Advanced Materials Research Vols. 396-398 (2012) pp 1506-1512
`Online available since 2011/Nov/22 at ,,;,n,.·.scien,ific.11el
`© (2012) Trans Tech Publications, Switzerland
`.,
`do i: 10. 4 0 2 8/www.scientific.net/ AMR. 3 96-3 98.1506
`
`Exhibit A
`
`3
`
`The comparison of physicochemical properties of four Cross-linked
`sodium hyaluronate gels with different cross-linking agents
`Yu jia Cui 1
`, Wei guo Wang 1
`,b (Correspondent), Pen9 Li 1
`'
`Yong liang Zhao1
`,d, Ya' Nan Gu 1
`, Jia Ii Wan 2
`'
`'
`1 Bioengineering College, Henan university of technology, Zhengzhou, Henan 450001, China
`2Henan Universe IOL Research&Manufacturing Co.LTD, Zhengzhou, Henan 450001, China
`acyj19870402@yahoo.com.cn, b wwgwangwwg@qq.com, clipengmail@163.com,
`dzylzhao@yahoo.com. en, 0 17575528@qq.com ,f82434853@qq.com
`
`8
`
`,c,
`
`Keywords: cross-linked sodium hyaluronate gels, different cross-linking agents, dynamic viscosity,
`intrinsic viscosity, enzyme-resistant degradation properties in vitro
`
`Abstract: Purpose The physicochemical properties of four cross-linked sodium hyaluronate gels
`(CHA) with different cross-linking agents were compared in order to research out the different
`stability and Enzyme-resistant degradation properties of these CHA gels. Methods The CHA
`hydrogels were prepared with different cross-linking agents, such as PEG20000, PDE, BDDE and
`ADH. The optimal reaction conditions were determined by single factor experiment. Dynamic
`viscosity was tested by Stabinger method. Intrinsic viscosity was determined by Uzziah's viscosity
`method. The enzyme-resistant degradation properties in vitro of CHA-gels were analysed by
`carbazole and spectrophotometry. Results The concentrations of N aOH/HCl, concentrations of HA
`and the ratio of cross-linking agent to HA are major factors of conditions which influenced the
`physicochemical properties of CHA gels. PDE-CHA and PEG20000-CHA gels possess better
`Dynamic viscosity, PDE-CHA gel has also better intrinsic viscosity, ADH-CHA and BDDE-CHA
`gels get better Enzyme-resistant degradation properties than PEG20000-CHA and PDE-CHA gels.
`Conclusion The CHA-gels prepared under optimal reaction conditions have different physical and
`chemical properties, which set foundation for developing double cross-linked or multifunctional
`gels with both excellent stability and enzyme-resistant degradation properties.
`
`Introduction
`
`Hyalouronic acid(HA), a high molecular weight, acidic polysaccharide typically composed of the
`alternating disaccharide GlcUAW1 ~3)GlcNAc[l]. It has wonderful biocompatibility, but natural
`hyaluronic acid is easy to degrade by hyaluronidase(HAase) in vivo[2,3].
`At present, at home and abroad, researches carried out by cross-linking HA gel with different
`cross-linking agents in order to get better viscoelasticity and Enzyme-resistant degradation
`properties, and maitain the original biocompatibility [ 4-7]. The marketted products of CHA gels
`are Allergan's Hylaform® gel of U.S.A[8], CORNEAL's SKGEL® gel of France[9], Q-Med AB's
`Restylane® gel of Sweden[ 1 O] and so on. The cross-linking agents of these CHA gel products such
`as biscarbodimide (EDC) , divinyl sulfone(DVS) [ 11-13], are all allergenic and toxic more or little.
`PEG20000, PDE, ADH and BDDE are cross-linking agents with
`the characteristics of
`lower-toxicity and will have broad application in pharmaceutics and cosmeceuticals [ 14-16].
`Different physicochemical properties of four CHA gels preparing with PEG20000, PDE, ADH and
`BDDE were compared in this paper.
`
`All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of TTP,
`www.ttp.net. (ID: 162.116.29.38-24/04/12, 19:14:53)
`
`Exhibit 1025
`Prollenium v. Allergan
`
`

`

`Advanced Materials Research Vols. 396-398
`
`1507
`
`MATERIALS AND METHODS
`MATERIALS
`ether,
`acid dihydrazide, ADH(SIGMA); Poly diglycidyl
`PEG20000(SIGMA); Adipic
`PDE(SIGMA); 1,4-Butanediol diglycidyl ether, BDDE(Alfa Aesar); Hyaluronidase (SIGMA);
`Medical level sodium hyaluronate dry powder(0912081 0)(Shandong FuRuiDa biological chemical
`Co., LTD); Carbazole(Chinese medicine group Shanghai chemical reagent company); The standard
`glucuronic acid(Shanghai biological technology Co., LTD); Folin-Ciocalteu's phenol(Shanghai Ruji
`biological technology development Co., LTD); NaOH; HCl; Na2HPO4; NaH2PO4; Anhydrous
`alcohol; phenol;
`FA1204B electronic balance (Shanghai precision &scientific instrument Co., LTD); 85-type 1
`constant temperature and heating magnetic blender (changzhou guohua instrument plant); 723 N
`uv-vis spectrophotometer; HH-4 display constant temperature water-bath pot; TGL-16 C high-speed
`centrifuge; LDZX-50 a KBS vertical pressure steam sterilization pot; Mooney viscometer having
`rotor syringe; Ubbelohde viscometer.
`METHODS
`Preparation of Cross-linked hyaluronate gels
`The cross-linked hyaluronate gels were prepared by the following process: A certain weight of
`medical level sodium hyaluronate dry powder was dissolved in the solution of 0.00 lmol/mL--o 0.1
`mol/mL NaOH or HCl, then different cross-linking agents which are PEG20000, PDE, BDDE and
`ADH were added
`into the solution. Churned the mixtures for hours to make the CHA-gels
`homogeneous. The pH of CHA-gels was adjusted at 7.0~7.3. At the end, CHA-gels were
`heat-treated at 115°C ~ 121 °C for 8 min~30min.
`Comparisons of stability of CHA gels under different conditions
`Taking dynamic viscosity and intrinsic viscosity[ 18] as the evaluation standards of stability of CHA
`gels, the factors which influenced the stability of CHA were chosen, such as the different
`cross-linking agents, the time of reaction, the reaction temperatures, the concentrations of HA, the
`concentrations of NaOH and HCl, the ratios of cross-linking agents to HA (g/g).
`Comparison of stability of CHA gels with different time of reaction
`The reacting time of preparing CHA-gel was set from 2h~36h. The selected cross-linking agents
`were PEG20000, PDE, BDDE and ADH. Others conditions were set with definite parameters.
`Comparison of stability of CHA gels by different temperatures
`The choosing temperature scope of preparing CHA-gel were from 0°C~ 100°C. The selected
`cross-linking agents were PEG20000, PDE, BDDE and ADH. Others conditions of preparation
`were set with definite parameters.
`Comparison of stability of CHA gels by different concentrations of NaOH and HCl
`The concentrations of N aOH/ HCl agents were set from 0.00 lmol/mL--o 0.1 mol/mL. The selected
`cross-linking agents were PEG20000, PDE, BDDE and ADH. Others conditions were set with
`definite parameters.
`Comparison of stability of CHA gels under different concentrations of HA
`The choosing concentrations of HA was from 0.5%~2.5%.The selected cross-linking agents were
`PEG20000, PDE, BDDE and ADH. Others conditions were set with definite parameters.
`Comparison of stability of CHA gels with the different ratios of cross-linking agent to HA
`The ratio of cross-linking agent to HA was set froml:20~1:2.5(g/g). The selected cross-linking
`agents were PEG20000, PDE, BDDE and ADH. Others conditions were set with definite
`parameters.
`
`

`

`1508
`
`Advances in Chemical Engineering
`
`Comparison of stability of CHA gels by different conditions of sterilization
`The prepared PEG20000-CHA, PDE-CHA ,BDDE-CHA and ADH-CHA gels were categorized into
`two groups, one was treated at 121 °C for 8 min. The other one was treated at 115°C for 30min.The
`parameters of dynamic viscosity and intrinsic viscosity were compared respectively.
`The determination of viscoelasticity of CHA-gels.
`The dynamic viscosity of CHA-gels prepared by different cross-linking agents were tested by
`Stabinger method. The intrinsic viscosity of CHA-gels prepared by different cross-linking agents
`were determined by Uzziah's viscosity method[18].
`Examination of enzyme-resistant degradation properties of CHA-gels in vitro
`Carbazole method[18]. 4g different CHA-gels which were prepared as above were analysed
`respectively. The gels were added with 2 ml of 856 U/ml HAase, at 37 °C, reacting
`for 65hr. Let
`the volume to 5 ml with PBS buffer solution(pH 7.1 ),then pipetted 1ml of the gel-extract mixtures
`adding with 4ml of absolute ethyl alcohol respectively to centrifugate for 15 min,at the speed of
`10000 r/ min. Pipetted 2 ml supernatant liquid which let the volume to 5mL by PBS as solution I.
`On the other hand, 15g different CHA-gels which were prepared as above were hydrolyzed by
`10ml sulfuric acid solution (0.5 mol/L) for 20 min in the boiling water bath. Metered the volume to
`100 ml with PBS buffer solution (pH 7.1) as solution II. lmL of second solution was used to be
`analyzed.
`The Carbazole and sulphuric acid spectrophotometry was used to analysed the content of
`GlcA,the numerical value of resistance to enzymatic degradation in vitro(R) was computed by the
`following formula:
`
`R =l - 15/64xA/B
`A-content of GlcA in the I solution. (mg/ml);
`B-content of GlcA in the II solution. (mg/ml).
`
`RESULTS and DISCUSSION
`Stability of CHA gels under different times of reaction
`The researched results of reaction time of CHA-gel indicate that the stability of the CHA-gel is
`increasing with the time of reaction. The reaction probably ends by 4hr because of the possession of
`the approximate data of viscoelasticity of the gel by the end of 4hr and 6hr. Results are shown in
`Figure 1.
`
`~
`
`~ 80
`
`(I.. 160
`6 t 40
`
`0
`0
`
`0
`
`A---~---~_-_::t::::.::l::---~---:~~.:;:~::i:o::~
`
`I,.
`
`·· ·-&· -·ffGZ
`0000-m.1
`
`--♦--PD!:-m.l
`
`····"'-·····BDDl!-m.l
`
`-
`
`-x- - .IDH-m.l
`
`-~ 20
`E f 0
`
`C,
`
`·/~-~-~-~-~~~~-~
`Oh
`2h
`4h
`6h
`8h
`12h
`24h 36h
`Figure 1 Dynamic viscosity (xl04mPa.s) of CHA gels at different times
`Stability of CHA gels under different reaction temperatures
`The stability of different CHA-gels sharply go up between 0°C and 20°C,they remain steady
`between 20°C~40°C,and then drop at 60°C~l00°C. PEG20000-CHA and PDE-CHA gels have better
`heat-stablility than BDDE-CHA and ADH-CHA All the process of CHA preparation should be
`below 40°C in order to get gels of excellent stability or physicochemical properties. Comparative
`results are shown in Figure 2.
`
`

`

`Advanced Materials Research Vols. 396-398
`
`1509
`
`0
`
`~ 80
`en
`&_ 70
`~ 60
`E_ 50
`z,
`·u; 40
`0
`~ 30
`">
`-~ 20
`E
`~ 10
`>,
`0
`0
`
`O'C
`
`4000
`
`3500
`~
`3000 6
`~
`2500 ·u;
`0 u
`2000 -~
`u
`1500 -~
`·;:::
`1000 c
`
`500
`
`lOO'C
`
`SO'C
`
`40'C
`
`60'C
`
`30'C
`20'C
`~PEG20000-CHA
`~PDE-CHA
`IZZZZZIBDDE-CHA
`c::===iADH-CHA
`-Natural HA gel
`---+• Intrinsic viscosity(cm3/g) of PEGZ0000C-HA gels
`"""~¾===Intrinsic viscosity(cm3/g) of PDE-CHA gels
`"""'@"'" Intrinsic viscosity(cm3/g) of BDDE-CHA gels
`~Intrinsic viscosity(cm3/g) of ADH-CHA gels
`" + " Intrinsic viscosity(cm3/g) of Natural HA gel
`
`Figure 2 Dynamic viscosity (xl04mPa.s) and Intrinsic viscosity(cm3/g) of CHA gels at different temperatures
`
`Influence of different concentrations of Na OH and HCI on stability of CHA gels
`The stability of the CHA-gels are decreasing along with the increasing concentration of NaOH or
`HCl, for the simple reason that CHA-gels are easily degradable in the high pH as well as natural
`hyaluronic acid.
`The dynamic viscosity of PEG20000-CHA prepared with NaOH solution is higher than with HCl
`solution,The dynamic viscosity of PDE-CHA, BDDE-CHA and ADH-CHA gels prepared with
`HCl solution are higher than with NaOH solution. The results for the dertermination of intrinsic
`viscosity show that: PDE-CHA> PEG20000-CHA and BDDE-CHA> ADH-CHA.
`The optimal conditions of preparing PEG20000-CHA gel is 0.00lmol/mL NaOH, The optimal
`conditions of PDE-CHA, BDDE-CHA and ADH-CHA gel preparing is 0.00lmol/mL HCl.
`Comparison results are shown in Figure 3 and Figure 4.
`
`80
`70
`60
`50
`40
`30
`20
`10 0 .__ __ ...._ __ __._ ______ ..._ __ ..._ __ ....,
`
`0. 001
`0. 005
`0. 01
`"""''~ """ PEG20000 CHA
`"' ~ "' EDDE-CHA
`
`0. 05
`
`0. 1
`PDE CHA
`-X-ADH-CHA
`
`0. 1
`0.001
`0.005
`0.01
`0.05
`·'""~ """PEG20000-CHA ~PDE-CHA
`-X-ADH-CHA
`~ ~ " BDDE-CHA
`
`Figure 3 Dynamic viscosity (xl04mPa.s) of CHA
`under different concentrations ofNaOH(mol/mL)
`
`Figure 4 Dynamic viscosity (x 104mPa.s) of CHA gels
`under different concentrations ofHCl(mol/mL)
`
`

`

`1510
`
`Advances in Chemical Engineering
`
`Influence of the different ratios of cross-linking agent to HA on stability of CHA gels
`The dynamic viscosity of the CHA-gel steadily increased between 1:20 and 1:10,but the figures
`fallen at the ratio of 1:2.5. The examination results of intrinsic viscosity are PDE-CHA>
`PEG20000-CHA and BDDE-CHA> ADH-CHA.
`Comparison of stability of CHA gels with different concentrations of HA
`The dynamic viscosity and intrinsic viscosity of CHA-gels are increasing along with the growing
`concentration of HA, dynamic viscosity rising from about 105mPa.s to over 106mPa.s, intrinsic
`
`
`viscosity rising from 2517 cm3 / g to 5241 cm3 / g.
`Stability of CHA gels under different conditions of sterilization
`The dynamic viscosity of CHA gels after sterilization are just about 1/3-1/2 persentage of
`non-sterilizated CHA gels, but much high than natural HA gel.The keeping proportions of intrinsic
`viscosity are at most 80% percentage after sterilization. The steady dynamic viscosity numbers of
`gels indicate that PEG20000-CHA, PDE-CHA and BDDE-CHA gels have better heat- stability than
`ADH-CHA and natural HA gels. After 115°C sterilization for 30min, the dynamic viscosity of
`ADH-CHA gels, along with the intrinsic viscosity of BDDE-CHA and ADH-CHA gels went down
`to the bottom. Comparison results are shown in Figure 5, Figure 6.
`
`4100
`
`Ct')
`
`OJ -- 3600
`E
`(.) 3100
`~
`Cl) 2600
`0
`(.)
`Cl) 2100
`s
`(.) 1600
`Cl)
`C
`·c 1100
`c
`600
`
`-+- Intrinsic
`viscosity(cm3/g)
`of CHA gels
`before
`sterilization
`---~---- Intrinsic
`viscosity(cm3/g)
`of CHA gels
`sterilized at 121
`~C, 8min
`···,~'-··· Intrinsic
`viscosity(cm3/g)
`of CHA gels
`sterilized at 115
`~C, 30min
`
`PEG20000-CHA
`
`PDE-CHA
`
`BDDE-CHA
`
`ADH-CHA
`
`Natural HA
`gel
`
`
`
`Figure 5 The Intrinsic viscosity( cm3 / g) of CHA gels before and after sterilization
`
`88.12 97
`
`.4
`
`3 90.01 86.74
`
`120
`100
`80
`60
`40
`20
`0
`
`60.69 67·34 58.53
`
`120
`100
`80
`60
`40
`20
`0
`
`The Dynamic viscosity ( X 104mPa. s) before
`sterilization
`i:::I EDDE-CHA
`□ PDE-CHA
`[]Natural HA gel
`
`C5I PEG20000-CHA
`CADH-CHA
`
`The Dynamic viscosity ( X 104mPa. s) after
`sterilization
`i:::I EDDE-CHA
`□ PDE-CHA
`□ Natural HA gel
`
`C5I PEG20000-CHA
`CADH-CHA
`
`Figure 6 Dynamic viscosity (xl04inPa.s) of CHA gels before and after sterilization
`
`The dynamic viscosity and intrinsic viscosity decreased after sterilization indicate that the
`preparing process of CHA still should be optimized, in order to have better stability of
`cross-linked sodium hyaluronate gels.
`
`

`

`Advanced Materials Research Vols. 396-398
`
`1511
`
`Enzyme-resistant degradation properties in vitro of CHA-gels prepared by different
`cross-linking agents.
`The regression equation of the standard curve for the determination of GlcA content is
`Y=0.5955x+0.0035, R2=0.9992. The contents of GlcA in solution I are ADH-CHA> BDDE-CHA >
`PDE-CHA> PEG20000-CHA>Natural HA gel. The enzyme-resistant properties (R) of CHA gels
`are much higher than natural HA-gel. Researched results indicate that the gels prepared by BDDE
`and ADH have better enzyme-resistant degradation properties in vitro than PEG20000 and PDE.
`These crossed-linked gels have better enzyme-resistant degradation properties than natural HA-gel
`apparently. Results are shown in table 1.
`
`Extinction value at Extinction value at Enzyme-resistant
`530nm/ solution I 530nm/ solution II properties /R(%)
`
`Table 1 Extinction value at 530nm ofGlcA of the gels prepared by different cross-linking agents
`CHA gels with different
`cross-linking agents /
`contents of HA
`PEG20000-CHA gel/1.5%
`PDE-CHA gel/1.5%
`BODE-CHA gel/1.5%
`ADH-CHA gel/1.5%
`Natural HA-gel/1.5%
`
`0.265
`0.273
`0.224
`0.205
`1.348
`
`0.425
`0.425
`0.424
`0.424
`0.485
`
`85.4%
`84.9%
`87.6%
`88.7%
`34.8%
`
`Conclusions
`The CHA hydrogels prepared with different cross-linking agents have different physical and
`chemical properties. PEG20000-CHA and PDE-CHA gels have excellent heat-stability while
`ADH-CHA gel does not. ADH-CHA gel has better Enzyme-resistant degradation properties than
`PEG20000-CHA, PDE-CHA and BDDE-CHA gels.
`The gels with both excellent stability and enzyme-resistant degradation properties can be
`prepared based on this research using different cross-linking agents such as PEG20000, PDE, ADH
`and BDDE. They can be used in Adjuvant therapy of Eye Surgery and arthritis treatment, as well as
`preventing adhesion of surgery. They can be used for a new drug delivery system.
`
`Acknowledgements
`
`The research work was funded by the Commission of Science and Technology of Henan Province
`(CSTHP), the Program for Tackling Key Problems, programs No.112102310326 and the
`Commission of Education of Henan Province (CEHP), the Science Foundation Program, programs
`No. 2011A180007; Henan University of Technology, special program of introducing persons of
`ability No.2007BS023.
`Correspondent: Weiguo Wang (1962-
`), male, Professor of bioengineering, Master of D.,
`Researching interests: biopharmaceuticals, bioengineering and bioprocess. Tel.: 03 71-6062123 8,
`13838298531. Email: wwgwang@yahoo.com.cn.
`
`

`

`1512
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`Advances in Chemical Engineering
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