research article healing efficacy of an egf impregnated...

Research ArticleHealing Efficacy of an EGF Impregnated Triple Gel BasedWound Dressing In Vitro and In Vivo Studies

Najmeh Khanbanha1 Fatemeh Atyabi12 Azade Taheri1 Fatemeh Talaie2

Mirgholamreza Mahbod3 and Rassoul Dinarvand12

1 Department of Pharmaceutics Faculty of Pharmacy Tehran University of Medical Sciences PO Box 14155-6451 Tehran Iran2Nanotechnology Research Center Faculty of Pharmacy Tehran University of Medical Sciences PO Box 14155-6451Tehran Iran

3Noor Ophthalmology Research Center Noor Eye Hospital PO Box 19393-3475 Tehran Iran

Correspondence should be addressed to Fatemeh Atyabi atyabifatumsacir

Received 5 February 2014 Revised 29 April 2014 Accepted 23 May 2014 Published 8 July 2014

Academic Editor Aijun Wang

Copyright copy 2014 Najmeh Khanbanha et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

To accomplish an ideal wound healing process which promotes healthy tissue growthwith less scaring a novel gel based topical drugdelivery system composed of 3 different polymers chitosan dextran sulfate and polyvinylpyrrolidone K30 (CDP) was preparedThe physicochemical properties of the prepared gels were investigated in vitro Gels showed a maximum swelling ratio of 50 plusmn 195times of dried gel in PBS at pH 74The swelling ratios increase in acidic and alkaline pH to 553 plusmn 175 and 655 plusmn 242 respectivelyIn the rheological test prepared gels revealed viscoelastic properties and a small linear viscoelastic region of 0166 In vivo woundhealing promoting activities of CDP gels containing 20 120583gmL EGF were evaluated on surgically induced dermal wounds in ratsusing pathologic examination The application of CDP gel with incorporated EGF significantly reduced the defect on the ratrsquos skinand enhanced epithelial healing compared with the topical application of the EGF-free CDP gelThe results clearly substantiate thebeneficial effects of the topical application of CDP containing EGF in the acceleration of healthy wound healing process with lessscarring

1 Introduction

As a fundamental response to tissue injury wound healing isa normal complex process including four general phases ofhemostasis (clot formation) inflammation cell proliferationextracellular matrix production and remodeling which usu-ally in each phase occurs consequently in a regulated manner[1 2] The clot and the surrounding wound tissue secretea variety of cytokines and growth factors such as plateletderived growth factor (PDGF) and epidermal growth factor(EGF) that simulate wound repair process [3 4] Severalstudies showed that topical use of growth factors such asEGF on the wound could accelerate the rate of the epidermalregeneration and reepithelialization of chronic wounds [56] To achieve an appropriate efficacy of EGF therapy inwound repair the local bioavailability of the EGF should beprolonged using a suitable drug delivery system To increase

the low bioavailability of peptides and proteins such as EGF inconventional topical solutions viscous drug delivery systemssuch as sponge have been explored [7] Gel based naturalpolymers such as chitosan and dextran are used for treatmentof dermal wounds [8]Wound dressing prepared with naturalpolymers is found to prolong the retention time of thera-peutic peptides on the wound and increase the therapeuticeffects of these peptides [9] Dextrans are polysaccharideswhich are found in bacterial extracellular matrix [10] have agood solubility level in water and have been investigated forprotein delivery in pharmaceutical studies [11 12] Dextransulfate is a negatively charged polysaccharidewhich is derivedfromdextran via sulfation andwas shown to have appropriatecharacteristics for the treatment of wounds [13] Chitosan is anatural polymer derived from chitin known to stimulate cellproliferation and wound healing process through its hemo-static properties [14] Moreover bacteriostatic properties of

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 493732 10 pageshttpdxdoiorg1011552014493732

2 BioMed Research International

chitosan could be useful in relation to wound treatment [14]Chitosan and its derivatives have been used successfully forcontrolled delivery of drugs peptides and proteins [15ndash20]Wound dressing prepared using chitosan and dextran couldabsorb thewound exudates and hold themoisture providing asuitable moisturized environment at the wound site [21] Butthe electrostatic interaction between dextran and chitosancould reduce the stability of dextran-chitosan gels We usedpolyvinylpyrrolidone K30 (PVP K30) as suitable polymer toreduce the dextran-chitosan instability (CDP gel) In thiswork we prepared CDP gel as a suitable gel based drugdelivery system to increase the bioavailability of therapeuticpeptides and proteins such as EGF to wound tissueThen weimpregnated EGF in CDP gel EGF impregnated CDP gel wasused as suitable wound dressing to accelerate and facilitatewound healing process Physical properties of wound dress-ing and the effects of EGF on healing process in comparisonwith controls were studied in full thickness excision woundmodel in rats in vivo

2 Material and Methods

21 Materials Already characterized low and mediummolecular weight chitosan samples (deacetylation degreeapproximately 95) were purchased from Chitoclear (Siglu-jordur Iceland) Dextran sulfate (MW 8 and 10 kDa) waspurchased from Sigma-Aldrich (St Louis MO USA)Polyvinylpyrrolidone K30 (PVP K30) was purchased fromBASF (Ludwigshafen Germany) Recombinant human epi-dermal growth factor (rhEGF) was obtained from SichuanHuamai Technology Co (Sichuan China) All other reagentsused were of analytical grade

22 Preparation of Gels

221 Preparation of Chitosan Solutions Chitosan solutionsof low andmediummolecular weight were prepared in diluteacetic acid (1ww) and themixtures were heated and stirredat 50∘C for 24 h until clear solutions were achieved Chitosansolutions were neutralized with 1N solution of NaOH topH 51 and then were degassed at 25∘C for 15min using asonicator (Elmasonic Elma Hans Schmidbauer GmbH amp CoKG) to remove air bubbles

222 Preparation of Dextran-PVP Solution Dextran sulfate(MW8 and 10 kDa) was dissolved in deionized water Knownamount of PVPK30was added to the above solution to obtaindextran-PVP solution

223 Preparation of CDP-EGF Gels For preparation ofchitosan-dextran-PVP (CPD) gels freshly prepared chitosansolution was added dropwise to dextran-PVP solution underslow stirring to achieve the final composition of gel for-mulations as given in Table 1 For preparation of CDP-EGFgels EGF was added to prepared CDP gels to obtain aconcentration of 20120583gmL

23 Swelling Study Swelling of gels was studied by a gravi-metric procedure CDP gels were cast into 13 diameter times 45height (approximately 059mm3) molds and then vacuumdried at 60∘C for 12 h Dried gels were accurately weightedand swollen in PBS pH 74 at 25∘C The swollen gels wereremoved from the buffers at certain times (5 10 20 30 4560 and 120min) then blotted to remove excess water andreweighed Each experiment was performed in triplicateTheswelling ratio was calculated using the following equation

SR =119882119904

119882119889

(1)

where119882119904is the weight of the swollen gel and119882

119889is the weight

of the dried gelTo characterize the swelling behavior of the gels the

following experiments were conducted To determine theeffect of pH on the swelling ratio of gels CDP gels wereimmersed in acetate buffer pH 45 and phosphate buffer pH9 at 25∘C for 5 10 20 30 45 60 and 120min The swellingratio was calculated as mentioned above The effect of ionicstrength on the swelling ratio of gels wasmeasured in 1N PBSpH 74 4N PBS pH 74 and 8N PBS pH 74 at 25∘C for 5 1020 30 45 60 and 120min as mentioned above

231 Gel Fraction Different formulations of gels were castinto 059mm3 molds and then vacuum dried at 50∘C for 12 huntil a constant weight was reached (119882

119889)The dried gels were

immersed in 40mL distilled water at 50∘C for 12 h undervacuum The sol part of immersed gels was extracted fromthe water The remaining part was vacuum dried at 50∘C for12 h till constant weight was achieved (119882

119903) The gel fraction

percentage was determined using the following equation

Gel Fraction () = (119882119903

119882119889

) times 100 (2)

24 Differential Scanning Calorimetry (DSC) Analysis Thephysical or chemical interaction between polymers wasanalyzed by differential scanning calorimetry (DSC) studiesSamples were analyzed using a differential scanning calori-metric (DSC) analyzer (DSC 823 Mettler-Toledo Germany)5mg of the mixture of chitosan and dextran sulfate orchitosan-dextran sulfate dried gels were placed in aluminumpans For thermogram acquisition the samples were heatedover a range of 30∘C to 500∘C at a rate of 10∘C per min andthe thermal behavior of samples was recorded

241 Rheological Studies To study the plastic deformationstress and the viscoelastic properties of the gel the rheologicalanalysis of the prepared gels was performed using a stresscontrolled Paar Physica MCR 300 (Anton Paar GmbHAustria) with a plate-plate measurement system PP25 (thegap between the plates was 1mm) The temperature was25∘C for all tests The data were collected using Rheoplus32Service V340 software (Anton Paar GmbH Austria) Gelswere placed between the plates of the measuring system andtwo sets of experiments were performed

BioMed Research International 3

Table 1 Composition of CDP gel formulations

SamplesConcentration

Low molecularweight chitosan

Medium molecularweight chitosan

Dextran sulfate(MW 8kDa)

Dextran sulfate(MW 10 kDa) PVP

F1 133 033 033F2 15 025 025F3 16 02 02F4 16 02 02F5 16 02 02F6 16 02 02

The amplitude sweep test (oscillatory condition) wasperformed (25∘C) in the range of 001ndash100 Pa for the gelsThestorage modulus (1198661015840) the loss modulus (11986610158401015840) and the linearviscoelastic (LVE) deformation range were investigated usingthis test

Further gel flow measurement was performed using aplate-plate measurement system PP25 (the gap between theplates was 1mm) with a shear stress range of 1ndash100 Pa Theyield point of the gel was determined

242 In Vivo Studies All in vivo experiments were con-ducted according to the guidelines of Ethical Committee ofthe Pharmaceutical Research Centre Faculty of PharmacyTehran University of Medical Sciences Iran 15 male Wistarrats (275 plusmn 25 g) were purchased from Pasteur Institute(Tehran Iran) Animals had free access to food and waterThe animals were divided into three groups There were 5animals in each group Ratsrsquo dorsal hair was depilated whilethe animals were under ketaminexylazine anesthesia Skintissue (15 cm long times 15 cm) was surgically removed usingsterile surgical tools to create full thickness wound on theback of the animal The animals were housed individuallyunder standardized environmental conditions (day 0) In firstgroup 04mL of CDP gel containing 20120583gmL EGF waslocally applied to the full thickness skin wounds from day1 once a day for 11 days The second group received the sameCDP gel formulation without EGF and the third group wasthe control group in which the animal was wounded but notreatment was applied

Before using CDP gel with EGF (20 120583gmL) all theanimalsrsquo wounds in all groups were washed with sterilenormal saline solution and photographed and then the gelswere applied on the wounds Wounds were covered withnonwoven retention tape using adhesive 3M tape After 8experimental days three rats of each group were sacrificedby chloroform inhalation Other remaining animals weresacrificed after 11 experimental days The images of woundswere captured after rising of wounds with normal salineusing a digital camera The images of wounds were analyzedfor the wound area using Photoshop (versus CS5) softwareTissue specimens from the wound site of individual rats wereexcised after animal euthanization The skin samples werefixed in 10 formaldehyde solution dehydrated with gradedalcohol cleared in xylene and then embedded in paraffinblock 5 120583m sections have been cut from blocks stained

with Haematoxylin and Eosin and observed under a lightmicroscope

243 Statistical Analysis Statistical significance was deter-mined by a one way analysis of variance (ANOVA) followedby a post hoc test for multiple comparisons using SPSS Vr3software 119875 lt 005 was considered statistically significant

3 Results

31 Preparation of CDPGels Theviscosity of 3 (and higher)of chitosan solutions was too high and thus these solutionswere not suitable for gel preparation process Moreover theviscosity of 1 (and lower) of chitosan solutions was too lowand thus these solutions were not suitable for gel preparationprocess Thus chitosan solutions with concentration of 2ww were selected for gel preparation For prevention ofthe formation of chitosan-dextran precipitation (because ofelectrostatic interaction) PVP K30 was used as a bulkingpolymer to slow down the electrostatic interaction andprecipitation

32 Swelling Behavior of CDP Gels Water uptake of CDP gelswas followed gravimetricallyThe swelling curves of CDP gelsin PBS pH of 74 at 25∘C are given in Figure 1 CDP gels in PBSat pH 74 show high swelling ratios from 1684 plusmn 195 for F5to 5003 plusmn 106 for F4 F4 showed the highest swelling ratio(5003) in PBS at pH 74 after 45minutesThus F4 containing16 low molecular weight chitosan 02 dextran sulfateand 02 PVP was chosen as an optimum sample to conductfurther experiments Swelling behavior was carried out for F4at different pH values (45 74 and 9) (Figure 2) Maximumswelling ratio of F4 was observed at pH 90 whereas lesser butsignificantly different swelling ratio was observed in other pHvalues in this study Effect of ionic strength on the swellingratio of the candidate gel (F4) was investigated at differentconcentration of PBS (pH 74) at 25∘C (Figure 3) It wasobserved that as the concentration of PBS (pH 74) increased(ionic strength increased consequently) a decrease in gelswelling ratio occurred

33 Gel Fraction Thegel fractions of F1 F2 F3 F4 F5 and F6CDP gel formulations were 5474 5321 5856 67924776 and 4950 respectively F4 showed the highest gel


0

10

20

30

40

50

60

0 20 40 60 80 100 120 140

Swel

ling

ratio

Time (min)

F1F2F3

F4F5F6

Figure 1 Measurement of swelling ratio obtained from differentchitosan-dextran-PVP gels presented in Table 1 in PBS (pH 74) at25∘C at predetermined time points (5 10 20 30 45 60 and 120min)(119899 = 3 mean plusmn SD)

fraction among other formulations (Figure 4) Thus accord-ing to the results of swelling ratio and gel fraction studies F4formulation was chosen to perform further studies

34 DSC Analysis The differential scanning calorimetry(DSC) thermograms of CDP gels are shown in Figure 5An exothermic peak was observed in DSC thermogram ofphysical mixture of chitosan dextran and PVP at about 220ndash240∘C The thermograms of gels gave no intrinsic peak butshowed an approximately similar pattern to thermogram ofphysical mixture of chitosan dextran and PVP (Figure 5)

35 Rheological Properties The strain sweep test shows largedifferences between the elastic 1198661015840 and the loss 11986610158401015840 moduliwith 1198661015840 much higher than 11986610158401015840 indicating higher viscoelasticproperties of the prepared gel as shown in Figure 6 Howeverthe rapid decrease in modulus resulted in a very smallLVR (linear viscoelastic region) Flow measurement wasconducted to determine the plastic deformation stress thatis the yield point of gels (Table 2) The portion of 11986610158401015840 to1198661015840 is defined as tangent 120575 angle from 0 degrees indicating

an absolute elastic gel to 90 degrees indicating an absoluteviscous gel The results show that as the strain increases 1198661015840decreases more rapidly than 11986610158401015840 illustrating a more elastic geltransforms into a more viscous gel Consider

11986610158401015840

1198661015840

= tan 120575 (3)

36 In Vivo Wound Healing Study According to dataobtained from gel fraction and swelling studies F4 formu-lation was chosen for the treatment of full thickness woundson rats Macroscopic appearance of wounds treated with F4formulation of CDP gel with EGF F4 formulation of CDP

Swel

ling

ratio

0

10

20

30

40

50

60

70

80

pH 45pH 74pH 9

0 20 40 60 80 100 120 140Time (min)

Figure 2 Measurement of swelling ratio of chitosan-dextran-PVPgel (F4) in acetate buffer (pH 45) PBS (pH 74) and phosphatebuffer (pH 9) at 25∘C at predetermined time points (119899 = 3 meanplusmn SD)

1X PBS4X PBS8X PBS

0 20 40 60 80 100 120 140Time (min)

0

10

20

30

40

50

60

Swel

ling

ratio

Figure 3 Measurement of swelling ratio of chitosan-dextran-PVPgel (F4) in 1N PBS (pH 74) 4N PBS (pH 74) and 8N PBS (pH 74)at 25∘C (119899 = 3 mean plusmn SD)

01020304050607080

f1 f2 f3 f4 f5 f6

Gel

fra

ctio

n

Formulation

Figure 4 Gel fraction () of chitosan dextran and PVP gels


Table 2 Rheological properties of gel

Oscillatory test Flow testFlow point 119866

1015840

11986610158401015840 tan 120575 Angle Yield point

Stress Strain Crossover strain Shear stress001 100 001 100 001 100 001 100

F1 180 79 162 423 13 118 14 03 11 156 471 386 PaF2 192 47 289 614 123 166 175 03 14 151 549 389 PaF3 No No No 569 20 134 17 024 09 133 407 NoF4 188 758 175 245 13 129 146 05 11 278 481 642 PaF5 413 96 303 916 286 168 296 02 1 104 46 676 PaF6 187 356 37 386 121 143 211 04 17 203 602 374 Pa

Physical mixture

F3

F4

20 60 140100 180 220 260 300 340 380

50

420 460

F5

F6

(∘C)

Figure 5 DSC thermogram of physical mixture of chitosan-dextran(F3 F4 F5 and F6)

gel without EGF and untreated group (control group) areshown in Figure 7 Each wound was observed for a periodof 1 4 8 and 11 days after operation All rats survivedin the postoperative period until sacrifice There were noevidences of necrosis The formulation of CDP gels with andwithout EGF inducedno infection of thewoundswhereas thecontrol group showed wound infection at 4 and 8 days afteroperation The relative size reduction of the wounds treatedwith CDP gel with EGF CDP gel without EGF and untreatedwounds is illustrated in Figure 8 At 8 and 11 days afteroperation a significant size reduction in the wounds treatedwith CDP gel with EGF compared to CDP gel without EGFtreated groups and control groupwas observed Furthermorethe CDP gels with EGF gave about 90ndash95 of wound sizereduction at 11 days after operation

4 Histological Examination

As shown in Figure 9 at the end of day 8 there is not anysignificant difference in diameter of ulcers in different groupsof study (119875 ge 005) However the group treated by CDPgel with EGF reveals the highest rate of new epitheliumformation The greatest diameter of ulcer in CDP gel withEGF treated group at day 11 (392plusmn16) was significantly lesserthan CDP gel without EGF treated group (46 plusmn 132) andcontrol group (68 plusmn 18) (119875 le 005)

The reepithelialization diameter of the groups (Figure 11)treated with gels containing EGF was 11 plusmn 065 and 1925 plusmn153 at 8 and 11 days after operation while the control grouprevealed 096 plusmn 02 and 1175 plusmn 055 cm at 8 and 11 days afteroperation In days 8 and 11 of reepithelialization improvementin diameter in group treated with CDP gel containing EGFwas highest (not statistically significant) The microscopicphotos of the wounds at day 11 are shown in Figure 10Woundepithelium thickness was of quite similar values in all groups(approximately 150 120583m) data not shown

5 Discussion

In this study a novel drug delivery system composed ofa combination of three gels carrying EGF was preparedcharacterized and studied in vivo Our experiments showthat CDP gel could be used as a novel improved carrierfor EGF EGF could induce the wound healing processand minimize the scar formation EGF could induce cellproliferation and accelerate wound healing process and skinrepair through regulation of extracellularmatrix proliferationand epidermal stem cells differentiation Thus the localincrease in concentration of EGF at the wound site couldaccelerate the wound healing process [22 23] Followingtrauma the level of endogenous EGF increases to acceleratetissue repair process but the amount of endogenous EGF isnot sufficient for suitable wound repair Thus the applicationof exogenous EGF on the wound site could accelerate thewound healing process [24 25] Tanaka et al showed that thecontinuous use of EGF is a necessary factor for maximumwound healing [25] Moreover they demonstrated that theuse of EGF in gel formulations was more effective for woundhealing promotion than the use of EGF in solutions Thegel formulations could remain on the wound surface fora long time and provide a suitable concentration of EGFon the wounded area while solutions have fast drainage[26] Moreover gel based wound dressings are suitableformulations for the treatment of wounds and burns becausethey absorb the exudates of wounds and maintain suitablemoisture on the wound surface [27 28] In practice suitablegel based wound dressings could be applied easily [29 30]and could be removed easily since the skin is injured [31] Inthe present novel drug delivery system we were able to takeadvantage of the different properties of different polymers


103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(a)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(b)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(c)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(d)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(e)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(f)

Figure 6 The strain sweep test of gel (a) F1 (b) F2 (c) F3 (d) F4 (e) F5 (f) F6


(a) (b) (c)

Figure 7 Photographs of the relative size reduction of the woundsin rat (a) Wound treated with CDP gel containing EGF (b) woundtreated with CDP gel without EGF and (c) untreated group (controlgroup) on days 1 4 8 and 11 after operation (119899 = 3 mean plusmn SD)

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8 9 10 11 12

Relat

ive w

ound

size

Time (day)

Hydrogel with EGF treated groupHydrogel treated groupControl group

Figure 8 The relative size reduction of the wounds treated withCDP gel with EGF CDP gel without EGF and untreated wounds(119899 = 3 mean plusmn SD)

(chitosan and dextran) in wound healing while preventinginstability due to electrostatic interactions between chitosanand dextran polymers A mixture of chitosan and dextransulfate forms a gel at room temperature but electrostatic inter-actions between amino groups of chitosan and sulfate groups

0

1

2

3

4

5

6

7

8

9

10

DH1 H1 C

Wou

nd d

iam

eter

Day 8Day 11

Figure 9 Histopathological wound diameter evaluation of thewounds treated with CDP gel with EGF (DH1) CDP gel withoutEGF (H1) and untreated wounds (c)

Figure 10 Photographs of treatedwound areas (A11) wound treatedwith CDP gel containing EGF (B11) wound treated with CDP gelswithout EGF and (C11) untreated group (control group) on day 11after operation Scale bar 1mm

0

05

1

15

2

25

Reep

ithel

ializ

atio

n

DH1 H1 C

Day 8Day 11

Figure 11 Histopathological reepithelialization diameter evaluationof the wounds treated with CDP gel containing EGF (DH1) CDP gelwithout EGF (H1) and untreated wounds


of dextran sulfate could cause the formation of chitosan-dextran precipitation To prevent formation of chitosan-dextran sulfate precipitate PVP was used to decrease thechitosan and dextran interactions Similar pattern of theDSC thermograms obtained from CDP gel and chitosandextran and PVP gels suggests that no chemicalmodificationoccurs in CDP gel [32] The presence of PVP is probably alsopreventing a potential gel destabilization due to the change inratio of the two other components (chitosan and dextran) inthe final gel which could likely leave one of the components inexcess with respect to the otherMoreover EGF has a negativecharge at pH above 46 (isoelectric pH of EGF) [33] thus theelectrostatic interaction between negatively charged EGF andamino groups of chitosan could also sufficiently entrap EGFin the CDP gel

In the present study the results obtained from gel fractionstudies show that F4 CDP gel formulation has the highest gelfraction An ideal wound dressing should be able to absorbthe exudates of wound and decrease the risk of microbialinfection that could initiate through the increased accumula-tion of wound exudates Moreover a suitable wound dressingshould maintain the moisture on the surface of the woundduring treatment to prevent scabbing The conventionalwound dressings absorb the wound moisture and fully dryup the damaged site causing the bandaging to stick to thewound leading to further damage Therefore the swellingbehavior and absorption capacity of gels could be importantcriteria in the design of a suitable gel based wound dressingThe high swelling ratio of F4 formulation which could inpart be contributed to lower percentage of PVP as comparedto F1 formulation shows that this CDP gel could provide anadequate drainage of wound exudates and suitable levels ofmoisture on the wound surface Moreover the combinatoryantimicrobial effects of chitosan and dextran could protectthe wound from the external contamination Thus CDP gelhas different complimentary properties that make it suitablefor promotingwound healing Generally as the gel fraction ofgel increases the strength of the gel increases [34 35] whichmakes F4 formulation a suitable gel for in vivo studies

The prepared gel showed appropriate rheological prop-erties In strain sweep tests large differences were observedbetween the elastic 1198661015840 and the loss 11986610158401015840 moduli with 1198661015840being much higher than 11986610158401015840 which could indicate the higherviscoelastic properties of the prepared gel A small viscoelas-tic ldquoplateaurdquo and a rapid decrease in modulus resulting ina very small LVR (linear viscoelastic region) indicate thatthe structure is a ldquoweak gelrdquo and ready to flow at higherstrain [36 37] The CDP gel revealed viscoelastic propertieswith a small LVR (linear viscoelastic region) of 0166 Thisnonlinear rheological behavior can be seen in gels based onphysical interaction (eg hydrogen bond ionic interactionand entanglement of polymeric chain) This in turn couldbe contributed to partial and not complete cross linking thatallows the slidingmovement of polymeric domains over eachother [38 39] and indeed this property seems to be importantdue to the nature of the skin injury Flow measurementwas conducted to determine the plastic deformation stressthat is the yield point of gels The higher the yield pointis the more deforming resistant the gel is Although all

prepared gels are found to have physical and weak ionicinteraction F4 prepared with low molecular weight chitosan(16) 02 dextran sulfate (10 KD MW) and 02 of PVPhas a higher yield point around 64 Pa and is therefore moreresistant to deformation Mechanical properties of gels arevery important for pharmaceutical applications For examplemaintaining the integrity of a drug delivery device during thelifetime of the application is crucial

The results of our study showed a significant promotionin the wound healing process in the group given CDP gelcontaining EGF treatment compared to treatment with thesame gel without EGF and control group Furthermorepathological studies showed that epidermis length had alsoincreased significantly in the CDP gel containing EGF treatedgroup compared to CDP gel treated group or controlgroup The results indicate that CDP gel combined withEGF could significantly improve epidermal cell proliferationand differentiation thereby promoting wound healing Itis though important to note that in our experiments thecontrol group showed infection a phenomenon that can leadto delay in the healing process In healing process of skinwounds both epithelial and mesenchymal cells (fibroblasts)are involved Fibroblasts are in charge of repair of dermis andthey proliferate and release extracellular matrix These twolead to repair of dermis with keloid formation and resultantcontracture of wound On the other hand epithelial cellsproliferate and cover the keloid to make a complete repair Ifthey fail to cover all the surface of keloid scar of woundwouldremain On the other hand animals receiving the EGF-freeF4 gel showed less infection whichmight be due to the higherpercentage of chitosan EGF especially might be reactingwith polyelectrolytes and thus further increasing the amountof available chitosan fortifying the antibacterial propertiesFinally CDP gels containing EGF accelerated the woundhealing process in vivo Prepared gels could be easily appliedon the wound and removed from the siteMoreover rising theconcentration of EGF at the wound site following applicationof the gels could promotefacilitate wound healing processwith a low chance of scar formation

6 Conclusions

In this study chitosan-dextran-PVP gel (CDP gel) prepared asa novel and suitable gel based drug delivery system intendedto increase the bioavailability of therapeutic peptides andproteins such as EGF toward wound tissue Wound dressingprepared using chitosan and dextran could absorb the woundexudates and hold the moisture providing a suitable mois-turized environment at the wound site But the electrostaticinteraction between dextran and chitosan could reduce thestability of dextran-chitosan gels In comparison with othermarketed gel based wound dressing products prepared withchitosan and dextran in this study polyvinylpyrrolidone K30(PVP K30) was applied as a suitable polymer to reduce thedextran-chitosan instability (CDP gel) CDP gels containingEGF accelerated the wound healing process in vivo whileeasily applied to or removed from the wound As a con-clusion the present gel can be applied as a suitable wound


dressing to increase EGF concentration at wound site and topromotefacilitate wound healing with a low chance of scarformation

Abbreviations

PVP K30 Polyvinylpyrrolidone K30CDP gels Chitosan dextran sulfate and

polyvinylpyrrolidone K30 gelsPDGF Platelet derived growth factorEGF Epidermal growth factorrhEGF Recombinant human epidermal growth

factor

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank the kind technical helpof the colleagues in the nanomedicine lab N Khanbanhaconducted the experimental work and helped in drafting thepaper F Atyabi conceived the study and is the correspondingauthor of the work R Dinarvand supervised the work MMahbod performed the histology experiments A Taherihelped with the interpretation of the data analysis F Talaiereviewed and edited the paper All authors read and approvedthe final paper

References

[1] AGosain and LADiPietro ldquoAging andwoundhealingrdquoWorldJournal of Surgery vol 28 no 3 pp 321ndash326 2004

[2] D Mathieu J C Linke and F Wattel ldquoNon-healing woundsrdquoin Handbook on Hyperbaric Medicine pp 401ndash428 2006

[3] W K Stadelmann A G Digenis and G R Tobin ldquoPhysiologyand healing dynamics of chronic cutaneous woundsrdquo TheAmerican Journal of Surgery A vol 176 no 2 supplement 1 pp26Sndash38S 1998

[4] G Broughton II J E Janis andC E Attinger ldquoThebasic scienceof wound healingrdquo Plastic and Reconstructive Surgery vol 117no 7 pp 12Sndash34S 2006

[5] G L Brown L B Nanney J Griffen et al ldquoEnhancement ofwound healing by topical treatment with epidermal growthfactorrdquoTheNew England Journal of Medicine vol 321 no 2 pp76ndash79 1989

[6] L B Nanney ldquoEpidermal and dermal effects of epidermalgrowth factor during wound repairrdquo Journal of InvestigativeDermatology vol 94 no 5 pp 624ndash629 1990

[7] A Buckley J M Davidson C D Kamerath T B Wolt andS C Woodward ldquoSustained release of epidermal growth factoraccelerates wound repairrdquo Proceedings of the National Academyof Sciences of the United States of America vol 82 no 21 pp7340ndash7344 1985

[8] L L Lloyd J F Kennedy P Methacanon M Paterson and CJ Knill ldquoCarbohydrate polymers as wound management aidsrdquoCarbohydrate Polymers vol 37 no 3 pp 315ndash322 1998

[9] Q Bi Q Zhang J Ma et al ldquoEffect of combination therapywith alginate dressing and mouse epidermal growth factoron epidermal stem cells in patients with refractory woundsrdquoChinese Medical Journal vol 125 no 2 pp 257ndash261 2012

[10] M J MMeddens JThompson P C J Leijh and R Van FurthldquoRole of granulocytes in the induction of an experimental endo-carditis with a dextran-producing Streptococcus sanguis andits dextran-negative mutantrdquo British Journal of ExperimentalPathology vol 65 no 2 pp 257ndash265 1984

[11] P Yousefpour F Atyabi R Dinarvand and E Vasheghani-Farahani ldquoPreparation and comparison of chitosan nanopar-ticles with different degrees of glutathione thiolationrdquo DARUJournal of Pharmaceutical Sciences vol 19 no 5 pp 367ndash3752011

[12] P Yousefpour F Atyabi E V Farahani R Sakhtianchi andR Dinarvand ldquoPolyanionic carbohydrate doxorubicin-dextrannanocomplex as a delivery system for anticancer drugs invitro analysis and evaluationsrdquo International Journal of Nano-medicine vol 6 pp 1487ndash1496 2011

[13] L Frank C Lebreton-Decoster G Godeau B Coulomb andJ Jozefonvicz ldquoDextran derivatives modulate collagen matrixorganization in dermal equivalentrdquo Journal of BiomaterialsScience Polymer Edition vol 17 no 5 pp 499ndash517 2006

[14] G Biagini A Bertani RMuzzarelli et al ldquoWoundmanagementwith N-carboxybutyl chitosanrdquo Biomaterials vol 12 no 3 pp281ndash286 1991

[15] S Saremi F Atyabi S P Akhlaghi S N Ostad and RDinarvand ldquoThiolated chitosan nanoparticles for enhancingoral absorption of docetaxel preparation in vitro and ex vivoevaluationrdquo International Journal of Nanomedicine vol 6 no 1pp 119ndash128 2011

[16] M Mahmoodi M E Khosroshahi and F Atyabi ldquoLaserthrombolysis and in vitro study of tpa release encapsulatedby chitosan coated plga nanoparticles for amirdquo InternationalJournal of Biology and Biomedical Engineering vol 4 no 2 pp35ndash42 2010

[17] E B Denkbas E Ozturk N Ozdemi K Kececi and MA Ergun ldquoEGF loaded chitosan sponges as wound dressingmaterialrdquo Journal of Bioactive and Compatible Polymers vol 18no 3 pp 177ndash190 2003

[18] S P Akhlaghi S Saremi S N Ostad R Dinarvand andF Atyabi ldquoDiscriminated effects of thiolated chitosan-coatedpMMApaclitaxel-loadednanoparticles ondifferent normal andcancer cell linesrdquo Nanomedicine Nanotechnology Biology andMedicine vol 6 no 5 pp 689ndash697 2010

[19] S Abashzadeh R DinarvandM Sharifzadeh G HassanzadehM Amini and F Atyabi ldquoFormulation and evaluation of anin situ gel forming system for controlled delivery of triptorelinacetaterdquo European Journal of Pharmaceutical Sciences vol 44no 4 pp 514ndash521 2011

[20] F Atyabi F A Moghaddam R Dinarvand M J Zohuriaan-Mehr and G Ponchel ldquoThiolated chitosan coated poly hydrox-yethyl methacrylate nanoparticles synthesis and characteriza-tionrdquo Carbohydrate Polymers vol 74 no 1 pp 59ndash67 2008

[21] SThomas ldquoA structured approach to the selection of dressingsrdquoWorld Wide Wounds 1997

[22] S K Repertinger E Campagnaro J Fuhrman T El-AbaseriS H Yuspa and L A Hansen ldquoEGFR enhances early healingafter cutaneous incisional woundingrdquo Journal of InvestigativeDermatology vol 123 no 5 pp 982ndash989 2004


[23] D Woodbury E J Schwarz D J Prockop and I B BlackldquoAdult rat and human bone marrow stromal cells differentiateinto neuronsrdquo Journal of Neuroscience Research vol 61 no 4pp 364ndash370 2000

[24] X Fu X Li B Cheng W Chen and Z Sheng ldquoEngineeredgrowth factors and cutaneous wound healing success andpossible questions in the past 10 yearsrdquo Wound Repair andRegeneration vol 13 no 2 pp 122ndash130 2005

[25] A Tanaka T Nagate and H Matsuda ldquoAcceleration of woundhealing by gelatin film dressings with epidermal growth factorrdquoThe Journal of VeterinaryMedical Science vol 67 no 9 pp 909ndash913 2005

[26] E Eriksson and J Vranckx ldquoWet wound healing from labora-tory to patients to gene therapyrdquo American Journal of Surgeryvol 188 no 1 pp 36ndash41 2004

[27] J BergerM Reist J MMayer O Felt and R Gurny ldquoStructureand interactions in chitosan hydrogels formed by complexationor aggregation for biomedical applicationsrdquo European Journalof Pharmaceutics and Biopharmaceutics vol 57 no 1 pp 35ndash522004

[28] T Kiyozumi Y Kanatani M Ishihara et al ldquoMedium (DMEMF12)-containing chitosan hydrogel as adhesive and dressing inautologous skin grafts and accelerator in the healing processrdquoJournal of BiomedicalMaterials Research BApplied Biomaterialsvol 79 no 1 pp 129ndash136 2006

[29] J K Francis Suh andHWTMatthew ldquoApplication of chitosan-based polysaccharide biomaterials in cartilage tissue engineer-ing a reviewrdquo Biomaterials vol 21 no 24 pp 2589ndash2598 2000

[30] T Kiyozumi Y Kanatani M Ishihara et al ldquoThe effect ofchitosan hydrogel containing DMEMF12 medium on full-thickness skin defects after deep dermal burnrdquo Burns vol 33no 5 pp 642ndash648 2007

[31] A D Sezer E Cevher F Hatıpoglu Z Ogurtan Bas A L andJ Akbuga ldquoPreparation of fucoidan-chitosan hydrogel and itsapplication as burn healing accelerator on rabbitsrdquo Biological ampPharmaceutical Bulletin vol 31 no 12 pp 2326ndash2333 2008

[32] G Carpenter and S Cohen ldquoepidermal growth factorrdquo AnnualReview of Biochemistry vol 48 pp 193ndash216 1979

[33] T Dai M Tanaka Y Huang and M R Hamblin ldquoChitosanpreparations for wounds and burns antimicrobial and wound-healing effectsrdquo Expert Review of Anti-Infective Therapy vol 9no 7 pp 857ndash879 2011

[34] J O Kim J K Park J H Kim et al ldquoDevelopment of polyvinylalcohol-sodium alginate gel-matrix-based wound dressing sys-tem containing nitrofurazonerdquo International Journal of Phar-maceutics vol 359 no 1-2 pp 79ndash86 2008

[35] Z Ajji I Othman and J Rosiak ldquoProduction of hydrogelwound dressing using gamma radiationrdquo Aalam Al-Zarra pp58ndash62 2006

[36] S S Anumolu A S DeSantis A R Menjoge et al ldquoDoxy-cycline loaded poly(ethylene glycol) hydrogels for healingvesicant-induced ocularwoundsrdquoBiomaterials vol 31 no 5 pp964ndash974 2010

[37] M J Moura M M Figueiredo and M H Gil ldquoRheologicalstudy of genipin cross-linked chitosan hydrogelsrdquo Biomacro-molecules vol 8 no 12 pp 3823ndash3829 2007

[38] H D Lu M B Charati I L Kim and J A Burdick ldquoInjectableshear-thinning hydrogels engineered with a self-assemblingDock-and-Lock mechanismrdquo Biomaterials vol 33 no 7 pp2145ndash2153 2012

[39] S R van Tomme M J van Steenbergen S C de Smedt C Fvan Nostrum andW E Hennink ldquoSelf-gelling hydrogels basedon oppositely charged dextranmicrospheresrdquo Biomaterials vol26 no 14 pp 2129ndash2135 2005

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


chitosan could be useful in relation to wound treatment [14]Chitosan and its derivatives have been used successfully forcontrolled delivery of drugs peptides and proteins [15ndash20]Wound dressing prepared using chitosan and dextran couldabsorb thewound exudates and hold themoisture providing asuitable moisturized environment at the wound site [21] Butthe electrostatic interaction between dextran and chitosancould reduce the stability of dextran-chitosan gels We usedpolyvinylpyrrolidone K30 (PVP K30) as suitable polymer toreduce the dextran-chitosan instability (CDP gel) In thiswork we prepared CDP gel as a suitable gel based drugdelivery system to increase the bioavailability of therapeuticpeptides and proteins such as EGF to wound tissueThen weimpregnated EGF in CDP gel EGF impregnated CDP gel wasused as suitable wound dressing to accelerate and facilitatewound healing process Physical properties of wound dress-ing and the effects of EGF on healing process in comparisonwith controls were studied in full thickness excision woundmodel in rats in vivo

2 Material and Methods

21 Materials Already characterized low and mediummolecular weight chitosan samples (deacetylation degreeapproximately 95) were purchased from Chitoclear (Siglu-jordur Iceland) Dextran sulfate (MW 8 and 10 kDa) waspurchased from Sigma-Aldrich (St Louis MO USA)Polyvinylpyrrolidone K30 (PVP K30) was purchased fromBASF (Ludwigshafen Germany) Recombinant human epi-dermal growth factor (rhEGF) was obtained from SichuanHuamai Technology Co (Sichuan China) All other reagentsused were of analytical grade

22 Preparation of Gels

221 Preparation of Chitosan Solutions Chitosan solutionsof low andmediummolecular weight were prepared in diluteacetic acid (1ww) and themixtures were heated and stirredat 50∘C for 24 h until clear solutions were achieved Chitosansolutions were neutralized with 1N solution of NaOH topH 51 and then were degassed at 25∘C for 15min using asonicator (Elmasonic Elma Hans Schmidbauer GmbH amp CoKG) to remove air bubbles

222 Preparation of Dextran-PVP Solution Dextran sulfate(MW8 and 10 kDa) was dissolved in deionized water Knownamount of PVPK30was added to the above solution to obtaindextran-PVP solution

223 Preparation of CDP-EGF Gels For preparation ofchitosan-dextran-PVP (CPD) gels freshly prepared chitosansolution was added dropwise to dextran-PVP solution underslow stirring to achieve the final composition of gel for-mulations as given in Table 1 For preparation of CDP-EGFgels EGF was added to prepared CDP gels to obtain aconcentration of 20120583gmL

23 Swelling Study Swelling of gels was studied by a gravi-metric procedure CDP gels were cast into 13 diameter times 45height (approximately 059mm3) molds and then vacuumdried at 60∘C for 12 h Dried gels were accurately weightedand swollen in PBS pH 74 at 25∘C The swollen gels wereremoved from the buffers at certain times (5 10 20 30 4560 and 120min) then blotted to remove excess water andreweighed Each experiment was performed in triplicateTheswelling ratio was calculated using the following equation

SR =119882119904

119882119889

(1)

where119882119904is the weight of the swollen gel and119882

119889is the weight

of the dried gelTo characterize the swelling behavior of the gels the

following experiments were conducted To determine theeffect of pH on the swelling ratio of gels CDP gels wereimmersed in acetate buffer pH 45 and phosphate buffer pH9 at 25∘C for 5 10 20 30 45 60 and 120min The swellingratio was calculated as mentioned above The effect of ionicstrength on the swelling ratio of gels wasmeasured in 1N PBSpH 74 4N PBS pH 74 and 8N PBS pH 74 at 25∘C for 5 1020 30 45 60 and 120min as mentioned above

231 Gel Fraction Different formulations of gels were castinto 059mm3 molds and then vacuum dried at 50∘C for 12 huntil a constant weight was reached (119882

119889)The dried gels were

immersed in 40mL distilled water at 50∘C for 12 h undervacuum The sol part of immersed gels was extracted fromthe water The remaining part was vacuum dried at 50∘C for12 h till constant weight was achieved (119882

119903) The gel fraction

percentage was determined using the following equation

Gel Fraction () = (119882119903

119882119889

) times 100 (2)

24 Differential Scanning Calorimetry (DSC) Analysis Thephysical or chemical interaction between polymers wasanalyzed by differential scanning calorimetry (DSC) studiesSamples were analyzed using a differential scanning calori-metric (DSC) analyzer (DSC 823 Mettler-Toledo Germany)5mg of the mixture of chitosan and dextran sulfate orchitosan-dextran sulfate dried gels were placed in aluminumpans For thermogram acquisition the samples were heatedover a range of 30∘C to 500∘C at a rate of 10∘C per min andthe thermal behavior of samples was recorded

241 Rheological Studies To study the plastic deformationstress and the viscoelastic properties of the gel the rheologicalanalysis of the prepared gels was performed using a stresscontrolled Paar Physica MCR 300 (Anton Paar GmbHAustria) with a plate-plate measurement system PP25 (thegap between the plates was 1mm) The temperature was25∘C for all tests The data were collected using Rheoplus32Service V340 software (Anton Paar GmbH Austria) Gelswere placed between the plates of the measuring system andtwo sets of experiments were performed








F1 133 033 033F2 15 025 025F3 16 02 02F4 16 02 02F5 16 02 02F6 16 02 02







3 Results





0

10

20

30

40

50

60

0 20 40 60 80 100 120 140

Swel

ling

ratio

Time (min)

F1F2F3

F4F5F6






11986610158401015840

1198661015840

= tan 120575 (3)


Swel

ling

ratio

0

10

20

30

40

50

60

70

80

pH 45pH 74pH 9

0 20 40 60 80 100 120 140Time (min)


1X PBS4X PBS8X PBS

0 20 40 60 80 100 120 140Time (min)

0

10

20

30

40

50

60

Swel

ling

ratio


01020304050607080

f1 f2 f3 f4 f5 f6

Gel

fra

ctio

n

Formulation





1015840




Physical mixture

F3

F4

20 60 140100 180 220 260 300 340 380

50

420 460

F5

F6

(∘C)






5 Discussion



103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(a)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(b)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(c)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(d)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(e)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(f)



(a) (b) (c)


0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8 9 10 11 12

Relat

ive w

ound

size

Time (day)




0

1

2

3

4

5

6

7

8

9

10

DH1 H1 C

Wou

nd d

iam

eter

Day 8Day 11



0

05

1

15

2

25

Reep

ithel

ializ

atio

n

DH1 H1 C

Day 8Day 11








6 Conclusions




Abbreviations



factor



Acknowledgments


References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of








F1 133 033 033F2 15 025 025F3 16 02 02F4 16 02 02F5 16 02 02F6 16 02 02







3 Results





0

10

20

30

40

50

60

0 20 40 60 80 100 120 140

Swel

ling

ratio

Time (min)

F1F2F3

F4F5F6






11986610158401015840

1198661015840

= tan 120575 (3)


Swel

ling

ratio

0

10

20

30

40

50

60

70

80

pH 45pH 74pH 9

0 20 40 60 80 100 120 140Time (min)


1X PBS4X PBS8X PBS

0 20 40 60 80 100 120 140Time (min)

0

10

20

30

40

50

60

Swel

ling

ratio


01020304050607080

f1 f2 f3 f4 f5 f6

Gel

fra

ctio

n

Formulation





1015840




Physical mixture

F3

F4

20 60 140100 180 220 260 300 340 380

50

420 460

F5

F6

(∘C)






5 Discussion



103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(a)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(b)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(c)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(d)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(e)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(f)



(a) (b) (c)


0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8 9 10 11 12

Relat

ive w

ound

size

Time (day)




0

1

2

3

4

5

6

7

8

9

10

DH1 H1 C

Wou

nd d

iam

eter

Day 8Day 11



0

05

1

15

2

25

Reep

ithel

ializ

atio

n

DH1 H1 C

Day 8Day 11








6 Conclusions




Abbreviations



factor



Acknowledgments


References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

10

20

30

40

50

60

0 20 40 60 80 100 120 140

Swel

ling

ratio

Time (min)

F1F2F3

F4F5F6






11986610158401015840

1198661015840

= tan 120575 (3)


Swel

ling

ratio

0

10

20

30

40

50

60

70

80

pH 45pH 74pH 9

0 20 40 60 80 100 120 140Time (min)


1X PBS4X PBS8X PBS

0 20 40 60 80 100 120 140Time (min)

0

10

20

30

40

50

60

Swel

ling

ratio


01020304050607080

f1 f2 f3 f4 f5 f6

Gel

fra

ctio

n

Formulation





1015840




Physical mixture

F3

F4

20 60 140100 180 220 260 300 340 380

50

420 460

F5

F6

(∘C)






5 Discussion



103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(a)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(b)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(c)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(d)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(e)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(f)



(a) (b) (c)


0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8 9 10 11 12

Relat

ive w

ound

size

Time (day)




0

1

2

3

4

5

6

7

8

9

10

DH1 H1 C

Wou

nd d

iam

eter

Day 8Day 11



0

05

1

15

2

25

Reep

ithel

ializ

atio

n

DH1 H1 C

Day 8Day 11








6 Conclusions




Abbreviations



factor



Acknowledgments


References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




1015840




Physical mixture

F3

F4

20 60 140100 180 220 260 300 340 380

50

420 460

F5

F6

(∘C)






5 Discussion



103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(a)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(b)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(c)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(d)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(e)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(f)



(a) (b) (c)


0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8 9 10 11 12

Relat

ive w

ound

size

Time (day)




0

1

2

3

4

5

6

7

8

9

10

DH1 H1 C

Wou

nd d

iam

eter

Day 8Day 11



0

05

1

15

2

25

Reep

ithel

ializ

atio

n

DH1 H1 C

Day 8Day 11








6 Conclusions




Abbreviations



factor



Acknowledgments


References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(a)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(b)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(c)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(d)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(e)

103

102

101

G998400

G998400998400

001 01 1 10 100

G998400

G998400998400

(Pa)

Strain 120574

(f)



(a) (b) (c)


0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8 9 10 11 12

Relat

ive w

ound

size

Time (day)




0

1

2

3

4

5

6

7

8

9

10

DH1 H1 C

Wou

nd d

iam

eter

Day 8Day 11



0

05

1

15

2

25

Reep

ithel

ializ

atio

n

DH1 H1 C

Day 8Day 11








6 Conclusions




Abbreviations



factor



Acknowledgments


References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


(a) (b) (c)


0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8 9 10 11 12

Relat

ive w

ound

size

Time (day)




0

1

2

3

4

5

6

7

8

9

10

DH1 H1 C

Wou

nd d

iam

eter

Day 8Day 11



0

05

1

15

2

25

Reep

ithel

ializ

atio

n

DH1 H1 C

Day 8Day 11








6 Conclusions




Abbreviations



factor



Acknowledgments


References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of







6 Conclusions




Abbreviations



factor



Acknowledgments


References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



Abbreviations



factor



Acknowledgments


References













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

research article healing efficacy of an egf impregnated...

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