases one hour is enough for completion of the cross(cid:173)
`linking reaction.
`Another method of controlling the swelling ratio of 50
`cross-linked HA gels involves adding neutral salt to the
`reaction mixture. We have found that the swelling ratio
`of the gels obtained in the presence of water soluble
`neutral salts, such as the chlorides, sulfates, phosphates
`and acetates of alkali metals, decreases with the increase 55
`of salt concentration. A salt can be used in concentra(cid:173)
`tion up to 20 wt. % and higher, depending upon the
`nature of the salt and its effect on the solubility of HA
`in the reaction mixture.
`To obtained cross-linked gels of other hydrophillic 60
`polymers the same reaction conditions as for HA can be
`used. The swelling ratio of these gels can be conve(cid:173)
`niently controlled by incorporating HA into the gel
`structure. When the mixed gels are obtained, the com(cid:173)
`position of the polymer mixture can vary over a broad 65
`range depending on the swelling ratio of the cross(cid:173)
`linked gel desired. The preferred content of HA in the
`mixture is from 5 to 95 wt. %.
`
`4
`Cross-linked gels of HA or other polymers or mixed
`cross-linked gels filled with inert substances are ob(cid:173)
`tained by incorporating these substances into the reac(cid:173)
`tion mixture before the addition of DVS. These inert
`substances are, preferably, water-insoluble liquids or
`solid substances. Examples of such substances are petro(cid:173)
`latum and kaolin. To obtain a filled cross-linked gel, a
`chosen substance (based on a consideration of the de(cid:173)
`sired properties of the gel) is emulsified or dispersed in
`an alkaline solution of HA or other polymer or mixture
`of HA with other polymer or polymers and DVS is
`added to the mixture. The amount of DVS and the
`other parameters of the reaction are selected depending
`upon the desired properties of the gel. The relative
`amount of filler in the gel can vary over a broad range
`and is from 1 to 95 wt. % calculated on the total amount
`of polymers and filler, preferably from 5 to 90 wt. %.
`Cross-linked gels containing low molecular weight
`substances such as drugs, dyes and others covalently
`attached to the macromolecular network are obtained,
`preferably by incorporating the named substances into
`an HA or HA and other polymers solution before the
`addition of DVS. An example of such a substance is
`carminic acid, an FDA approved substance for use in
`food and drug preparations.
`It is probably the presence of a glucosidic moiety of
`· the carminic molecule which takes part in the cross(cid:173)
`linking reaction with DVS. It should be understood that
`a great number of substances can be used to obtain a
`modified cross-linked gel of this type. The only essential
`feature of these substances is that they contain chemical
`groups with active hydrogen atoms reactive to DVS.
`The amount of such low molecular weight substances
`which can be used in the reaction depends upon the
`desired level of that substance in the gel. This amount
`can be in the range of from 1 to 99 wt. % as calculated
`on polymer content in the gel, preferably, 5 to 90 wt. %.
`The cross-linked HA and mixed gels obtained ac(cid:173)
`cording to the present invention can be used for many
`purposes. We have found that these highly swollen gels
`are very useful in cosmetic formulations and can be
`considered as water-retaining and water-delivering in(cid:173)
`gredients in these formulations.
`As HA is known to be a biologically tolerable poly(cid:173)
`mer in the sense that it does not cause any immune or
`other kind of response when introduced into a human
`body, the cross-linked HA gels can be used for various
`medical applications. The cross-linked gels modified
`with other polymers or low molecular weight sub(cid:173)
`stances can be used as drug delivery devices. For exam(cid:173)
`ple, we have found that heparin introduced in a cross(cid:173)
`linked HA gel retains its antithrombogenic activity.
`We have also found that cross-linked gels of HA can
`slow down the release of a low molecular weight sub(cid:173)
`stance dispersed therein but not covalently attached to
`the gel macromolecular matrix.
`The domain of the cross-linked hyaluronic acid
`(alone or co-polymerized with other polyanionic or
`neutral polymers) forms a molecular cage. In this cage,
`hydrophilic or hydrophobic molecules of various phar(cid:173)
`macological or biological activity can be dispersed.
`Thus, the cage constitutes a depot for these substances
`of various molecular size. The substances contained in
`the domain of the molecular cage will be delivered into
`the environment by diffusion. The delivery process is
`controlled by such factors as the exclusion volume ef(cid:173)
`fect and the pore size of the molecular cage and by the
`molecular interaction between the polymeric network
`
`

`

`6
`was obtained which had a swelling ratio in water of
`2910.
`
`EXAMPLE2
`This example illustrates the effect of alkali concentra(cid:173)
`tion on the cross-linking of HA.
`A sample of HA with a MW of about 3 X 1Q6 was
`dissolved in a calculated amount of 0.2M NaOH solu(cid:173)
`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(cid:173)
`tion was 0.0IM. 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.
`EXAMPLE3
`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(cid:173)
`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(cid:173)
`lated amount ofDVS 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.
`
`20
`
`4,605,691
`5
`and the substance contained therein. Thus, the molecu(cid:173)
`lar cage forms a depot for the controlled delivery of
`drugs or other substances to the skin or other tissues.
`There is one additional property of the cross-linked
`HA gels which makes them potentially very useful as 5
`drug delivery devices. The swelling ratio of these gels
`in water depends substantially upon the salt concentra(cid:173)
`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- IO
`duced into the body (because of the normal salt content
`of the body fluids and tissues), thus delivering its con(cid:173)
`tents, i.e., an incorporated drug, into the body tissue.
`The cross linked gels filled with various substances
`can also be used in cosmetic formulations. For example, 15
`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.
`DETAILED DESCRIPTION OF THE
`INVENTION
`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 25
`given merely by way of illustration and are not intended
`to limit the invention as set forth in the claims.
`EXAMPLE 1
`This example illustrates the effect of varying HA 30
`molecular weight on the cross-linking reaction.
`0.3410 g. of sodium hyaluronate obtained from
`rooster combs (intrinsic viscosity in 0.1 SM solution of
`NaCl [71] 3850, MW about 2.5 X 106) was mixed with
`8.1840 g. of 0.2M NaOH solution to give a 4% by 35
`weight solution after stirring for 30 minutes. Then,
`0.0721 g. of DVS was stirred into the solution. The
`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 40
`gel was left to swell in water overnight. Then it was
`broken into small particles by vigorous stirring in water.
`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 45
`sample weighing about 1 g. was centrifuged in a glass
`filter at 3,000 rpm for two hours. Then the particles left
`on the filter were hydrolyzed with 2 ml of IN H2SO4
`solution for three hours at 95°-98° C. The clear solution
`obtained was neutralized upon cooling with 2 ml of IN 50
`NaOH solution and the glucuronic acid content was
`determined by the carbazole method (An Automated
`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- 55
`ing ratio was expressed as 100/[HA]%, where [HA]%
`is a percent of HA in the swollen gel.
`The swelling ratio in water of the gel obtained was
`820.
`This example was repeated with the exception that 60
`the solution of HA in alkali was kept at room tempera(cid:173)
`ture for 24 hours. This led to a HA hydrolysis. The
`intrinsic viscosity [71) of the polymer was 1064 which
`corresponded to a MW of about 0.5 X 1Q6. A cross(cid:173)
`linked gel could not be obtained from this polymer at 65
`the HA/DVS ratio used above.
`The example with the degraded HA was repeated but
`the HA/DVS ratio used was about 2. A cross-linked gel
`
`EXAMPLE4
`This example illustrates the effect of varying the
`HA/DVS ratio on the swelling behavior of the result-
`ing gel.
`.
`Six solutions of sodium hyaliironate 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/(cid:173)
`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.
`EXAMPLES
`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(cid:173)
`pared with the use of the above described procedure.
`Sodium hyaluronate concentration in 0.2M sodium hy(cid:173)
`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.
`
`EXAMPLE6
`This example illustrates the cross-linking of hyrox(cid:173)
`yethyl cellulose with the use of DVS.
`
`

`

`7
`0.4312 g. of air-dry hydroxyethyl cellulose (Cellosize
`OP-100000@, Union Carbide) was dissolved with stir(cid:173)
`ring in 10.3 g. of 0.2N sodium hydroxide to give 4% by
`weight. 0.0855 g. of DYS was stirred into this solution
`(polymer/DYS ratio was about 5:1 by weight) and the 5
`mixture was left for one hour at room temperature. A
`cross-linked gel was obtained which was processed as
`described in Example l. To determine the polymer
`concentration in the gel and, hence, the swelling ratio, a
`weighed sample of the gel was put into acetone, kept 10
`overnight, washed several times with acetone and dried
`in a vacuum oven at 50° C. to a constant weight. The
`swelling ratio of the gel obtained was 43 which is sub(cid:173)
`stantially less than for cross-linked HA gel obtained
`under the same reaction conditions.
`
`I 5
`
`4,605,691
`
`8
`In each example, sodium hyaluronate and carboxy(cid:173)
`methyl cellulose 9H4F were dissolved in 0.2M sodium
`hydroxide solution in such amounts as to provide spe(cid:173)
`cific ratios of the two polymers In all cases the total
`polymer concentration was 4% by weight and the poly(cid:173)
`mer/DYS ratio was about 5:1. The gels were obtained
`and processed as described above. The polymer content
`in the gels was determined as described in Example 1,
`with the exception that the hexosamine concentration
`(instead of glucoronic acid) was determined by a known
`method (A Rapid Procedure for the Estimation of
`Amino Sugars on a Micro Scale, Analytical Biochemis(cid:173)
`try 15, 167-171 [1966]) in the hydrolyzate. The polymer
`content was calculated from the HA concentration and
`the ratio of the two polymers.
`
`EXAMPLE7
`This example illustrates the cross-linking of xanthan
`gum with the use of DYS.
`0.4935 g. of air-dry xanthan gum (Kelzan@, Kelco) 20
`was dissolved in 11.3 g. of 0.2M sodium hydroxide
`solution to give a 4% by weight solution. To this solu(cid:173)
`tion 0.0718 g. of DYS was added (the polymer/DYS
`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 30
`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.
`
`25
`
`35
`
`EXAMPLES
`This example illustrates the cross-linking of a cationic
`water-soluble cellulose polymer with the use DYS.
`0.5483 g. of a cationic cellulose polymer obtained by
`chemical modification of hydroxyethyl cellulose (Poly(cid:173)
`mer Deare JR@, Union Carbide) was dissolved in 40
`13.71 g. of 0.2M sodium hydroxide solution to give a
`4% by weight solution to which 0.0849 g. of DYS was
`added (the polymer/DYS 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 45
`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.
`
`50
`
`EXAMPLE9
`This example illustrates the cross-linking of carboxy(cid:173)
`methyl cellulose with the use of DYS.
`0.4703 g. of carboxymethyl cellulose sodium salt (9H
`4F, Hercules) was dissolved in 11. 76 g. of 0.2M NaOH 55
`to give a 4% by weight solution to which 0.0651 g. of
`DYS was added (the polymer/DYS ratio was about
`7:1). The reaction mixture was kept for an hour at room
`temperature and the gel obtained was processed and
`analysed as described in. the preceding example. The 60
`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 1~13
`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
`
`HA Content in the Starting
`Mixture, Wt. %
`
`Swelling Ratio
`in Water
`
`Example 10
`Example II
`Example 12
`Example 13
`
`70
`50
`20
`0
`
`8196
`6757
`1117
`623
`
`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(cid:173)
`tained from HA and collagen. 0.2531 g. of dry sodium
`hyaluronate was dissolved in 2.5 ml of O. lM sodium
`hydroxide solution. 0.063 g. of collagen obtained from
`human umbilical cord was dissolved in 2.3 ml of O. IM
`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
`KC! was dissolved in the mixed solution and DVS was
`stirred into the reaction mixture in an amount providing
`a polymer/DYS ratio of about 5:1. The reaction mix(cid:173)
`ture was kept at room temperature for an hour and the
`gel obtained was treated as described above. The poly(cid:173)
`mer content in the swollen gel 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(cid:173)
`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(cid:173)
`solved in 1.5 ml of 0.2M acetic acid solution and the
`solutions were combined. The total polymer concentra(cid:173)
`tion was 7.5 wt. % and the weight ratio HA/collagen
`was about 2: 1. 0.05 g. of sodium chlori4e was dissolved
`in the mixed solution to which 0.1189 g. of DYS was
`added, thus providing a polymer/DYS 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.
`
`

`

`10
`hydroxide solution to give an approximately 4 wt. %
`solution of polymer. 0.40 g. of DYS was added to the
`solution (polymer/DYS ratio was 1:2) and the mixture
`was left for an hour at room temperature. The gel ob-
`5 tained was processed as described in the preceding
`examples
`Red colored transparent gel particles were obtained
`and the color did not disappear after extensive washing
`with water. The swelling ratio in water determined by
`10 the weight method was 115.
`EXAMPLE21
`This example illustrates the effect of salt concentra(cid:173)
`tion in water on the swelling behavior of a cross-linked
`HA gel.
`A cross-linked HA gel was obtained as described in
`the preceding examples such an HA concentration in
`0.2M NaOH of 4 wt. %; HA/DYS ratio 5:1, at room
`temperature for one hour. The gel particles were put
`into water and aqueous sodium chloride solution of
`different concentrations and the swelling ratios were
`determined. The following results were obtained:
`
`NaCl Concentration, M
`Swelling Ratio
`Water
`990
`0.05
`413
`0.15
`384
`0.50
`219
`1.00
`176
`30 - - - - - - - - - - - - - - - - - - - - -
`
`4,605,691
`9
`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 0.0503 g. of heparin was added.
`The heparin content calculated on the basis of the total
`amount of polymers was 14.5 wt. %. 0.0590 g. of DYS
`was stirred into the mixture. The reaction was carried
`out for an hour at room temperature. The obtained gel
`was processed as described in the preceding examples.
`The swelling ratio of the gel was 625.
`EXAMPLE 17
`This example illustrates a cross-linked hydroxyethyl
`cellulose gel filled with petrolatum.
`0.5292 g. of dry hydroxyethyl cellulose was dissolved
`in 10.58 g. of IM sodium hydroxide solution and 1.058 15
`g. of white petrolatum was stirred into the solution. The
`petrolatum/polymer ratio was about 2. A solution of
`0.1771 g. of DYS in 1.0 g. of IM sodium hydroxide
`solution was added to the emulsion with vigorous stir(cid:173)
`ring. The reaction mixture was left for an hour at room 20
`temperature and the gel obtained was treated as de(cid:173)
`scribed in the above examples. To find the petrolatum
`content in the gel, a gel sample was digested with 2 ml
`of IN H2SO4 at 95° C. for three hours. Then 2 ml of IN
`NaOH was added to the mixture followed by 4 ml of 25
`xylene to extract the petrolatum. The extract was dried
`off in vacuum and the residue was weighed. The calcu(cid:173)
`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
`9.1 g. of0.2N sodium hydroxide solution to give a 4 wt. 35
`% solution of polymer. 0.3660 g. of petrolatum was
`stirred into the solution and 0.0730 of DYS was added
`to the resulting emulsion with vigorous stirring. The
`polymer/DYS ratio was about 5:1. The reaction mix(cid:173)
`ture was left for an hour at room temperature. The 40
`obtained gel was processed as described in the preced(cid:173)
`ing example. The swelling ratio of the gel determined
`through hexosamine content was 738 and the petrola(cid:173)
`tum content determined as in the preceding example
`was 0.1 wt. %.
`
`45
`
`EXAMPLE22
`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 Exam